Electrophysiological and atomic force microscopy experiments indicate that Ca2+ stabilizes the hemichannel closed state. However, structural data show that Ca2+ binding induces an electrostatic seal preventing ion transport without significant structural rearrangements. In agreement with the closed-state stabilization hypothesis, we found that the apparent Ca2+ sensitivity is increased as the voltage is made more negative. Moreover, the voltage and Ca2+ dependence of the channel kinetics indicate that the voltage sensor movement and Ca2+ binding are allosterically coupled. An allosteric kinetic model in which the Ca2+ decreases the energy necessary to deactivate the voltage sensor reproduces the effects of Ca2+ and voltage in Cx46 hemichannels. In agreement with the model and suggesting a conformational change that narrows the pore, Ca2+ inhibits the water flux through Cx hemichannels. We conclude that Ca2+ and voltage act allosterically to stabilize the closed conformation of Cx46 hemichannels.The opening of connexin (Cx) hemichannels in the membrane is tightly regulated by calcium (Ca Gap Junction channels connect the cytoplasm of two adjacent cells and are involved in electrical coupling. Hemichannels, on the other hand, connect the cell interior with the extracellular milieu allowing the influx and release of signaling molecules for autocrine and paracrine cell communication. These proteins control many cellular processes and play an important role in human pathophysiology, as they are expressed in almost every tissue of the human body and are related to several hereditary diseases5. Both gating mechanisms are regulated by voltage. Each gate is also differentially regulated by several factors. Ca2+ and several other polyvalent cations8 and extracellular pH affect the slow gate9, while intracellular pH11, post-translational modification of the C-terminal domain12 and metabolic inhibition13 modulate the activation of the fast gate. Regulation of Cx hemichannels by extracellular cations has been extensively studied18. Among these cations, Ca2+ is key for the regulation of Cxs. The extracellular media contains Ca2+ in the millimolar range, concentrations which have been reported to maintain Cx hemichannels mainly closed at resting potentials16. Thus, regulation of hemichannels by Ca2+ can be important to prevent their opening under physiological conditions, and subsequent leakage of the intracellular content19. The affinity of connexins for divalent cations has been measured by analyzing the steady-state currents at different Ca2+ concentration and fitting the normalized ionic currents to a Hill equation of inhibition16. Using this methodology, the half maximal inhibitory concentration (IC 50) of Ca2+ for Cx32 and Cx26 are 1.3 mM and 0.33 mM, respectively.Cx based channels display two gating mechanisms termed “loop” or “slow” gating and “Vj” or “fast” gating2+ generates a shift in the activation of Cx hemichannels to higher voltages20. In the case of Cx46, the effects of voltage on the channel sensitivity to Ca2+ and Mg2+ was first interpreted as a voltage-dependent block followed by a voltage-dependent stabilization of the blocked state6. However, Verselis and Srinivas found that Cx46 hemichannels close even in absence of divalent cations, ruling out the possibility of a divalent block as the gating mechanism, and proposed that external divalent cations stabilize the slow gate closures induced by hyperpolarization18. Moreover, AFM imaging of Cx hemichannels shows that in the presence of Ca2+ the extracellular mouth of the channel narrows21. Taken together, these results support the hypothesis that voltage-dependent gating is an intrinsic property of the slow gate and divalent cations stabilize the closed state(s). However, in human Cx26 the results indicate that extracellular Ca2+ destabilizes the open state of hemichannels facilitating its closure, a mechanism that would require that Ca2+ binds to the open state of the channel16. A new crystallographic structure of the Cx26 gap junction channel in a Ca2+ bound configuration shows no major structural changes, raising the hypothesis of an electrostatic mechanism by which the binding of Ca2+ into the pore generates a large and positive electrostatic potential that hinders cation flux15. Recently, Lopez and colleagues show that the small charged MTSES (2-sulfonatoethyl methanethiosulfonate) reagent and cadmium ion can pass through the putative Ca2+ binding