In our search for species-dependent functional TPC1 channel variants with different luminal Ca2+ sensitivity, we found in total three acidic residues present in Ca2+ sensor sites 2 and 3 of the Ca2+-sensitive AtTPC1 channel from Arabidopsis thaliana that were neutral in its Vicia faba ortholog and also in those of many other Fabaceae. When expressed in the Arabidopsis AtTPC1-loss-of-function background, wild-type VfTPC1 was hypersensitive to vacuole depolarization and only weakly sensitive to blocking luminal Ca2+. When AtTPC1 was mutated for these VfTPC1-homologous polymorphic residues, two neutral substitutions in Ca2+ sensor site 3 alone were already sufficient for the Arabidopsis At-VfTPC1 channel mutant to gain VfTPC1-like voltage and luminal Ca2+ sensitivity that together rendered vacuoles hyperexcitable. Thus, natural TPC1 channel variants exist in plant families which may fine-tune vacuole excitability and adapt it to environmental settings of the particular ecological niche.To fire action-potential-like electrical signals, the vacuole membrane requires the two-pore channel TPC1, formerly called SV channel. The TPC1/SV channel functions as a depolarization-stimulated, non-selective cation channel that is inhibited by luminal Ca However, the physiological relevance of a potential Ca2+ permeability in planta is highly controversial  identified the TPC1 mutant fou2 (fatty acid oxygenation upregulated 2) (fou2 channel behaves like a hyperactive TPC1 channel (fou2 TPC1 channel toward voltage confers hyperexcitability to the vacuole and results from a mutation of the negatively charged glutamate at position 454 to the uncharged asparagine (D454N)  . Interes channel  and open channel . In cont (D454N) . This TPr site 1 . Therefo2 mutant . Consequasmonate . Interesynthesis . Howeverynthesis . Functior site 2 . Neutral opening .Vicia faba and A. thaliana are historically established prototypes of the SV channel. Nevertheless, only AtTPC1 but not VfTPC1 has been identified at the molecular level so far. To fil this gap, we cloned VfTPC1 and analyzed its electrical properties in the background of the AtTPC1-loss-of-function Arabidopsis mutant. Despite similarities of the fava bean channel with its Arabidopsis homolog, both TPC1s showed also astonishing differences in voltage- and luminal Ca2+-dependent properties. In search for the structural reasons for this divergence, we could pinpoint three polymorphic sites within two luminal Ca2+ coordinating regions. When these polymorphic sites were implemented into the Arabidopsis TPC1 channel, AtTPC1 was converted into a hyperactive SV/TPC1 channel desensitized to luminal Ca2+, mimicking the properties of VfTPC1 and the AtTPC1 mutant fou2. Natural polymorphic TPC1 channel variants, like VfTPC1, with differences in key properties might be employed by some plant species to better meet their needs in specific environmental situations.The TPC1 channels from V. faba guard cell vacuoles even under unnatural high luminal Ca2+ loads  ) were determined for both TPC1 channel variants  and AtTPC1 . These results confirm that VfTPC1, unlike AtTPC1, activates near the vacuolar resting membrane voltage   but abseh AtTPC1 —4A, B. Ah AtTPC1 . This povariants . They we C1⇆ O) . From th derived , showing voltage .2+ concentration was increased from 0 to 10 mM Ca2+, the activation kinetics of AtTPC1 was strongly slowed down ), indicated by a 1.7- and 1.6-fold rise in V1 and V2, respectively (2+ (G/Gmax(V)) could not be resolved reliably. Together these findings document that VfTPC1 is much less susceptible to inhibitory luminal Ca2+ than AtTPC1.When the luminal Cawed down . Howeverwed down . Even monal Ca2+ , the connal Ca2+ . In conttivation . Compareectively . A rise ectively . AtTPC1 vely ) and even more so the activation voltage curves (G/Gmax(V)) of the double and triple AtTPC1 mutants appeared shifted to more negative voltages by about 30 mV compared to wild-type AtTPC1 , G/Gmax(V)) of the AtTPC1 mutants and VfTPC1, however, showed similar strong SV channel activity even at this extreme luminal Ca2+ level  were shifted to more positive values. Thus, the hyperactivity of the native V. faba TPC1 channel is most likely related to the altered Ca2+ sensor site 3 rather than to Ca2+ sensor site 2. Considering further the similar luminal Ca2+ sensitivity of E605A/D606N and E457N/E605A/D606N  as a template (fou2 (2+ sensor (site 3) that is coupled to the functional VSD2. In the wild-type and fou2 structures, the three luminal acidic residues  of site 3 line the conduction pathway upstream of the selectivity filter  clearly alters the electrostatics of the pore. These substitutions almost certainly prevent the pore entrance from binding the inhibitory Ca2+ observed in the wild-type structure . Higher luminal Ca2+ increases the probability of Ca2+ to occupy the coordination site which stabilized the E605/D606 motif in the pore-facing conformation. Neutralizing E605A/D606N mutations would reduce electrostatic interactions and result in channels that open with less activation energy. This effect could explain how an altered Ca2+-binding site at the luminal pore entrance of VfTPC1 – compared with AtTPC1 – shifts the voltage activation threshold to more negative values. The difference in the voltage activation profile between AtTPC1 and VfTPC1 may have been further enhanced by a luminal Mg2+ effect on the intact luminal pore E605/D606 motif of AtTPC1 wild type. Like Ca2+, also luminal Mg2+ inhibits the AtTPC1 channel but with a much lower efficiency than Ca2+ . The cation selectivity of AtTPC1 was first studied under bi-ionic conditions in the animal HEK cell expression system (2+ conditions (2+ sensor site 1 in the presence of a wild-type pore region was used. The relative permeability ratio PK/Ca/PNa of AtTPC1 was not affected by disruption of Ca2+ sensor site 3 (E605A/D606A) or Ca2+ sensor site 1 (D240A/D454A/E528A)  , the lat2+-activated K+ channels with large conductance, similar to TPC1 (+ conditions (100 mM) a single-channel conductance of about 80 pS was determined for AtTPC1 wild type  during the entire subsequent recording period of 10 s, regardless of the stimulus intensity , the voltage activation threshold of both, VfTPC1 and the AtTPC1 triple mutant E457N/E605A/D606N, was close to the resting membrane voltage. In comparison, AtTPC1 wild type showed a significant shift of the voltage activation threshold toward positive voltages  was found to depend on the stimulus strength (tplateau. A negatively shifted TPC1 activation curve (tplateau and stimulus strength was reduced (2+) tune the sensitivity of the vacuole membrane and shape the duration of the excited state. In comparison to AtTPC1-expressing vacuoles, those equipped with VfTPC1 or the AtTPC1 triple mutant, which both activate more negatively, are hyperexcitable . Here, we have now found that E605 and D606 play the most important role within this triad of site 3 in gating control in AtTPC1  are part of a Ca2+-induced Ca2+ release (CICR) network  prevent retarded growth and (2) benefit from a TPC1 channel variant with a greatly reduced luminal Ca2+ sensitivity for potentially better adaptation to their respective ecological niche.In well agreement with experiments with vacuoles , our catNa ≈ 5:1  may sugg 5:1  and attpc1-2 mutant  according to the manufacturer’s instructions. For guard cell RNA extraction epidermal fragments were isolated using the blender method , VfTPC1 and LjTPC1 were amplified from the cDNA libraries, essentially as described by After the total RNA was extracted from mature leaves of tpc1-2 Arabidopsis mutant plants and transiently transformed with the different TPC1 channel constructs. For channel expression, protoplasts were then stored in W5 solution ethanesulfonic acid)/Tris, pH 5.6, 50 μg ml−1 ampicillin) at 23°C in the dark for usually 2 days.Essentially following the protocols from The 293 embryonic renal epithelial cell line authenticated by STR profiling was acquired from ATCC . During laboratory research use the cell line identity has been authenticated by microscopy and the cell culture was negatively tested for mycoplasms by Eurofins . HEK293 cells were transfected with the respective eYFP-TPC1-fused constructs with Lipofectamine 2000  according to the manufacturer’s instructions. Cells were seeded 18–24 hr after transfection on glass coverslips (diameter 12 mm). Protein expression was verified on the single-cell level by the appearance of eYFP fluorescence in the plasma membrane upon excitation with a 473-nm laser.Vacuolar membrane localization of the expressed eGFP/eYFP-fused TPC1 channels was verified by imaging the fluorescence signal of transformed protoplasts and vacuoles with a confocal laser scanning microscope  . eGFP anA. thaliana wild-type protoplasts after their enzymatical isolation, essentially as described in attpc1-2 mutant in the recording chamber 2 days after transformation using a vacuole release (VR) solution (N-methyl-D-glucamine), 5 mM EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), 3 mM MgCl2, 10 mM HEPES(4-[2-hydroxyethyl]piperazine-1-ethanesulfonic acid)/Tris pH 7.5 and adjusted to 450 mOsmol kg−1 with D-sorbitol. The whole-vacuole configuration was then established either with wild-type vacuoles or TPC1-transformed vacuoles. The latter vacuoles were easily identified upon their eGFP- or eYFP-based fluorescence measured between 510 and 535 nm after excitation at 480 nm with a precisExcite HighPower LED lamp . Patch pipettes were prepared from Harvard glass capillaries  and typically had a resistance in the range of 1.4–3.1 MΩ. The membrane capacitance (Cm) of the individual vacuoles accessed and compensated in patch-clamp experiments ranged from 31.1 to 68.5 pF. When the seal was stable throughout the experiment, membrane currents and voltages were recorded with a sampling rate of 150 µs at a low-pass filter frequency of 2.9 or 3 kHz using an EPC10 or EPC800 patch-clamp amplifier, respectively (HEKA Electronic). Data were acquired with the software programs Pulse or Patchmaster (HEKA Electronic) and offline analyzed with IGORPro (Wave Metrics).Mesophyll vacuoles were liberated from solution . The VR tact-0.5) and steady-state current amplitudes (Iss). The half-activation time (tact-0.5) was the time at which 50% of the steady-state current amplitude was reached. As a normalization measure for the membrane surface of the individual vacuole, the determined steady-state currents were divided by the respective compensated membrane capacitance (Cm). Conductance/voltage curves (G/Gmax(V)) were quantified from tail current experiments as a measure for the relative voltage-dependent open-channel probability. Following pre-pulse voltages in the range of −80 to +110 mV, instantaneous tail currents were determined at −60 mV. The midpoint voltages  and the equivalent gating charges  were derived by fitting the G/Gmax(V) curves with a double Boltzmann equation (tdeact-0.5) were determined at which the initial tail current amplitude has declined by 50%.Voltage recordings were carried out with TPC1-transformed vacuoles in the current-clamp mode as described by equation . After p2, 10 mM HEPES (pH 7.5/Tris) and was adjusted with D-sorbitol to an osmolality of 520 mOsmol kg−1. The standard pipette solution at the luminal side of the tonoplast basically consisted of 150 mM KCl, 2 mM MgCl2, 10 mM HEPES (pH 7.5/Tris) and was adjusted with D-sorbitol to a final osmolality 500 mOsmol kg−1. The pipette solution was supplemented with 10 or 50 mM CaCl2 or adjusted to 0 mM Ca2 by addition of 0.1 mM EGTA. Current-clamp experiments (2 and the pipette solution was adjusted to 0.2 mM free Ca2+ by addition of 4.1 mM EGTA and 4.3 mM Ca2+ (maxchelator/webmaxc/webmaxcS.htm). It should be noted that voltage- and current-clamp experiments with non-transformed A. thaliana attpc1-2 vacuoles as negative controls have been published previously by In the voltage-clamp experiments of eriments  and correriments  were per–1 with D-sorbitol. Excised membrane patches with the cytosolic side of the tonoplast facing the bath medium were formed from the whole-vacuole configuration. Single-channel fluctuations were recorded with a sampling rate of 100 µs at a low-pass filter frequency of 1 kHz using an EPC10 patch-clamp amplifier. Single-channel current amplitudes were determined from all-point histograms of the current recordings and plotted against the respective voltages. The single-channel conductance for each membrane patch was derived from linear regression of the current–voltage plot. The bath medium contained 100 mM KCl, 0.5 mM CaCl2, 10 mM HEPES (pH 7.5/Tris). The pipette medium consisted of 100 mM KCl, 2 mM MgCl2, 2 mM EGTA, 10 mM MES (pH 5.5/Tris). Both solutions were adjusted to 400 mOsmol kg−1 with sorbitol.Vacuoles were isolated from transiently transformed protoplasts by perfusion with a solution containing 10 mM EGTA, 10 mM HEPES/Tris pH 7,5, adjusted to 200 mOsmol kg2, 0.3 mM free Ca2+ (adjusted by 4.3 mM CaCl2 and 4 mM EGTA), 10 mM HEPES (pH 7.4/Tris). The standard bath solution was composed of 150 mM NaCl, 10 mM HEPES (pH 7.4/Tris). Under these experimental conditions bath and pipette solutions mimicked the vacuolar and cytosolic solute conditions, respectively. To determine the relative permeability ratio on the same HEK cell under bi-ionic cation conditions, the Na+-based bath medium was replaced by either a K+- or Ca2+-based solution. The K+-based bath medium contained 150 mM KCl, 10 mM HEPES (pH 7.4/Tris), and the Ca2+-based one consisted of 15 mM CaCl2, 120 mM NMDG-Cl, 10 mM HEPES (pH 7.4/Tris). Only cells were included in the analysis that exhibited stable seal conditions throughout the whole set of solution exchange.Whole-cell current recordings were performed at a setup described previously . Data weVrev caused by the change of the external solution from either Na+ to K+ or Na+ to Ca2+ solution was used to estimate the relative permeability ratios PCa/PNa or PK/PNa essentially as described by + to Na+ permeability ratio (PK/PNa) the following For determination of the TPC1 ion selectivity, tail current experiments were conducted. After pre-activating the TPC1 channels by a voltage step from −70 mV  to +100 mV for 500–1000 ms, the relaxation of the outward currents in response to hyperpolarizing voltage pulses was recorded either with 10 mV intervals reaching from +60 to −120 mV or in 5 mV decrements reaching from +30 to −40 mV to visualize and to analyze the reversal potentials, respectively. After the double-voltage pulse, the membrane was clamped again to the holding voltage of −70 mV. The total time interval between the double-voltage pulses at holding voltage was 3.4 s. In order to clearly assign the reversal potential to TPC1 currents, which are not contaminated by leakage currents, the slope of the tail currents was determined and plotted against the corresponding voltages. The reversal potential for each solute condition was then determined by interpolation the smallest negative and positive slope values. The shift in the reversal potential ΔVrev = VK- VNa, with the reversal potentials VK and VNa in K+- or Na+-based bath solution, respectively, and α = RT/F = 25.42 mV at 22°C. The relative permeability ratio PCa/PNa was calculated using where ΔVrev = VCa - VNa with the reversal potentials VCa and VNa in Ca2+- or Na+-based bath solution, respectively, and α has same meaning as above.where Δ2+  and G/Gmax(V) curves for wild-type AtTPC1 acquired under 0 and 10 mM luminal Ca2+ were identical to those shown in 2+-induced shifts in the V1/2 values for significant differences, the V1 and V2 mean values determined in the absence of luminal Ca2+ were subtracted from the individual V1 and V2 values, respectively, derived under 10 mM luminal Ca2+ conditions  and SPSS 2020 .Patch-clamp experiments were conducted with individual vacuoles of ectively . Due to nditions . In analfou2) Ca2+-bound structure  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Leon D Islas as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Detlef Weigel as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Vivek Garg (Reviewer #2).Thank you for submitting your article "Vicia faba SV channel VfTPC1 is a hyperexcitable variant of plant vacuole Two Pore Channels" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:After consultation, the reviewers agree that the following experiments are needed:1. Reverse mutations in VfTPC1 need to be made and tested.2+ on open probability would help provide crucial insight into channel gating .2. Expanding the single channel data to include the effect of CaIn addition, please attend to the reviewer's individual comments, which are aimed to improve the clarity of the data presentation and the manuscript's conclusions.Reviewer #1 (Recommendations for the authors):1) The order of presentation of findings in the text and the order of the figures is confusing. It jumps back and forth. The figures should be presented in the same order as they are refered to in the text.2+ is puzzling, the tail currents are much slower than any other channels studied in the same condition and do not correspond to the findings in Figure 3 (High Ca2+ slows the tail current). Might this be an artifact of a poorly clamped vacuole with currents too big?2) Figure 1. The recording of VfTPC1 in 0 Ca2+ on the G-V curves for all channels, but, since the maximum conductance is not reached, it is impossible to understand to what extent the effect of ca2+ is on the V1/2 of activation or a reduction of the maximum conductance, indicating possible block by such high concentrations of Ca2+. This possible block of ca2+ should be explored in single-channel recordings.3) Figure 4 shows the effects of Ca4) Line 177. The authors state that VfTPC1 channels activate at the resting potential of the vacuole but no indication is given of this value.5) The model employed for generation of vacuole action potentials is presented in a manner that is too simplified. More details on the equations and numerical methods are needed to enhance reproducibility.6) Figure 4- Supplement 1. The tail currents of LjTPC1 channels are extremely slow. The current density indicates that the current magnitude is extremely large and the tail current shows signs of poor space clamp. A meaningful comparison between conditions  should be made with smaller currents.7) Structural Figures 6 and its supplement lack detail and spatial orientation to be useful. These figures should be remade in a clearer way for the reader to get the message.2+. The authors should explain if this is explained by reduced permeability to ca2+ ions as compared to Na+ and K+ or if it reflects a pore blocking effect of luminal Ca2+.8) Tail currents in Figure 7 show a reduction of their magnitude in the presence of Ca2+ concentration used? An experiment showing the effects of Ca2+ on single-channel behavior would be more informative (see comment 3 above).9) The single channel data presented in Figure 8 is interesting, but it lacks context. What is the CaReviewer #2 (Recommendations for the authors):2+-sensitivity of the double mutant (E605A/D606N). But the abstract and the discussion give the impression that three polymorphic residues are responsible for the above sensitivity. This needs to be clarified or the text might need some rewording to reconcile these statements.Line 246-252, authors acknowledge that E457N mutation does not add further to voltage and Ca2+ sensor sites and the two or three residues mediating luminal Ca2+ sensitivity can be depicted in a cartoon or topology model of the channel in one of the figure.To enhance clarity, the position of the three CaVoltage protocol should be clearly defined for each figure along with the pulse interval (either in the legend or methods) e.g. the voltage used for recording tail currents in Figure 1 is presumably the same as the holding potential. Since the deactivation is significantly different between AtTPC1 and VfTPC1, pulse interval can significantly modify the current activation kinetics.In the previous publication authors already tried some of the individual single point mutations for some of the polymorphic residues presented in this paper. It will be worthwhile to present that data in this paper for comparison (maybe as supplementary figures).E457 residue (site 2) is far away from the E605/D606 residues (site 3) in the model. This mutant was not tested before. It will be worth checking the current phenotype of the single mutant, E457N AtTPC1, and comparing it with the double mutant E605A/D606N.2+-sensitivity similar to AtTPC1. Is it possible that the luminal Ca2+ and the allosteric voltage sensor coupling mechanism be different in At versus Vf?When comparing AtTPC1 and VfTPC1 channels, I wonder if making the reverse or corresponding mutations in VfTPC1 (i.e. A607E/N608D) will result in a channel with CaReviewer #3 (Recommendations for the authors):My specific comments are as follows.2+ concentrations of plant vacuoles are between 1.5 and 2.3 mM, yet the lowest Ca2+ concentration used in this study was 10 mM. Are there any measurable differences between AtTPC1 and VfTPC1 at physiological ca2+ concentrations? Is there a plant growth phenotype when the Ca2+ sensors of AtTPC1 are mutated?1. The physiological luminal Ca2. In Figures 1 and 9, vacuoles from Arabidopsis tpc1-2 should be included as negative controls.3. For Figure 2, the activation is both time and voltage dependent. It might be helpful to compare the time courses of the close state vs. the fully activated state. According to the G-V curve in Figure 4, AtTPC1 is not fully activated at 90 mV. Hence, a stronger depolarization might be necessary for the demo trace in Figure 2A. Likewise, the deactivation in Figure 3 should also be studied with the fully activated AtTPC1. In Figure 3A, it is not clear which trace is for +80 mV or +100 mV.4. The authors suggested that three luminal residues are responsible for the distinct properties of TPC1 from different species. While it was shown that mutating the residues in AtTPC1 into those in VfTPC1 made the AtTPC1 channel resembling VfTPC1, how about the reversed mutations in VfTPC1? Do they make the VfTPC1 channel resembling AtTPC1?Lotus japonicus appeared to be much slower than that of VfTPC1. Given these residues  are the same for both channels, it is unlikely they are the primary determinants responsible for the slow deactivation.5. The deactivation of TPC1 from 6. In Figure 7A, the scale bars should be numbered, and the controls from non-transfected HEK293 cells should be shown. In addition, the selectivity data from AtTPC1, AtTPC1-E605A/D606N, and VfTPC1-A607E/N608D in HEK293 cells should be compared.2+.7. For E605A/D606N and E457N/E605A/D606N mutant channels, longer depolarizations might be needed to see the steady state currents in 50 mM ca8. In Figure 8A, the genetic background should be mentioned in the legend. Additionally, single channel recordings of non-transfected and VfTPC1-A607E/N608D -transfected cells should be provided.9. Indeed, all the experiment conditions should be clearly described in the figure legend, e.g., the steady state currents in Figure 1 and the genetic background of experiments in Figure 2, Figure 3, and Figure 9.2+ dose-dependence of the inhibition of TPC1 open probability?10. What is the Ca Essential revisions:After consultation, the reviewers agree that the following experiments are needed:1. Reverse mutations in VfTPC1 need to be made and tested.2+. The results are described below in the response to the last comment of reviewer 2.As requested, we tested the effect of reverse mutations (N458E/A607E/N608D) in VfTPC1 on its response to membrane voltage and luminal Ca2+ on open probability would help provide crucial insight into channel gating .2. Expanding the single channel data to include the effect of Ca2+ block. For this reason, and in view of published data on the TPC1 single channel conductance and open probability under Ca2+ loads, we did not examine the luminal Ca2+ effect at the single channel level. Please see our detailed response to comment 3 