ring22, suggesting that the gating of Cx26 hemichannels promoted by Ca2+ is not electrostatic. Hence, there are still some contradictions and open questions about the mechanism of Cx hemichannel regulation by extracellular Ca2+ and its relation to voltage. Can Ca2+ bind to the open state? Is Ca2+ binding voltage dependent? Can Ca2+ affect the ion flux through an electrostatic mechanism? What is the mechanism through which Ca2+ is stabilizing the closed states? To address these issues we tested the effects of voltage and Ca2+ in Cx46 hemichannels expressed in X. laevis oocytes. We found that Cx46 apparent affinity for Ca2+ changes with voltage, reaching a constant value at voltages that keep most of the channels closed and decreasing exponentially as we get to higher voltages. These observations can only be explained by a voltage-independent binding to the closed state by Ca2+. Further analysis of the channel kinetics reveals two distinguishable kinetic steps and these are directly regulated by voltage, with Ca2+ accelerating the deactivation kinetics which reaches a constant value at high Ca2+ concentrations. This cannot be explained by a linear mechanism and indicate that Ca2+ binding is allosterically coupled to the voltage sensor. The best kinetic model able to account for the equilibrium and kinetic data considers three voltage dependent steps and six independent Ca2+ binding sites. Analysis of the water permeability of Cx46 in hypotonic medium and under Ca2+-free and high Ca2+ conditions we found that Ca2+ binding to the Cx46 channel promoted a substantial reduction in the passage of water indicating that Ca2+ promotes the closing of a physical gate in the channel. This work supports that Ca2+ binds only to the closed state of the channel by a voltage independent mechanism. Remarkably, a detailed analysis of the activation and deactivation channel kinetics suggests that Ca2+ and voltage sensors are allosterically coupled to promote Cx46 hemichannel closing.Additionally, Ca6. The Cx46 hemichannel voltage dependence can be obtained by measuring the normalized tail current right after the pulse. We obtained the (Itail(V)/Imax) -voltage relationship and fitted this data to a Boltzmann function (equation V1/2) of −18 mV and an apparent number of gating charges (zδ) of 2.8 elementary charges . As the Ca2+ concentration in the bath increases, the current induced by the depolarizing pulse decreases  at the end of the pulse can be fitted using the Hill equation for inhibition (equation IC50 of 0.17 mM and a Hill slope (n) of 2 . Alternatively, we can also use a linear model consisting of three states O-C-CCan where Ca2+ binding to the closed state is described by a Hill equation. The Hill equation assumes highly cooperative binding steps. We fitted all the inhibition curves to these models and the resulting fitting parameters determined for three of them are presented in Table In kinetic scheme R1 only the closed-open transition, defined by It can be seen that the linear model using the Hill equation has the lowest errors in the parameters estimates compared to the other two linear sequential binding models. Increments in the number of parameters are associated with the error increase, as can be observed for the three binding sites model. Also, the linear model with two binding sites resembles the linear model using the Hill equation. In the two sites model the second binding has an affinity about 250 times greater than the first, thus binding to the first site will generate a highly cooperative binding to the second site. These are the reasons why the linear model using the Hill equation was preferred over the alternative linear models.2+ affinity  reaches a constant value of (1/n) Log (KD) when V ≪V1/2 and that the Log (IC50) increases linearly with voltage when V≫ V1/2  can be determined by two methods. First, we can determine the voltage dependence of the channel and later the affinity for Ca2+ at any voltage. With this data, we can extrapolate the Kd of the closed state. Second, we can determine the affinity of the channel using very low voltages that will keep most of the channels closed. Our results using the extrapolation from 0 mV gives a Kd of 3 µM and using the IC 50 at -50 mV a Kd of 8 µM. By fitting all the data points we obtain a Kd of 5 µM (95% CI: 3.9–6.8) (See Appendix 1 for details).Our model shows that using a voltage pulse that keeps most of the channels closed will result in an inhibition curve that resembles