of reviewer 1.Our data excludes the possibility that the current reduction is predominantly caused by a CaIn addition, please attend to the reviewer's individual comments, which are aimed to improve the clarity of the data presentation and the manuscript's conclusions.We have addressed each of the reviewer's comments, please see details below.Reviewer #1 (Recommendations for the authors):1) The order of presentation of findings in the text and the order of the figures is confusing. It jumps back and forth. The figures should be presented in the same order as they are refered to in the text.eLife’s policy for supplementary illustrations which is to link the supplemental figures to the main figures. However, as the previous ordering strategy seems to be difficult for the readership to read in some places in our manuscript, we have separated some figure supplements from the main figures and now show some of them as main figures or as Appendix figure in the order as they are referred in the main text. Accordingly, the numbering of the figures has been changed.We have tried to follow 2+ is puzzling, the tail currents are much slower than any other channels studied in the same condition and do not correspond to the findings in Figure 3 (High Ca2+ slows the tail current). Might this be an artifact of a poorly clamped vacuole with currents too big?2) Figure 1. The recording of vfTPC1 in 0 Ca2+ are significantly slower than those of the AtTPC1 wild type channel at higher negative membrane voltages  and becomes faster with an increase to 10 mM Ca2+ . These experiments were characterized by similar patch clamp parameters such as membrane capacitance (mC), series resistance (seriesR) and absolute current amplitudes (I) at the pre-activating voltage pulse applied before the deactivating voltage pulse, as given by the following mean values ± SE: AtTPC1-WT (n=5) mC = 42.7 ± 1.9 pF, seriesR = 2.6 ± 0.3 MΩ, I = 8.7 ± 1.6 nA; AtTPC1-D605A/E606N (n=4) mC = 47.0 ± 4.2 pF, seriesR = 2.3 ± 0.2 MΩ, I = 11.0 ± 1.1 nA; AtTPC1-D605A/E606N/E457N (n=5) mC = 48.0 ± 2.0 pF, seriesR = 2.7 ± 0.2 MΩ, I = 8.8 ± 0.6 nA; VtTPC1 (n=4) mC = 45.5 ± 4.3 pF, seriesR = 2.9 ± 0.2 MΩ, I = 10.3 ± 1.0 nA. In comparison, AtTPC1-WT, the double and triple AtTPC1 mutants  show comparable deactivation kinetics. We would also like to point out that such slow deactivation kinetics as found here for VfTPC1 is not uncommon among the TPC1 channels of other plant species. For example, the TPC1 channel variant from the moss Physcomitrella patens (PpTPC1a) also shows much slower deactivation than AtTPC1 . See also the response to comment 6 (reviewer 1) and to comment 5 of reviewer 3. In the revised manuscript, we now go into more detail about the different deactivation behavior.We have no reason to believe that the slower relaxation of the VfTPC1 tail currents compared with the AtTPC1 channel variants in Figure 1A  is an artifact due to poor clamping of the vacuole. This is supported by the mean half-deactivation times in Figure 3B : The mean half-deactivation times of VfTPC1 derived from current recordings at 0 mM Ca2+ on the G-V curves for all channels, but, since the maximum conductance is not reached, it is impossible to understand to what extent the effect of Ca2+ is on the V1/2 of activation or a reduction of the maximum conductance, indicating possible block by such high concentrations of Ca2+. This possible block of ca2+ should be explored in single-channel recordings.3) Figure 4 shows the effects of Ca2+-induced shift of the activation threshold shown in Figure 4 and also in Figure 1A  clearly excludes the possibility that the current reduction predominantly results from a block of Ca2+. Figure 4A  further shows that in 0 Ca2+ the G/V curves almost reached their maximum, in particular for VfTPC1, the AtTPC1 double and triple mutant. Thus, the slope of the curve at the midpoint activation voltage is rather well determined. With increasing Ca2+, this slope does not change markedly, which supports the notion that the dominant effect is on the midpoint activation voltage V1/2 and not on Gmax. Based on these considerations, we refrained from exhaustive single channel measurements to additionally disprove a fact that is rather unlikely. Nevertheless, a Ca2+ block might contribute to the observed current reduction. In current recordings on Vicia faba vacuoles, Ward and Schroeder , the single channel conductance of VfTPC1 was only 16 pS, but increased to 155 pS when K+ (100 mM) was the major cation on both sides of the vacuole membrane. Under symmetric K-based solute conditions (100 mM) on both sides of the vacuole membrane, the presence of 5 mM Ca2+ at the luminal side caused a decrease in the single channel conductance from 250 pS (see below 2+) to 155 pS (Ward and Schroeder 1994). Thus, luminal Ca2+ at a concentration of 10 or 50 mM under symmetric K conditions (150 mM) in our whole-vacuole current recordings probably affected not only the TPC1 voltage dependence but also the single channel conductance even though the negative Ca2+ effect on the ion transport capacity may be attenuated by the higher K+ concentration (150 vs 100 mM K). Note that, the Ca2+ effect on the ion transport capacity does not appear to correlate with Ca2+ sensor site 3, as this side is not functional in VfTPC1.The CaArabidopsis thaliana wild type and fou2 mutant plants in Beyhl et al. . It was demonstrated that a luminal Ca2+ concentration of 0.1 and 1 mM under symmetric 100 mM K+ only affected the single channel open probability and did not reduce the single current amplitude of both wild type and fou2 TPC1 channels , the latter being a mimic of our AtTPC1-D605A/E606N mutant.In addition, our group has studied this question also on 4) Line 177. The authors state that VfTPC1 channels activate at the resting potential of the vacuole but no indication is given of this value.The reviewer is right that the resting potential of the vacuole should be mentioned. We revised the manuscript accordingly in the introduction and discussion.5) The model employed for generation of vacuole action potentials is presented in a manner that is too simplified. More details on the equations and numerical methods are needed to enhance reproducibility.in silico experiments (see Supplementary File 3). Although they have already been described in our cited earlier paper Jaslan et al. 2019 (doi: 10.1038/s41467-019-10599-x.), for the convenience of the reader this additional information is now provided in the revised manuscript.We provided now further details on the 6) Figure 4- Supplement 1. The tail currents of LjTPC1 channels are extremely slow. The current density indicates that the current magnitude is extremely large and the tail current shows signs of poor space clamp. A meaningful comparison between conditions  should be made with smaller currents.2+ because the membrane currents were very large indeed. After re-inspection of our LjTPC1 experiments under 0 mM Ca2+, we now found an experiment with lower TPC1 currents at +110 mV. This current amplitude was comparable to those recorded at +110 mV with the other TPC1 channel variants (VfTPC1: 595±10 pA/pF (n=5); AtTPC1-WT 571±66 pA/pF (n=5); AtTPC1-D605AD606N 671±81 pA/pF (n=5); mean±SE). Nevertheless, the tail currents were still very slow as illustrated in the relabeled Appendix 1—figure 1. However, such extremely slow relaxation for TPC1 channel is not unusual. The At2HsTPC2 mutant, in which three selectivity filter residues in AtTPC1 were replaced by those of human HsTPC2, also showed very slow tail currents . In addition, see also response to comment 5 of reviewer 3.Thank you for the critical comment. We agree that space clamp problems cannot be fully excluded for this experiment with LjTPC1 channels shown under 0 mM luminal Ca7) Structural Figures 6 and its supplement lack detail and spatial orientation to be useful. These figures should be remade in a clearer way for the reader to get the message.We agree that the spatial orientations were lacking and rewrote the figure caption and reference to the supplementary material. We feel that this does define the orientations much more precisely and coherently. Please note that the former Figure 6 and its figure supplement was relabeled as Figure 9.In addition, also considering comment 2 of reviewer 2, we revised Figure 6—figure supplement 1  as follows:2+ bindings sites in comparison to Arabidopsis AtTPC1.We add a topology cartoon of the VfTPC1 protein showing positions of the three luminal Ca2+ bindings sites are highlighted in color in the 3D model of the VfTPC1 channel.The amino acid residues of the three luminal CaWe now show not only a front view of the 3D VfTPC1 model, but also an orthogonal top view.2+. The authors should explain if this is explained by reduced permeability to Ca2+ ions as compared to Na+ and K+ or if it reflects a pore blocking effect of luminal ca2+.8) Tail currents in Figure 7 show a reduction of their magnitude in the presence of CaFor clarity, we have slightly revised Figure 7  and show now the concentration of the relevant permeant cations on both sides of the HEK cell plasma membrane above the raw current traces in Figure 7C-E . Additionally, the dashed zero lines of the current responses in C-E were set to the same level, and the currents of nontransfected HEK293 cells are shown as a negative control for all three solute conditions.2+ condition compared with K+ or Na+. On the one hand the lower tail current amplitude in Ca2+ can be attributed to the 10-fold lower Ca2+ concentration (15 mM) compared with Na+ or K+ (each 150 mM). On the other hand, it also is in line with the recent mechanistic explanation that the relative permeability of TPC1 (PCa/PNa » 5:1) does not correlate with the actual Ca2+ conductivity . Or in other words, Ca2+ ions cannot permeate the channel better than K+ or Na+ ion which may additionally lead to a lower tail current amplitude. This is further supported by the very small single channel conductance under Ca2+based solute conditions compared to K+-based ones. Furthermore, the single channel conductance is reduced at a high luminal Ca2+ addition under K+-based solute conditions (see above the author response Tables 1 and 2 given in response to comment 3 from reviewer 1). This point is now addressed in the legend to Figure 7 .Yes, the tail currents recorded in Figure 7C-E  from the same HEK cell were reduced under extracellular Ca2+ concentration used? An experiment showing the effects of Ca2+ on single-channel behavior would be more informative (see comment 3 above).9) The single channel data presented in Figure 8 is interesting, but it lacks context. What is the Ca2, 10 mM Hepes (pH 7.5/Tris), and the pipette medium consisted of 100 mM KCl, 2 mM MgCl2, 2 mM EGTA, 10 mM MES (pH 5.5/Tris). Although this solute composition was mentioned in the Material and Methods under the section “patch clamp experiments with membrane patches”, for clarity and ease of comparison, the concentration of the relevant cations K+ and Ca2+ used for these recordings is now additionally given in the legend of Figure 8 . Regarding the Ca2+ effect on the single channel behavior, please see the response to comment 3 from reviewer 1 above.In Figure 8 , the single channel recordings were performed using the following K-based solutions: The bath medium contained 100 mM KCl, 0.5 mM CaClReviewer #2 (Recommendations for the authors):2+-sensitivity of the double mutant (E605A/D606N). But the abstract and the discussion give the impression that three polymorphic residues are responsible for the above sensitivity. This needs to be clarified or the text might need some rewording to reconcile these statements.Line 246-252, authors acknowledge that E457N mutation does not add further to voltage and Ca2+ sensor site 3 in the luminal pore entrance is most likely responsible for this gating behavior of VfTPC1, which is clearly distinct from the AtTPC1 channel of Brassicaceae.”We agree and revised the abstract accordingly. In the first paragraph of the discussion, it was already