that of the closed state. Thus, the apparent affinity for the closed state (2+ concentration (See Appendix 1). In fact, the deactivation and activation kinetics of the channel does not show a single exponential behavior they are better fitted by the sum of two exponentials . Since Cx hemichannels are hexamers we tested alternative models with 2, 3 and 6 binding sites, according to the symmetry of the channel. Based on the odds ratio . The correlation between parameters is usually low indicating a good exploration of the parameter space by the chain . Bayesian MCMC methods allow to determine parameter identifiability and errors2+ and not with the electrostatic barrier mechanism. To experimentally test whether Ca2+ binding stabilizes a conformation in which the Cx46 pore has contracted, we measured volume changes of oocytes injected with Cx46 in the presence or absence of Ca2+ under hypotonic conditions. If Ca2+ is stabilizing a closed configuration of the Cx46 hemichannel, we expect to detect a clear decrease of the water flow in the presence of the Ca2+. On the other hand, a purely electrostatic mechanism , being water a dipole, should not hinder water flow through the channel. Oocytes were placed in a hypotonic solution and the volume of oocytes was tracked. Volume relative to the initial volume (V/Vi) was calculated. After 900 s oocytes expressing Cx46 showed an increase in the volume of about 8% in conditions without Ca2+, in 5 mM of Ca2+ the volume change was about 3%   Fig. .Figure 72+ and voltage in the regulation of Cx46 hemichannels. Since both regulatory mechanisms are ubiquitous among Cx hemichannels we expect that our findings are pertinent to other Cx isoforms. The present study supports the idea that Ca2+ and voltage act synergistically on the slow gate. Under physiological conditions, the extracellular concentration of Ca2+ and typical membrane voltage of a resting cell would maintain most Cx hemichannels closed. Nevertheless, conditions that lower local extracellular Ca2+ concentration such as repetitive activity in neurons30 may promote the opening of hemichannels even at resting potentials.In this study, we address the interplay between Caet al. that Ca2+ inhibited Cx hemichannels through a voltage-dependent block, followed by a voltage-dependent binding to this blocked state6. The voltage-dependent stabilization of the closed state comes from an experiment where the divalent cation was added while keeping the channel closed, after the application a reduction of the current induced by depolarization was observed6. This experiment argues in favor of a stabilization of the closed state (or blocked state as defined in this first model). The voltage-dependent block was called into question by Verselis et al., who showed that Cx46 hemichannels did close even in the absence of divalent cations, and adding divalent cations produced longer-lived closed states18. These results indicate that divalent cations stabilize the closed state of the channel but whether this binding is voltage dependent is still unknown. Also, they do not discard the binding of Ca2+ to the open state. Moreover, a new structure of the gap junctional channel by Bennet et al. showed binding of Ca2+ to the open conformation, to which they propose an electrostatic barrier as the mechanism through which Ca2+ might affect the permeation15. We note here that this electrostatic mechanism is mechanistically equivalent to a block of the open state and is at odds with Verselis et al. results18.It was initially proposed by Ebihara 2+ bind to the open state? Is the Ca2+ binding voltage dependent? What is the mechanism through which Ca2+ may be stabilizing the closed states? Considering the new evidence we gathered here, we also re-examined the electrostatic barrier or closed state stabilization mechanism for Ca2+ inhibition. We relied on the ability of voltage to modify the open-closed equilibrium. We found that as we lower the voltage of the test pulse the apparent affinity of Ca2+ increases and saturates at negative potentials (−30 and −50 mV). Altogether the present results can only be explained by a voltage-independent stabilization of the closed state of the channel by Ca2+. A possible interference in our results can come from the presence of Mg2+ in the recording solution. The presence of Mg2+ is necessary to avoid endogenous currents that arise in the absence of divalent cations. The effect of Mg2+ in the channel is similar to Ca2+ but with a lower affinity18, so it is expected that Mg2+ will be displaced by Ca2+ in our experiments, especially at high Ca2+ concentrations.In this work, we addressed the following issues: Can Ca2+ binding to Cx46. The best model that accounts for Ca2+ affinity utilizes the Hill equation as an approximation for binding in the closed state. It has been shown that even for simple models with two binding sites it is not possible to obtain the parameters of the model from binding curves alone28. Thus, even though the Hill equation has no clear physical meaning due to the assumption of infinite cooperativity, it is useful to quantify Ca2+ affinity of the Cx46 hemichannel.We tested several linear binding models to describe Ca2+ dependence of the channel kinetics. From a linear model with only one voltage-dependent transition, we would expect that the kinetics show only one exponential in absence Ca2+. It is also expected that Ca2+ should not affect the kinetics at very negative potentials. In fact, Cx46 hemichannels kinetics are better explained by two exponentials, and both components are voltage-dependent. This has also been observed by other groups14. The kinetics at −100 mV also showed Ca2+ dependence, with saturation at high Ca2+ concentration. We could explain the voltage-dependent time constants using a linear model with two voltage-dependent steps, but the Ca2+ dependence of the deactivation should not be affected in this kind of model. If the time constants were determined by the rates of Ca2+ binding and unbinding we expect them to tend to zero at high Ca2+ concentration, which they don’t. Thus, we are certain that voltage and Ca+2 dependence of Cx46 cannot be explained by linear models, but a model in which Ca2+ binding and voltage sensing are allosterically coupled.To test the suitability of the linear model, we measured the voltage and Ca2+ and voltage dependence of the channel kinetics. In this model, the first transition is voltage dependent and not affected by Ca2+. The existence of this transition is important to explain the change in Ca2+ affinity with voltage. If this transition is not voltage-dependent one would expect a saturation of the inhibition curve at high potentials. We assume that the kinetics of this first transition is too fast, probably in the order of milliseconds, to be observed under our experimental conditions . The binding of Ca2+ is considered to be limited by diffusion and thus in equilibrium with respect to the transition between the voltage-dependent steps. This consideration allows us to collapse a model with 10 or more states into four states with rates depending on the relative occupation of all the states in the model given in Fig. 2+-dependent time constants and is the most parsimonious model able to account for the equilibrium and kinetic data presented in this study. It is possible that other models, such as binding to intermediate states could generate the same effects as the one observed, but we discard these models because they do not generate inhibition curves that saturates at negative voltages.An allosteric model is able to account for all the observed phenomena; the saturation of the IC50 at negative potentials, the decrease in the apparent affinity with higher voltages and the Ca2+ binding and voltage sensor movement are coupled. This coupling is reflected by the coupling constant C which is given by cb/cf and is 1.28 18. These observations are in reasonable agreement with the assumption that the open-closed transition is in equilibrium with respect to deeper closed transitions.Verselis 2+ dependence18. Lopez et al. interpret their data based on a two-states kinetic model in which Ca2+ binding destabilizes the open state, which is equivalent to a Ca2+-blocking model. However, the evidence against a Ca2+ block in Cxs provided by Verselis and the fact that the D50N mutation impairs Ca+2 inhibition without a strong effect on the voltage-dependence of the channel make this mechanism untenable24. In a block model, the Kd can be obtained from the inhibition or from the time constant vs Ca2+. Lopez et al. found in Cx26 that the Kd obtained from inhibition and kinetics are 0.33 and 0.72 respectively and they interpreted the difference due to effects of Ca+2 on the open and closed state16. Using the allosteric model, we do not need to appeal to these differential effects. The difference between the Kd obtained between the inhibition curves and the kinetics can be reproduced in