stated that “The dual polymorphism of two residues within the Ca2+ sensor sites and the two or three residues mediating luminal Ca2+ sensitivity can be depicted in a cartoon or topology model of the channel in one of the figure.To enhance clarity, the position of the three CaWe revised the Figure 6—figure supplement 1  accordingly. For further details, please see our response to comment 7 of reviewer 1.Voltage protocol should be clearly defined for each figure along with the pulse interval (either in the legend or methods) e.g. the voltage used for recording tail currents in Figure 1 is presumably the same as the holding potential. Since the deactivation is significantly different between AtTPC1 and VfTPC1, pulse interval can significantly modify the current activation kinetics.We agree and revised the relevant Figure legends and sections in Materials and methods accordingly. Since the time interval between the activation and deactivation voltage pulses at holding voltage (-60 mV) were very long , the pulse interval should not have affected the kinetics.In the previous publication authors already tried some of the individual single point mutations for some of the polymorphic residues presented in this paper. It will be worthwhile to present that data in this paper for comparison (maybe as supplementary figures).2+ sensor site 3 is sufficient. We have mentioned in the present manuscript that AtTPC1 wild-type data at 0 and 10 mM Ca2+ are identical to those shown in Dickinson et al., 2021. Therefore, it is easy to compare the results of both papers. Otherwise, we believe it will be too redundant to show already published data in a supplementary figure again.With due respect, we believe that the citation of our previous paper  presenting single point mutations for some of the polymorphic residues in the caE457 residue (site 2) is far away from the E605/D606 residues (site 3) in the model. This mutant was not tested before. It will be worth checking the current phenotype of the single mutant, E457N AtTPC1, and comparing it with the double mutant E605A/D606N.2+ sensitivity of these mutants was significantly reduced compared with AtTPC1 wild type, but without promoting voltage-dependent activation, as half-activation potentials  were shifted to more positive values and activation kinetics were slowed down. At Ca2+ sensor site 2, VfTPC1 now contains N458 instead E458 at the homologous site to AtTPC1-E457. With respect to the effect of E457 on the luminal Ca2+ sensitivity of AtTPC1, therefore we further examined whether a neutral residue at site 457 (E457N) intensifies the effects of the double mutations in Ca2+ sensor site 3 (E605A/D606N) on AtTPC1 channel gating under different luminal Ca2+ concentrations. As mentioned in the previous manuscript version at the end of the result section “Polymorphic AtTPC1 triple mutant mimics the VfTPC1 channel features”, we found and concluded the following:We have already tested the single mutants AtTPC1-E457Q and AtTPC1-E457N in a previous paper under identical ionic patch clamp solutions . The luminal CaVicia faba TPC1 channel is most likely related to the altered Ca2+ sensor site 3.” Since the reader, like the reviewer, may overlook the fact that we have already addressed this AtTPC1-E457 issue, we have revised this section as followed: “Thus, the E605A/D606N exchange is already sufficient to provide AtTPC1 with the voltage and Ca2+ sensitivity of VfTPC1. The additional E457N replacement in the AtTPC1 E605A/D606N mutant background, however, did not further affect the transition from an Arabidopsis-like to a VfTPC1-like gating behavior, nor did it further reduce luminal Ca2+ sensitivity. Dadacz-Narloch et al. (2011) reported that neutral residues at site 457 (E → N/Q) in AtTPC1 made the channel significantly less sensitive to luminal Ca2+ but attenuated voltage dependent activation compared to wild type channels, as midpoint voltages  were shifted to more positive values. Thus, the hyperactivity of the native Vicia faba TPC1 channel is most likely related to the altered Ca2+ sensor site 3 rather than to Ca2+ sensor site 2. Considering further the similar luminal Ca2+ sensitivity of E605A/D606N and E457N/E605A/D606N , the two Ca2+ sensor sites 2 and 3 with their residues E457 and E605/D606, respectively, do not have additive effects on channel gating.“An additional E457N replacement did not further affect the transition from an Arabidopsis-like to a VfTPC1-like gating behavior. Considering the attenuating effect of neutralized site 457 (E → N/Q) on voltage-dependent activation of AtTPC1 , the hyperactivity of the native 2+-sensitivity similar to AtTPC1. Is it possible that the luminal Ca2+ and the allosteric voltage sensor coupling mechanism be different in At versus Vf?When comparing AtTPC1 and VfTPC1 channels, I wonder if making the reverse or corresponding mutations in VfTPC1 (i.e. A607E/N608D) will result in a channel with Ca2+. The triple mutant still started to activate close to the vacuolar resting membrane voltage around -30 mV and similarly responded to an increase to luminal Ca2+ (10 mM) . Thus, the VfTPC1 triple mutant did not gain the high luminal Ca2+ sensitivity and high depolarization-dependent gating behavior of AtTPC1, indicating that other additional structural adaptations have evolved and counteract these “gain-of-function” efforts. The literature shows that in contrast to “loss-of-function” mutations (successfully applied here to AtTPC1), such reverse gain-of-function approaches with plant ion channels also failed before . There are many subtle structural details that are important for the particular mechanism, not all of which can be identified and transplanted.Using the VfTPC1 triple mutant (N458E/A607E/N608D), we tested whether such inverse mutations cause VfTPC1 to become AtTPC1-like. Unfortunately, the gating behavior of this VfTPC1 triple mutant was only slightly different from VfTPC1 wild type under 0 and 10 mM luminal CaThe VfTPC1 triple mutant data are now presented and discussed in the manuscript.Reviewer #3 (Recommendations for the authors):My specific comments are as follows.2+ concentrations of plant vacuoles are between 1.5 and 2.3 mM, yet the lowest Ca2+ concentration used in this study was 10 mM. Are there any measurable differences between AtTPC1 and VfTPC1 at physiological Ca2+ concentrations?1. The physiological luminal Ca2+ concentrations of plant vacuoles range from as low as 0.2 mM up to 2.3 mM as described in the cited review by Schönknecht . Therefore, we used this lowest physiological Ca2+ concentration of 0.2 mM measured in higher plants in our current clamp and also voltage clamp experiments to record TPC1-dependent membrane polarization and the corresponding TPC1 currents . The results have been described in the section: “Vacuoles with VfTPC1 and AtTPC1 triple mutant are hyperexcitable”. Briefly, in these current clamp experiments the vacuoles equipped with VfTPC1 channels were hyperexcitable; they remained depolarized at a voltage of about 0 mV  during the entire subsequent recording period of 10 s, regardless of the stimulus intensity. In contrast, when AtTPC1-equipped vacuoles were challenged with even the highest current stimulus (1 nA), the post-stimulus voltage remained depolarized for only a short period  before relaxation to the resting voltage occurred. In comparison to wild type AtTPC1, the AtTPC1-triple mutant vacuoles were also hyperexcitable, behaving very much like the VfTPC1-equipped vacuoles.We would like to point out that the physiological luminal Ca2+ concentration (0.2 mM), the voltage activation threshold of both, VfTPC1 and the AtTPC1 triple mutant E457N/E605A/D606N, but not AtTPC1 wild type was close to the resting membrane voltage of around -30 mV . This explains the hyperexcitable vacuoles when equipped with VfTPC1 and the AtTPC1 triple mutant E457N/E605A/D606N.The further analysis of the corresponding TPC1 currents of these same vacuoles revealed, that at this physiological luminal Ca2+ sensors of AtTPC1 are mutated?Is there a plant growth phenotype when the Ca2+ sensor site 1 was destroyed by the mutation at site 454 in AtTPC1 (D454N). This so-called fou2 mutant shows a retarded growth phenotype, appears to be chronically wounded and therefore produces large amounts of the wounding hormone jasmonate . Since the fou2 TPC1 channel behaves like a hyperactive TPC1 channel, the fou2-equipped vacuoles are hyperexcitable  similar to vacuoles equipped with VfTPC1 or the AtTPC1 triple mutant E457N/E505A/D606N . According to the resolved 3D structure of the fou2 channel  and our VfTPC1 model in this paper, the key residues E605, D606 of the Ca2+ sensor site 3 are now rearranged, preventing binding of luminal Ca2+, as electrophysiologically observed for the AtTPC1 mutants E505A/D606N and E457N/E505A/D606N. Therefore, we would expect that AtTPC1 mutant plants E505A/D606N show a similar growth phenotype as fou2.Yes, as mentioned in the introduction (third paragraph), there is a phenotype when the Ca2. In Figures 1 and 9, vacuoles from Arabidopsis tpc1-2 should be included as negative controls.attpc1-2 vacuoles for Figures 1 and 9  have been already presented in previous publications of our group such as Jaslan et al. 2016 (doi: 10.1111/plb.12478) and Jaslan et al. 2019 (doi: 10.1038/s41467-019-10599-x), respectively. Since all these experiments were performed under identical ionic conditions, we feel that the manuscript will not gain much in showing published negative control experiments once again. However, we understand the reviewer's concerns very well and certainly agree that negative controls are important. Therefore, to convince the reader about the quality of our electrophysiological data, we now mention in Material/Methods that negative controls have been published in these papers .Such control experiments with 3. For Figure 2, the activation is both time and voltage dependent. It might be helpful to compare the time courses of the close state vs. the fully activated state. According to the G-V curve in Figure 4, AtTPC1 is not fully activated at 90 mV. Hence, a stronger depolarization might be necessary for the demo trace in Figure 2A. Likewise, the deactivation in Figure 3 should also be studied with the fully activated AtTPC1. In Figure 3A, it is not clear which trace is for +80 mV or +100 mV.In order to ensure that the TPC1 channels are fully closed, the vacuole membrane was clamped for 4.3 s to the (channel-closing) holding voltage of -60 mV between the applied voltage pulses. Therefore, activation of the TPC1 channels elicited upon increasing depolarized membrane voltage pulses was always achieved from the fully closed channel state. In Figure 2A (now labeled as 3A), the normalized demo traces recorded at +90 mV illustrate the significant differences in activation kinetics of the different channel variants found at less and higher depolarized voltages as well . Given the presented data set, we do we do not think the reader gains more by displaying demo traces at different voltages.o> 0.85 at +80 and +100 mV; see G/V curves in Figure 5A). In addition, the deactivation kinetics at different membrane voltages were always investigated with a constant number of open TPC1 channels in each experiment by always pre-activating at the same prepulse voltage of either +80 or +100 mV.The legend of Figure 3A (now labeled as 4A) now indicates that the pre-pulse voltage of +100 and +80 mV was used for activation of the three AtTPC1 channel variants and VfTPC1, respectively, to study the deactivation process. Unfortunately, it was not possible to pre-activate at +100 mV the VfTPC1 channels in the same way as the AtTPC1 channel variants, since the vacuole got lost. A pre-activating voltage pulse of +100 mV combined with the multiple subsequent deactivating voltage pulses was obviously too stressful for the VfTPC1-equipped vacuoles. However, because of the negatively shifted relative open-channel probability of VfTPC1, deactivation of VfTPC1 was examined as for the other channel types with mostly “fully open” channels  are the same for both channels, it is unlikely they are the primary determinants responsible for the slow deactivation.5. The deactivation of TPC1 from Physcomitrella patens. The PpTPC1a channel shows much slower deactivation than AtTPC1 . Nevertheless, like AtTPC1, PpTPC1a harbors also functional Ca2+ sensor sites 1, 2 and 3 , as indicated by the AtTPC1-like luminal ca2+ sensitivity and voltage dependence of PpTPC1a . Additionally, in contrast to VfTPC1, the AtTPC1-mutant E6045E/D606N shows deactivation kinetics similar to AtTPC1 wild type while its Ca2+ sensitivity and voltage dependence are VfTPC1-like . Thus, we can indeed conclude that other functional domains than the Ca2+ sensor site 3 determines the deactivation behavior of TPC1 channels. We now address this point in the result section “Polymorphic AtTPC1 triple mutant mimics the VfTPC1 channel features” in the manuscript.We absolutely agree with the conclusion of the reviewer. This is also supported by the PpTPC1a channel variant from the moss 2+ sensor 3 in PpTPC1a (ESD) compared to AtTPC1 (EDD) will not affect the gating behavior and Ca2+ sensitivity as shown in Dickinson et al. 2021 (DOI: 10.1073/pnas.2110936119). This is in line with the AtTPC1-like luminal Ca2+ sensitivity and voltage dependence of PpTPC1a .Note that the single mutation of Ca6. In Figure 7A, the scale bars should be numbered, and the controls from non-transfected HEK293 cells should be shown. In addition, the selectivity data from AtTPC1, AtTPC1-E605A/D606N, and VfTPC1-A607E/N608D in HEK293 cells should be compared.2+ insensitivity of VfTPC1 into AtTPC1-like features , and (ii) the relative permeability ratios of AtTPC1 wild type and the VfTPC1-like AtTPC1 mutant E605A/D606A were very similar.In Figure 7A , we have revised the scale bar, and we now show also the controls from non-transfected HEK293 cells under the three different solute conditions. However, we did not examine the VfTPC1A607E/N608D mutant because (i) the reverse mutations N458E/A607E/N608D in VfTPC1 did not convert the hypersensitivity and luminal Ca2+.7. For E605A/D606N and E457N/E605A/D606N mutant channels, longer depolarizations might be needed to see the steady state currents in 50 mM ca2+ are in the quasi-steady state and thus very close to reaching the steady-state current level. We would also have liked to extend the activating voltage pulses further, but already the applied 1-s lasting voltage pulses were quite stressful for the patched vacuole. Therefore, we have applied a very long time-interval of 4 s between the voltage pulses at the holding voltage, so that the vacuoles could recover, and the currents could relax fully at holding voltage. Or in other words, the risk of losing the vacuole at an even longer voltage pulse time would greatly increase; therefore, the 1-s-lasting voltage pulses for TPC1 current activation was already a compromise between lifetime of the patched vacuole and the achievement of the steady-state current level. However, since we are near the steady-state current level, we feel our results to be quite solid already.We agree that the depolarization-induced TPC1 currents in the presence of 50 mM Ca8. In Figure 8A, the genetic background should be mentioned in the legend. Additionally, single channel recordings of non-transfected and VfTPC1-A607E/N608D -transfected cells should be provided.2+ insensitivity of VfTPC1 into AtTPC1-like features  and (ii) the single channel conductance of the AtTPC1 wild type and the VfTPC1-like AtTPC1 mutant E605A/D606A were very similar as well.We have revised the legend of Figure 8 , accordingly. However, we did not additionally examine the single channel conductance of the VfTPC1-A607E/N608D mutant because (i) the reverse mutations N458E/A607E/N608D in VfTPC1 did not convert the hypersensitivity and luminal Ca9. Indeed, all the experiment conditions should be clearly described in the figure legend, e.g., the steady state currents in Figure 1 and the genetic background of experiments in Figure 2, Figure 3, and Figure 9.We agree and have revised the figure legends accordingly.2+ dose-dependence of the inhibition of TPC1 open probability?10. What is the ca1/V2 under three luminal Ca2+ conditions  . The difference in the corresponding Ca2+-induced shift in the V1/V2 values points to a different Ca2+ dose dependency of the TPC1 channel variants tested. This can be supported when the V1 and V2 values are plotted against the corresponding Ca2+ concentrations (see 2+(10 mM)-induced increase in the midpoint activation voltages – particularly V1 – of the AtTPC1 wild type appears to be steeper than for the other three TPC1 channel variants . However, more Ca2+ concentrations below 10 mM need to be tested in the future to gain more insights into it. Therefore, we did not further address this issue in the manuscript.In this work we have determined the midpoint activation voltages Vions see . The slo


The lack of equipped healthcare infrastructure in isolated hard-to-reach zones exposes their population to a higher risk of complications in common diseases. With a timely diagnosis setting a life-altering difference, worldwide efforts have been conducted for the development of point-of-care testing (PoCT) with cost-effective devices. Among the most common interests in PoCT is the analysis of blood smear samples, as they can help to detect, diagnose, and monitor a wide range of diseases and disorders. With microscopy being the traditional tool for these analyses, a significative advance has been the development of cost-effective digital holographic microscopy systems, driven in part by its label-free imaging capabilities that waive the need for any sample preprocessing. Here, a robust and portable digital lensless holographic microscope, functionalized for the analysis of non-preprocessed blood smear samples in PoCT environments, is presented, and its viability is tested in the observation of red blood cells. The device uses an optical fiber with a cone-shaped tip instead of a pinhole, which ensures the sturdiness of the system and eliminates the need for challenging alignment. While the distances of the microscope can be tuned before fabrication, the herein-reported operational parameters are functionalized for the specific analysis of blood samples. A timely diagnosis can set the difference between a life-threatening illness and a controlled affliction. Yet, access to equipped healthcare infrastructure in developing countries and low-resource settings is commonly limited to central cities and urban areas, leaving the rural regions and isolated hard-to-reach zones exposed to a higher risk of complications in common diseases. To fight this global health challenge, worldwide efforts have been conducted for the development and validation of point-of-care testing (PoCT) with cost-effective and portable devices for personalized medicine Among such techniques, Digital Lensless Holographic Microscopy (DLHM) is one of the most simple architectures, constituting a modern realization of Gabor’s original invention Similar devices to the design herein proposed can be found in the literature. In particular, those reported by Tobón-Maya et al. The traditional DLHM recording design is shown in panel (a) of The modified setup is thus shown in panel (b) of To apply these factors effectively, the recording sensor must also be considered. The most common low-cost board-level cameras, such as the ones extracted from CCTV cameras, massive-production CMOS sensors, and even open-source-targeted cameras like the Raspberry Pi Camera module, implement formats between 1/2.3″ and 1/3.2″ with pixel sizes between 1.4 and 2.2 µm. Thus, an average sensor size of 6 mm side length with 2 µm pixels was considered for the design. For such a pixel size, the representation of an 8 µm RBC in at least 50 pixels would require a magnification factor of 12x. If the Panel (c) of •Uses a simple and compact lensless microscopy principle, allowing its cost-effective and robust implementation.•Employs a single USB connection for both digital communication and energy supply, simplifying its use.•Replaces the traditional pinhole-based DLHM point source with a conical-tipped fiber, waiving the daunting alignment and mechanical stability requirements.•Implements fixed axial distances and adjustable transversal position, thus functionalizing its operation for the desired sample case while still allowing scanning of the field of view.•Follows a modular design, easing its assembly and, if desired, customization.In summary, the proposed device:The components listed in All the required components for the fabrication of the device are summarized in {All the required components can be either commercially acquired or fabricated from the supplied design files. Cost-effective and 3D printable dip-etching device for cone-shaped optical fiber tip fabrication”, available at Ref. While the fiber-coupled laser diode (component i4) could be directly used as the illumination source, the resulting spherical wavefront would be limited by the numerical aperture (NA) of a plane fiber, which is commonly between 0.1 and 0.15. As mentioned in section 2, ensuring that the illumination fully encompasses the digital sensor under the proposed distances requires a numerical aperture of, at least, 0.25. This is also the lowest possible value to achieve a 1 µm resolution with 533 nm light. If the plane fiber were to be used, the digital sensor would be underfilled by the available illumination at the design distances, and the maximum possible resolution of the system would be limited to approximately 2 µm. Thus, ensuring the usability of the device for the observation of red blood cells requires the use of NA values between 0.3 and 0.5, with the range of 0.4 to 0.5 being desirable. To increase the NA value, and thus the resolution capability of the system WORD OF CAUTION:Before employing a chemical etching method, it is of utmost importance to verify and recognize the potential risks associated with manipulating HF. Solutions of HF are colorless, acidic, and highly corrosive. They represent a risk for potential poisoning and injury under inadequate manipulation procedures. When using HF, make sure to strictly follow all the procedures and guidelines for its manipulation, keeping at hand the Security Data Sheet (CAS 7664–39-3) and ensuring the safe use of Personal Protective Equipment.•Producing the conical tip on a separate single-mode optical fiber with an FC/PC connector and coupling both fibers together.•Producing the conical tip on a separate unconnected single-mode optical fiber, removing the FC/PC connector of component i4, and splicing both fibers together.