the allosteric model with a large backward coupling constant cb and a low Kd. We can also explain the single exponential behavior allowing to the transition between the C1 and C2 state to be the slowest rate of the system, this will mask the kinetic effects of the deeper transitions. The application of the allosteric model to Cx26 can help to explain how the D50N mutation has a strong effect on Ca+2 inhibition without impairing the voltage dependence24. As Sanchez et al. noticed, it is probable that D50N mutation uncouples Ca2+ binding from loop gating24.In Cx26 the deactivation time constants show a single exponential, voltage and Ca2+ bound structure of Cx2615 showed that Ca2+ binds to pore-lining residues that are part of the proposed gate and voltage sensor33. The structure obtained does not show any important structural differences between the Ca2+ bound and unbound structures. To explain this finding, Bennet et al.15 suggested that bound Ca2+ acts as the electrostatic barrier preventing cation flux. It is possible that the constrain imposed by the formation of the gap junction in the extracellular loops might prevent the conformational changes and formation of the Ca2+ binding sites present in hemichannels. It is possible that Ca2+ can affect the channel conductance through several mechanisms, regulation of the open-closed equilibrium and direct regulation of ion permeation both mechanisms having differential roles in the regulation of hemichannels and gap junction channels. Our results are consistent with those of Lopez et al. in which they show that modification of glycine-45 in Cx26 when mutated to cysteine (G45C), can be modified by the negatively charged MTSES and Cd2+, and that this modification is not affected by the presence of Ca2+22. The G45C mutant is inhibited by Ca2+ indicating that the presence of Ca2+ does not generate an electrostatic barrier near the glycine-45 residue in Cx26 hemichannels. In line with this, we performed water permeability assays in oocytes expressing Cx46. When Ca2+ was added to the bath, the level of water influx into the oocyte was decreased. If the electrostatic plug is present, water being a dipole should not be as much affected as ions in its flux through hemichannels. Therefore, our results indicate that the presence of Ca2+ promotes the closure of the pore to levels that significantly hinder the flux of water.The recent Ca2+ can inhibit the channel. By stabilizing the closed state of Cxs channels, Ca2+ acts as a safety valve, keeping hemichannels closed and avoiding the deleterious consequences of their opening. The simplest mechanism we can think of is one in which negative charges in the voltage sensor/pore of Cx hemichannels are responsible for Ca2+ binding but they are only reachable by Ca2+ when voltage sensors acquired their resting conformation. One can think of Ca2+ as a “foot in the door” but unlike the block of Kv channels by tetraethylammonium34, it would “immobilize” the gating charge in its “resting” conformation. Finally, we believe that the allosteric model presented in this work will be of importance as a conceptual framework to understand Cxs hemichannel gating.The proposed model has direct mechanistic implications since it implies that part of the voltage sensor of the slow gate must move before Cain vitro transcription kit SP6 mMessagemMachine systems (Ambion) following manufacturer’s instructions. Stage V and VI oocytes were selected, defolliculated and injected with 5 ng of the RNA plus Cx38 antisense to null endogenous expression of Cx3835. Oocytes were incubated at 18 °C in ND96 medium containing (in mM): 96 NaCl, 1 KCl, 1 MgCl2, 10 HEPES and 1.8 CaCl2. Experiments were done 24–48 hrs post injection.Rat Cx46 pSP64T was kindly provided by Dr. Lisa Ebihara, Rosalind Franklin University. Cx46 DNA was linearized using the EcoRI enzyme (Fermentas) and transcribed using the 2 and 10 HEPES adjusted to pH 7.4. Macroscopic currents were sampled at 1 kHz using an analog to digital converter “Digidata 1200” (Axon instruments). Ca2+ was added to the recording chamber to obtain the desired concentration.Oocytes were injected with 0.25 nmol of BAPTA before the experiments. Hemichannel currents were obtained and using the two-electrode voltage clamp technique. Currents were acquired and filtered to 1 kHz with an “Oocyte clamp” (Warner Instruments). Electrodes were filled with a 3 M KCl solution and exhibited resistances between 0.5 and 2 MΩ. The bath solution