•Removing the FC/PC connector of the commercial fiber and processing the stripped end to produce the conical tip directly on the i4 component.Commercially, fiber-coupled laser diodes are commonly found with an FC/PC connector at the output, but the chemical etching procedures require direct access to the uncoated fiber. One of the following considerations should thus be applied:For the presented device, the third case was applied as it allows the highest stability and compactness of the illumination system. Consequently, preparing the cone-shaped fiber tip requires the additional components listed in As summarized in panel (a) of After these alterations to component i4, the fiber connector can be used as a point source of high NA, allowing the simplification of the traditional DLHM setup shown in panel (a) of After the aforementioned preparations, general construction can be pursued. The device employs a modular design, which eases its assembly and, if desired, customization. Thus, the following steps consider the independent assembly of each module and their later connection.1.Take the CMOS sensor (component C2) and fix it into the Camera block (component C1) using the M3 screws (component C3) as shown in panel (a) of 2.As shown in panel (b) of 3.Use the magnetic attraction between the pieces to couple the Top cover and the now assembled Camera module as shown in panel (c) of Camera module:1.As seen in panel (a) of 2.Use the threaded end of the Fiber support (S8) to screw it into the Sample block (component S1), as shown in panel (b) of 3.Connect the X-Carrier (component S2) and Y-Carrier (component S3) by sliding the matching dovetail features as seen in panel (c) of 4.Place the connected carriers into the partially assembled Sample block in the direction shown in panel (d) of 5.Introduce both Carrier rods (component S4) into the Sample block (S1) passing through the Y-Carrier (S3) as shown in panel (a) of 6.Place one of the Restitution springs (component S6) between the Y-Carrier (S3) and the Sample block (S1), using the dimple cavity in the carrier, shown in panel (b) of 7.Put the Restitution pin (component S5) through the other Restitution spring (S6), and into the Sample block (S1) using the hole in the face opposite to the threaded feature for the insertion of the Positioning screw (component S7). While the illustration of this step in panel (c) of 8.As shown in Sample module:1.Place the Driver (component i5) and the Power LED (component A6) into the Laser cage (component i2) in the positions illustrated in panel (a) of 2.Place the partially assembled Laser cage (i2) into the Illumination block (component i1) as illustrated in panel (b) of 3.Introduce the diode portion of the Laser diode (component i4) through the lateral hole in the coiling feature of the Illumination block (i1) and into the Laser cage (i2), threading the fiber as illustrated in panel (c) of 4.Fix the laser diode in the laser cage as seen in panel (a) of 5.Introduce the Coiling support (component i3) into the Illumination block (i1), using it to safely wrap the optical fiber (i4) around the coiling feature of the block as shown in panel (b) of 6.Introduce the USB cable (component i6) through the assembled Illumination block (i1) and into the laser cage, as shown in panel (a) of 7.As shown in panel (b) of Illumination module:With each of the modules individually prepared, a full assembly can be pursued. This stage requires setting up all the electrical connections and the union of the modular blocks. When assembling the device, both operations must be done simultaneously. The electrical connections of the device are summarized in panel (a) of The data lines of the USB connection, commonly found in white for the positive signal and green for the negative one, are only needed for communication with the CMOS sensor (component C2). However, for this connection, the four USB lines must be brought from the Illumination module to the Camera module. To do so, a series of pass-through apertures are available at each block , allowing to pass the lines through the paths illustrated in panel (b) of To join the modules, start by introducing the four Assembly rods (component A5) into the Illumination module. The rods are magnetically attached to the 1/4″ magnets (component A1) placed at the bottom side of the block and act as a guide and support for the attachment of the other modules. Next, add the Sample module, ensuring that the pass-through holes of both blocks are aligned, such that the USB lines can pass unobstructed. Before setting the Sample module, introduce the FC/PC connector of the Laser diode (component i4) into the Fiber support (component S8) and screw it inside the Fiber connector (component S9). Alternatively, if this proves to be difficult, the support can be unscrewed from the Sample module for the attachment of the laser diode and re-connected in place. Finally, add the Camera module, once again validating that the pass-through holes are aligned. The 1/4″ magnets (component A1) at the top of the block should magnetically attach to the rods, thus completing the assembly. The top cover can be easily removed at any time to complete the connection of the camera to the USB lines. This modular assembly procedure is illustrated in After assembly, the device can be straightforwardly operated. Initially, it must be connected to the USB port of a computer, such that digital communication can be established to the camera and the energy supply to initiate the laser can be drawn. At any point, the correct energy connection of the device to the computer can be verified by the activation of the Power LED on the Illumination module. Once connected, the camera can be accessed from the computer from any compatible webcam application. While most manufacturers may suggest a specific software, any open-source alternative can also be used. However, according to the selected CMOS sensor, the computer may need additional or updated drivers; for such cases, it is suggested to check their availability with the manufacturer or supplier of the CMOS camera. Additionally, it is highly recommended to check whether the application allows modifying the exposure time of the capture; this property allows compensation for the laser’s intensity. If such a configuration is not available, the physical power delivery can be tuned with the potentiometer in the Laser driver board. Having access to the camera in the computer, and after verifying that the laser is illuminating the sensor, the device will be ready to record holograms from blood smear samples.WORD OF CAUTION: Avoid opening the device while the laser is powered on to prevent accidental eye exposure to the laser beam. The sample to be inspected must be placed, as in any other microscopy setup, on a glass slide. The slide is then introduced into the microscope via the lateral slots, as shown in panel (a) of Finally, the image can be brought to the open-source image processing software of ImageJ The proposed device was constructed following the steps and designs herein presented. To achieve the imaging of a peripheral blood smear sample, the system was equipped with a low-cost laser diode operating at 533 nm connectorized to a single-mode patch cord cable whose output connector was modified to have a cone-shaped tip of the fiber protruding from the ceramic ferrule. For the observation of RBCs, this cone tip was selected to have a semi-angle of 20°, which corresponds to a numerical aperture of 0.47 This device was then used for the observation of healthy RBCs in a blood smear without any additional preparation; that is, neither dilutions nor any staining was used. The results are summarized in To validate the capability of the device to detect and show alterations in the morphology of the RBCs, two additional blood smears were prepared in non-isotonic media to emulate unhealthy cells. Initially, a blood sample was partially diluted in a solution of <0.6 % NaCl in distilled water. This hypotonic solution was expected to induce swelling of the cells by the osmotic flow of the surrounding water into the cell. Indeed, as can be seen from the retrieved phase information of the RBCs in panel (c) of The obtained results validate the capacity of this robust and compact DLHM device for the successful analysis of red blood cells in blood smear samples. The recovered phase maps show the ability of the device to retrieve morphological information of the cells, thus allowing potential imaging-based analysis of the blood health state. While its aluminum-based design involves a higher cost than a fully 3D-printed system, both in materials  and fabrication techniques , it also ensures high mechanical stability, durability, and structural strength, allowing its potential use in PoCT applications. Furthermore, the use of fine-positioning systems greatly benefits the study of microscopic specimens, as they allow a controlled scan of the sample plane. The overall dimensions of 80 mm × 54 mm × 54 mm, which constitute the device as a portable and compact system, come at the cost of employing fixed axial distances, which, while significantly reducing the alignment requirements, do restrict the adaptability of a given device to new imaging cases. Nonetheless, this limitation is partially alleviated by the modular design, which allows the straightforward adaptation of new source, sample, or camera blocks with different properties or dimensions to an existing device. Finally, the device shows the effective use of a cone-shaped fiber tip as the point source for the DLHM setup. This element of the design is recognized to be the most difficult component of the proposed device, as it requires the use of hydrofluoric acid and a suitably-equipped laboratory for its fabrication. However, it is also one of the most versatile components of the design, as its numerical aperture can be tailored as needed changing only the immersion time, and the resulting cone-shaped fiber has a high installation flexibility, enhanced durability, and minimal weight addition to the device.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


CBLB, FCER1A, GCSAML, STAT6, TPSD1, ZFPM1), the innate immunity (C4), and NF-κB signaling. The most significant association was observed for the splice-donor variant rs56043070[A] (hg38: chr1:247556467) in GCSAML , P-value = 3.6 × 10-44). We assessed the effects of the variants on transcripts, and levels of proteins relevant to urticaria pathophysiology. Our results emphasize the role of type 2 immune response and mast cell activation in the pathogenesis of urticaria. Our findings may point to an IgE-independent urticaria pathway that could help address unmet clinical need.Urticaria is a skin disorder characterized by outbreaks of raised pruritic wheals. In order to identify sequence variants associated with urticaria, we performed a meta-analysis of genome-wide association studies for urticaria with a total of 40,694 cases and 1,230,001 controls from Iceland, the UK, Finland, and Japan. We also performed transcriptome- and proteome-wide analyses in Iceland and the UK. We found nine sequence variants at nine loci associating with urticaria. The variants are at genes participating in type 2 immune responses and/or mast cell biology ( A meta-analysis of GWAS studies identified nine sequence variants associated with urticaria. These results and multi-omics analyses support the role of mast cell activation and type 2 immune response in urticaria. These outbreaks can occur as a reaction to allergens and physical stimuli or in an autoimmune or hereditary disorder, but the majority of outbreaks are idiopathic2. While most outbreaks resolve within 24 h, acute urticaria is defined as wheals lasting or recurring for less than six weeks, and longer-lasting recurrent outbreaks are classified as chronic4. Urticaria in any form is estimated to affect 15-25% of individuals, with chronic urticaria representing a rare form (0.5% in Europeans and 1.4% in Asians)5, and can impact quality of life6.Urticaria, also known as hives, is a skin disorder characterized by outbreaks of raised pruritic wheals caused by the release of histamine, typically ranging from a few millimeters to a few centimeters in diameter8. Sera from human urticaria patients as well as anti-IgE and anti-FcɛRI antibodies have been shown to induce both basophil and mast cell degranulation9. Mast cells have been identified as the main effector cells in urticaria10. Low basophil count is also common in urticaria11. There is evidence that two mechanisms contribute to the pathogenesis of chronic urticaria; the development of autoantibodies to FcεRI or IgE that lead to degranulation of mast cells and basophils, reported to be responsible for 30–50% of cases, and dysregulation of intracellular signaling pathways2. Recent data indicate that both IgG- and IgE-specific autoantibodies, as well as autoantibodies against other surface antigens of mast cells and basophils may contribute to chronic urticaria12.Mast cells and basophils in the skin play a key role in the pathophysiology of urticaria through the release of histamine and other mediators, including tryptases, into the skin, followed by vascular dilation and infiltration of lymphocytes and granulocytes into the lesions2. High dose antihistamines are only effective in about half of patients with chronic urticaria. The anti-IgE monoclonal antibodies omalizumab and