contained (in mM): 88 choline chloride, 1 KCl, 1 MgClItail(V)/Imax –voltage data was fitted using a Boltzmann function:Itail(V) is the tail current at voltage V, Imax is the maximum tail current, zδ is the apparent number of gating charges z multiplied by a fraction of the field δ they cross, V1/2 the voltage at which the tail current is half maximal. R, T, and F have their usual meanings.The 2+ inhibition curves were fitted using the Hill equation:I(Ca) is the tail current at a given Ca2+ concentration, I(0Ca) is the tail current without Ca, [Ca2+] is the Ca2+ concentration and n the Hill slope. The actual values used for the fitting are the Log [Ca2+] and Log IC50. It must be noted that the Hill slope represents the number of binding sites only in the case of infinite cooperativity between the binding sites. Nevertheless, it is relevant to the phenomenological description of the inhibition curves.The Ca2+ we used several linear and nonlinear models which are explained in detail in Appendix 1.For the modeling of Cx46 inhibition by CaGraph figures and fittings of Figs 2+ all transitions from O to C3 are voltage dependent and controlled by the slow gate. If we assume that the binding of Ca2+ and the first open-closed transition are in equilibrium with respect to the movement of the voltage sensors in the other closed states, this model can be collapsed into a four-states model, each one comprising several substates. The first state is called O and considers only the open state, this is in equilibrium with the C1 unbounded state. The C1 state comprises the Ca2+ bounded and unbounded C1n state, where n denotes the number of Ca2+ bounds. The states C2 and C3 comprise Ca2+ bounded and unbounded states of C2n and C3n. This model is treated in depth in Appendix 1.The model presented in Fig. 36.The global fitting of all the experimental data to a single mechanistic model with several calcium-binding sites and several voltage-dependent steps was done using a custom-made MCMC script in Mathematica (Wolfram 10.3). MCMC methods are a class of algorithms that allows us the sampling from a distribution based on constructing a Markov chain that has the desired distribution as its equilibrium distribution. Among the advantages of this methodology is the ability to assess parameter identifiability and calculation of parameter errors in complex models such as the one presented hereP(θ/D)represents the probability or probability density function of a set of parameters θ given the new information  also called the posterior probability, P(θ) is the prior probability of the parameters , P(D/θ) is the likelihood of the data given the set of parameters θ and P(D) is the probability of the data itself. It can be though more empirically as an integration constant that makes the integral of P(θ/D) over all the parameter space equal to one. A detailed explanation of the approach utilized can be found in Appendix 2.This methodology is based on the Bayes’ theorem, which for the parameters of a model can be written as:38. Oocytes injected with Cx46 and Cx38 antisense or Cx38 antisense only (CT) were left in a hypotonic solution containing (in mM) 44 NaCl, 0.5 KCl, 0.5 MgCl2 and 5 HEPES adjusted to pH 7.4, 5 mM CaCl2 was added to promote hemichannel closing. Videos of the oocytes were acquired using a Logitech® Webcam C920 HD Pro mounted on an Olympus BX51WI microscope with a 5X objective using Oasis software version 1.00 (Oasis Scientific). Each frame was converted to images using Virtual Dub software version 1.10.4. The images were analyzed in Mathematica 10.3 (Wolfram) using a custom made script. The script calculated the volume of the oocyte based on the area at the focal plane and assuming spherical symmetry. Relative volume (V/Vi) was calculated in 1 frame for each second. The rate of volume change was calculated using the last 50 seconds of the recording.Analysis of volume changes of oocytes was based on the methodology described for aquaporin water channelsThe datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Information