ligelezumab are effective in antihistamine resistant chronic and spontaneous urticaria, but only increase treatment response to 65–80%14. Achieving full urticaria remission has been identified as an important clinical goal6.Treatment of urticaria involves avoidance of external stimuli, and/or drug treatment, where antihistamines are the most commonly prescribed medicationTo search for sequence variants affecting risk of urticaria, we performed a meta-analysis of genome wide association studies (GWAS) on individuals with ICD-10 diagnostic code L50 and all sub-codes using data from Iceland, the UK, Finland, and Japan. The GWAS signals were further interpreted in the context of large-scale transcriptomics and proteomics data from Iceland and the UK.15. In the meta-analysis we detected associations of common sequence variants at nine loci with urticaria,  and 1,230,001 controls from Iceland, the UK, Finland, and Japan. The meta-analysis was performed on associations under the additive model using a total of up to 26.7 million sequence variants. To adjust for multiple testing, we used a weighted Bonferroni correction based on variant annotationGCSAML (formerly C1orf150),  with GCSAM17, an important factor for B cell receptor signaling19. The splice-donor variant correlates with a single variant in the intron of GCSAML; rs74227709[A] . At the GCSAML locus, we demonstrate co-localization of disease, biological trait, splice-QTL (sQTL), and protein-QTL (pQTL) signals from datasets generated in Iceland and the UK .We tested the nine urticaria-associated variants with four inflammatory phenotypes  in meta-analyses of cases and controls from Iceland, the UK, Finland, and Japan , where the minor allele associates with protection against these phenotypes in addition to urticaria    driven by homozygotes , we found a positive genetic correlation between urticaria (ICD-10 code L50) and asthma (ICD-10 code J45)  (Supplementary Data We explored the genetic correlation between urticaria ICD-10 code L50) diagnosis and 1985 case-control phenotypes defined from ICD codes and 18 quantitative phenotypes in the UK dataset (Serum IgE levels were not available from the UK). After correcting for multiple testing (significance thresholds:  code L50r2 > 0.80) between the urticaria-associated variants and coding or splice variants, top cis- pQTLs , top cis-eQTLs , and top sQTLs (based on same Icelandic RNA sequencing set).In order for us to gain insight into which genes and proteins are involved in the pathogenesis of urticaria, we looked for a strong correlation   with asthma, FCER1A RNA levels, and IgE levels26. The variant rs6703348[G] is also correlated with top cis-eQTLs for RNA levels of FCER1A in other tissues , a protein that binds to the Fc region of IgE and mediates the activation of mast cells and basophils27.In the Icelandic data, the urticaria-associated variant rs6703348[G] at STAT6 locus, is the top trans-pQTL in the Icelandic data for plasma levels of interleukin 5 receptor alpha subunit, encoded by IL5RA , where it is implicated in alternative splicing of STAT6 on RNA-sequencing  associating with increased plasma levels of TPSAB1 (pQTL), and higher RNA expression levels of TPSD1 (eQTL), and TPSB2 (eQTL) in blood, all at the same locus coding tryptases .The urticaria risk allele of the upstream gene variant rs4410077[T], at NFKB1, the risk allele for urticaria positively correlates with variants associated with higher RNA expression in neutrophils and adipose (r2 > 0.95) and increased plasma NFKB1 protein levels   with top sQTL, depicted in increased proportional usage of NFKB1 transcript ENST00000505458 (Supplementary Data NFKB1 encodes a Rel protein-specific transcription factor (p105), which is also a precursor for a subunit (p50) of the ubiquitous NF-κB transcription factor. NF-κB is known to be involved in inflammatory pathways and in pathogenesis of auto-immune disorders32.At Using a three-layered approach, including genomics, transcriptomics, and proteomics, we have discovered associations of nine sequence variants associating with urticaria in a meta-analysis of GWAS from Iceland, the UK, Finland, and Japan, and characterized their effect on other phenotypes.CBLB, FCER1A, GCSAML, STAT6, TPSD1, ZFPM1)35, one in the complement innate immunity (C4A), and one involved in NF-κB signaling, a transcription factor known to contribute to the pathogenesis of various inflammatory diseases when deregulated36.Six of our urticaria-associated variants are pointing to genes that are directly implicated in type 2 immune response and/or mast cell physiology  in FANTOM539. Expression levels were shown to be up to 20 times greater in mast cells than in basophils, the cell type with the second highest expression39. The association of the splice-donor variant in GCSAML with urticaria is thus congruent with the high GCSAML RNA expression seen in human mast cells.Mast cells have been described as the primary effector cells in urticariaGCSAML splicing, and demonstrated an alternative transcript usage. The variant leads to a significant increase in expression of a novel GCSAML transcript lacking exon 2, an exon that overlaps the first 4 amino acids of a 25 amino acid coiled-coil motif in GCSAML40. This increase is accompanied by a concomitant decrease in the levels of the primary transcript. It is unresolved, however, whether the associations we observe are caused by the reduced number of the primary transcript or by generation of the novel transcript. We note that the effects of rs56043070[A] on RNA splicing in adipose tissue gives limited information. The relevance in urticaria pathology is therefore not obvious. However, our data show that the splice-donor variant is capable of altering the splicing of GCSAML transcripts. It would be of interest to study this further in more relevant tissues or cell lineages, such as skin tissue and mast cells10 to assess the relevance to urticaria pathology.We have confirmed the predicted effect of rs56043070[A] on GCSAML urticaria risk-allele associates with greater plasma levels of five proteins; TPSAB1, TPSB2, KIT, SIGLEC6, and SELP. TPSAB1 and TPSB2 both encode tryptases, while KIT encodes mast/stem cell factor receptor (SCFR). Tryptases are produced and released by both mast cells and basophils, but as the overall contribution of basophils is small, tryptases are considered markers of mast cell activation41. Similarly, the ligand of SCFR has been shown to play a key role in mast cell differentiation and activation43. SIGLEC6 encodes an inhibitory motif (ITIM)-bearing receptor inhibiting mast cell activation through crosslinking with FcɛRIα44. SELP encodes P-selectin, a cell adhesion molecule expressed on endothelial cells to attract leukocytes to site of inflammation, and is upregulated during mast cell activation45. TPSAB1, TPSB2, and KIT transcripts were shown to be enriched in mast cells (two-fold or greater expression than in any other cell type)46. Furthermore, the transcriptome-wide analysis of the FANTOM5 dataset identified GCSAML, FCER1A, TPSAB1, TPSB2, KIT, and SIGLEC6 as either exclusively or preferentially expressed in mast cells39. The association profile of this variant, including all protein products significantly affected by it, indicates that GCSAML plays a role in mast cell regulation, with the splice-donor variant leading to greater mast cell activation.Taking advantage of proteomics data, we show that the GCSAML locus. The splice-donor variant is consistently the variant at this locus that most significantly associates with urticaria, basophil percentage, platelet count, RNA splicing of GCSAML, and levels of the five mast cell proteins. Together, these results demonstrate that GCSAML itself is important in the pathogenesis of urticaria.Testing the same set of variants across all datasets allows us to demonstrate co-localization of signals at the 13, further study of these pathways may help in addressing this unmet pharmacological need.Furthermore, our data are consistent with the causative nature of IgE in urticaria, i.e., the variants associating with decreased IgE levels also associated with decreased risk of urticaria. However, seven of the urticaria-associated variants did not associate with IgE levels, suggesting that its effect on urticaria may be mediated through another mechanisms. As current anti-histamine and anti-IgE monoclonal antibody treatments are ineffective in up to 35% of casesIn this study, we combine genomics, transcriptomics, and proteomics in an unprecedented manner. Taken together, our results underpin the role of mast cells and type 2 inflammation in the pathogenesis of urticaria. We demonstrate how these powerful -omics tools can aid in linking variants to disease mechanisms, and more definitively profile the consequences of human sequence variation.All Icelandic data were collected through studies approved by the National Bioethics Committee  following review by the Icelandic Data Protection Authority. Participants donated blood or buccal samples after signing a broad informed consent allowing the use of their samples and data in all projects at deCODE genetics approved by the NBC. All personal identifiers of the participants' data were encrypted by a third-party system, approved, and monitored by the Icelandic Data Protection Authority.The UK Biobank data were obtained under application number 56270. All phenotype and genotype data were collected following an informed consent obtained from all participants. The North West Research Ethics Committee reviewed and approved UK Biobank’s scientific protocol and operational procedures (REC Reference Number: 06/MRE08/65).Samples and phenotype data were collected from the Finnish biobanks and the national health registers, respectively, for the FinnGen database. The Coordinating Ethics Committee of the Hospital District of Helsinki and Uusimaa evaluated and approved the FinnGen study protocol. The project complies with existing legislation . The official data controller of the study is University of Helsinki.Biobank Japan collected phenotype data  and serum samples at participating medical institutions in Japan. All participants provided written, informed consent and the study was approved by ethics committees of the Institute of Medical Sciences, the University of Tokyo and RIKEN Center for Integrative Medical Sciences.To identify urticaria cases in Iceland, we searched for patients with International Classification of Diseases (ICD-10) diagnosis code L50 and all subcodes at Icelandic primary and secondary care centers. L50 covers various types of urticaria, i.e. allergic, physical, and idiopathic urticaria. Records spanned from 1985-2018.The UK Biobank study is a large prospective cohort study of ~500,000 individuals in the age range of 40–69 from across the UK. UK urticaria cases were identified by extracting cases with ICD-10 code L50 and all subcodes from UK Biobank GP clinical event records (Field ID 42040) and hospital diagnoses made during inpatient admissions (Field IDs 41270 and 41271). Only British individuals of European ancestry were included in the study. Individuals in the urticaria cohort were excluded from the control group.http://r7.finngen.fi).The phenotype data from the FinnGen study was produced from several national health registries. All urticaria cases were diagnosed by a physician and categorized using ICD-10 code L50 and all subcodes, ICD-9 code 708 and all subcodes, and ICD-8 code 708 and all subcodes. The summary statistics for available phenotypes, including urticaria, were imported on June 19th 2022 from a source available to consortium partners .The phenotype data from Biobank Japan was gathered at 12 medical institutions in Japan where all study participants had been diagnosed with one or more of 47 target diseases by physicians at the cooperating hospitals and classified using ICD-10 codes. For urticaria cases, individuals with ICD-10 codes L50 and all subcodes were extracted from the data. The summary statistics from GWAS from Biobank Japan, including urticaria, were imported on October 10, 2020 from the Biobank Japan’s website  using protein-coding transcripts from RefSeq. We carried out chip typing of 155,250 Icelanders (around 50% of the population) using Illumina SNP arrays. The chip-typed individuals were long-range phased48, and the variants identified in the whole-genome sequencing of Icelanders were imputed into the chip-typed individuals. In addition, genotype probabilities for 285,644 untyped close relatives of chip-typed individuals were calculated based on Icelandic genealogy50.The Icelandic part of the study is based on testing 32,542,746 variants identified in whole-genome sequence (WGS) data from 28,075 Icelanders participating in various disease projects at deCODE genetics, sequenced using Illumina standard TruSeq methodology to an average genome-wide coverage of 37X. SNPs and indels were identified and their genotypes called using joint calling with Graphtyper51, and with the Affymetrix UK Biobank Axiom array52. Imputation was performed by the Wellcome Trust Centre for Human Genetics using variants identified from 32,488 WGS individuals in the Haplotype Reference Consortium (HRC), UK10K haplotype resources, and 1000Genomes phase 3 panels53. This yielded a total of 96 million imputed variants, although only 40 million variants were imputed into 408,658 participants using the HRC reference set due to quality issues with the remaining variants.Genotyping of UK biobank samples was performed using a custom-made Affymetrix chip, UK BiLEVE AxiomFinnGen samples of 321,464 individuals were genotyped using Illumina and Affymetrix arrays  . Genotype imputation of ~17 million variants was performed using a population-specific imputation reference panel comprised of 3775 whole genomes.n = 2504) and Japanese whole-genome sequencing data (n = 1037).Biobank Japan genotyped 178,726 participants of East Asian ancestry using a combination of Illumina arrays. The genotypes were imputed using ~13.5 million variants from WGS individuals in the 1000 Genomes Project Phase 3 version 5 genotype data , blood sample availability for the individual and an indicator function for the overlap of the lifetime of the individual with the time span of phenotype collection were included as covariates in the logistic regression model. We used county of birth as a proxy covariate for the first principal components in our analysis because county of birth has been shown to be in concordance with the first principal components in Iceland54. For the UK, sex, age, and the first 40 principal components were included as covariates. In Iceland and the UK, quantitative traits were tested using a linear mixed model implemented in BOLT-LMM55. The quantitative traits were adjusted for age and sex in both populations and also for 40 principal components in the UK Biobank. The traits were transformed to a standard normal distribution. Only British individuals of European ancestry were included from the UK Biobank. To account for cryptic relatedness and population stratification in the Icelandic and UK data, we used linkage disequilibrium (LD) score regression56.Logistic regression was used to test for association between variants and disease in Iceland and the UK using software developed at deCODE genetics2 statistic from our GWAS scan by dividing it with the intercept (correction factor) from the LD score regression analysis. The correction factors that we obtained are consistent with the ones reported by others57 . The Finngen association analysis for binary traits was adjusted for sex, age, the genotyping batch, and the first 10 principal components. In the association analysis from Biobank Japan, both binary and quantitative traits were adjusted for age, sex, and the top 20 principal components as covariates.We adjusted the χ58. Sequence variants were mapped to NCBI Build38 and matched on position and alleles to harmonize the four datasets.For the meta-analysis, we used a fixed-effects inverse variance method based on effect estimates and standard errors from the four study groups for the additive model. For the full model from Iceland and the UK, we meta-analyzed using sample size and P-values59 to the meta-analysis summary statistics to identify independent association signals, using a stepwise model selection procedure. LD between variants was estimated using a set of 5000 whole-genome sequenced Icelandic individuals.We applied approximate conditional analysis, implemented in the GCTA software15. The adjusted significance thresholds are 2.9 × 10−7 for variants with high impact (N = 6263), 5.8 × 10−8 for variants with moderate impact , 5.2 × 10-9 for low-impact variants , 2.6 × 10−9 for other variants in Dnase I hypersensitivity sites  and 8.7 ×10-10 for all other variants .We applied genome-wide significance thresholds corrected for multiple testing using adjusted Bonferroni procedure weighted for variant classes and predicted functional impact. With 26,738,679 sequence variants being tested in the meta-analysis, the weights given in Sveinbjornsson et al., were rescaled to control the family-wise error rate (FWER)https://data.broadinstitute.org/alkesgroup/LDSCORE/eur_w_ld_chr.tar.bz2).We calculated genetic correlations between urticaria (ICD-10 code L50) diagnoses and other traits as follows: We used cross-trait LD score regression and summary statistics for urticaria (ICD-10 code L50) in the deCODE dataset and from the UKB dataset for other traits. In these analyses, we used results for about 1.2 million well imputed variants, and for LD information we used precomputed LD scores for European populations (downloaded from: n = 750) and whole-blood  from Icelanders. RNA sequencing reads were aligned to personalized genomes using the STAR software package60, for splice-junction quantification and qualitative assessment of alignments  with the urticaria-associated GWAS variants.RNA sequencing was performed on subcutaneous adipose tissue  and transcripts retaining intron 2 with and without rs56043070[A] variant  of the skipping of exon 2 using LeafCutter71 .72. Samples were selected stratified by age, sex, and recruitment center. Day of week of collection, participant ethnicity, and deprivation index were confirmed as representative of cohort distributions. Sample measurement and quality control were performed at Olink’s facilities producing Normalized Protein eXpression (NPX) values for each protein per participant. NPX is Olink’s relative protein quantification unit on log2 scale73. The plasma level measurements were standardized and adjusted for age, sex, and sample age.From the UK we used measurements of 2938 protein levels from 48,684 UK Biobank participants measured using the Olink Explore 3072 platformFurther information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1-16Reporting Summary


Recent work demonstrated that TRPA1 channel protein is up-regulated in several cancer types and that TRPA1-mediated Ca2+ signals can either engage an antiapoptotic pro-survival signaling pathway or to promote mitochondrial Ca2+ dysfunction and apoptosis. Herein, we sought to assess for the first time the outcome of TRPA1 activation by ROS on primary cultures of metastatic colorectal carcinoma (mCRC cells). We found that TRPA1 channel protein is up-regulated and mediates enhanced hydrogen peroxide (H2O2)-induced Ca2+ entry in mCRC cells as compared to non-neoplastic control cells. The lipid peroxidation product 4-hydroxynonenal (4-HNE) is the main ROS responsible for TRPA1 activation upon mCRC cell exposure to oxidative stress. TRPA1-mediated Ca2+ entry in response to H2O2 and 4-HNE results in mitochondrial Ca2+ overload, followed by mitochondrial depolarization and caspase-3/7 activation. Therefore, targeting TRPA1 could represent an alternative strategy to eradicate metastatic CRC by enhancing its sensitivity to oxidative stress.Colorectal carcinoma (CRC) represents the fourth most common cancer worldwide and is the third most common cause of malignancy-associated mortality. Distant metastases to the liver and lungs are the main drivers of CRC-dependent death. Pro-oxidant therapies, which halt disease progression by exacerbating oxidative stress, represent an antitumour strategy that is currently exploited by chemotherapy and ionizing radiation. A more selective strategy to therapeutically exploit reactive oxygen species (ROS) signaling would consist in targeting a redox sensor that is up-regulated in metastatic cells and is tightly coupled to the stimulation of cancer cell death programs. The non-selective cation channel, Transient Receptor Potential Ankyrin 1 (TRPA1), serves as a sensor of the cellular redox state, being activated to promote extracellular Ca Involvement of secondary organs, such as the liver and lungs, is the main driver of CRC-dependent death: 25% of CRC patients show metastatic disease at diagnosis, whereas ≈50% of the patients develop disease recurrence within 5 years from surgery or adjuvant treatment i rise on cell fate, as distinct Ca2+-permeable channels can be selectively coupled to different Ca2+-dependent decoders in cancer cells i in mCRC cells , which represents the most widespread used TRPA1 inhibitor i  of 35.37 µM, and a maximal response at 200 µM  induced low-amplitude intracellular Ca2+ oscillations  larger in mCRC cells as compared to non-neoplastic cells  and genetic (via the selective siTRPA1) blockade of TRPA1  (5 mM)    [2+]i that was sensitive to TRPA1 inhibition with HC-030031 (30 µM)  Fig. , F, a th signals , 27, andmL) Fig. , F mito) was evaluated in mCRC cells loaded with Rhod-2/AM, the most widely employed fluorophore to monitor mitochondrial free Ca2+ levels [2+]mito that was abolished by blocking TRPA1 with either HC-030031 (30 µM) or the selective siTRPA1 and by inhibiting mitochondrial Ca2+ uptake with the specific antagonist Ru360 (5 µM) i that was sensitive to both pharmacological (via HC-030031) and genetic blockade of TRPA1 activity (via a selective siTRPA1). The waveform of this Ca2+ response is quite different from the repetitive oscillations in [Ca2+]i evoked by TRPA1 activation in lung and breast cancer cells [2+ oscillations in cancer cells have long been known to stimulate cell proliferation and survival [2+]i lead to apoptotic cell death i, which was inhibited by blocking TRPA1-mediated Ca2+ entry via either HC-030031 or the selective siTRPA1. Reactive lipid mediators generated by lipid peroxidation of polyunsaturated fatty acids in the plasma membrane, such as 4-HNE, are involved in cancer initiation and progression i are nicely suited to recruit Ca2+-dependent effectors that promote cancer cell proliferation and survival, including Pyk2 i were monitored in mCRC and non-neoplastic cells loaded with the Ca2+-sensitive ratiometric indicator, Fura-2 acetoxymethyl ester (Fura-2/AM)  is the H2O2 concentration, and EC50 is the half-maximal effective concentration.All the data have been generated by mCRC and non-neoplastic cells expanded from three distinct patients. Each experiment has been carried out three times by using cells obtained by each patient in three separate days. The amplitude of cytosolic and mitochondrial Cat-test for unpaired observations, whereas Differences between multiple groups were evaluated by using one-way ANOVA analysis followed by the post hoc Dunnett’s or Bonferroni tests as appropriate. p < 0.05 indicated statistical significance. No statistical methods were used to predetermine the sample size.Pooled data are presented as mean ± SE. The number of cells analyzed for each condition is indicated in the corresponding bar histograms. Normality of the data was tested with Shapiro–Wilk test. If the data distribution was normal, differences between two groups were evaluated by using the Student’s Fura-2/AM and Rhod-2/AM were purchased from Invitrogen (Life Technologies). All the chemicals were purchased from Sigma-Aldrich .Supplemental MaterialOriginal Data File_Figure 1AOriginal Data File_Figure 1A_Beta-actinOriginal Data File_Figure S3A_TRPA1 silencingOriginal Data File_Figure S3A_Beta-actin silencing