We also observe ultrahigh ON/OFF ratio of >104 and modulation strengths ~200% V−1 of the nonlinear signal. The unusual nonlinear behavior, including super-quadratic voltage and power dependence, is ascribed to the high-field domain, which can be further controlled by near-infrared optical excitation and electrical gating. The ability to electrically control nonlinear optical signals in nanostructures can enable optoelectronic devices such as optical transistors and modulators for on-chip integrated photonics.Dynamic control of nonlinear signals is critical for a wide variety of optoelectronic applications, such as signal processing for optical computing. However, controlling nonlinear optical signals with large modulation strengths and near-perfect contrast remains a challenging problem due to intrinsic second-order nonlinear coefficients via bulk or surface contributions. Here, via electrical control, we turn on and tune second-order nonlinear coefficients in semiconducting CdS nanobelts from zero to up to 151 pm V Dynamically controlling nonlinear signals with large modulation amplitudes is a challenging task. Here, Ren et al. demonstrate high-contrast electric switching of the second-order nonlinear coefficient in CdS nanobelts and explain the observed transistor-like behavior based on high-field domains. Two most commonly studied phenomena for controlling optical signals via applied electric fields are: electro-optical effect3 and electric field-induced second-harmonic generation (EFISH)7. EFISH was first observed in a bulk calcite crystal in 19626, in which a strong electric field (Fl) was applied to break structural inversion symmetry and induce effective second-order nonlinear coefficients 7 and metal–semiconductor8 interfaces, metal-oxide-semiconductors9, polymer-filled plasmonic nanoslits10, and metamaterials11. For most reports, the initial SHG signal at zero applied voltage was not zero and only intrinsic non-zero second-order nonlinear coefficients were typically modified via the electric field. Typically EFISH is a weak effect due to the small third-order nonlinear coefficients, and consequently the field-induced second-order nonlinear coefficients are small (<10 pm V−1)6, and optical modulation is weak (<10% V−1) with a small tuning range due to the presence of large intrinsic second-order nonlinear coefficients (~10 pm V−1)11. Even if a strong electric field is applied to an insulating bulk material or an interface such as semiconductor/electrolyte, it is challenging to switch bulk second-order nonlinear coefficients from zero to very-large values , let alone in semiconductors, which typically have a large density of free carriers. It would be desirable if new nonlinear coefficients could be obtained in semiconductors with perfect contrast and strong modulation strengths, via controlling the unique electric field profile, to enable their use for practical applications and integration with other on-chip electronic/photonic devices.Interest in electrically controlled optical functionalities have been motivated by applications in linear and nonlinear optical systemsd11) in individual semiconducting CdS nanobelts to a value as high as 151 pm V−1 (approximately two-times larger than other intrinsic tensor elements of CdS) by applying a symmetry-breaking electric field. The field-induced second-order nonlinear coefficients, in contrast to intrinsically non-zero coefficients, are intrinsically zero and are produced by applying an external field, which breaks certain relevant mirror symmetries along the field direction. The voltage-dependence of the SHG signal corresponding to d11 (d11-SHG) exhibits very-high modulation strengths of ~200% V−1, and, importantly, with an error function-like shape (rapid rise followed by saturation), which is different from the linear or quadratic response typically observed in EFISH11 and important for device applications, such as nonlinear optical transistors. These unusual properties are attributed to the formation of a high-field domain in the material, which initiates at the electrical contacts and the domain wall moves with the applied field leading to very-high optical nonlinearities to turn on tensor elements that are otherwise zero. The high-field domain can be manipulated by infrared (IR) excitation and gate voltage, which further allows for SHG signal modulation in addition to applied electric fields.In this paper, we report extremely large tunability of an intrinsically zero second-order nonlinear coefficient  of the sample in Fig. 3. Asymmetric contacts (Ti/Au for Ohmic and Au/Al for Schottky) were fabricated in order to apply strong electric field to the sample via the Schottky field (see section Methods) at one junction. A femtosecond pulsed Ti:sapphire laser, tuned from 680 to 1080 nm with ~140 fs pulse width and 80 MHz repetition rate was focused to a spot size of ~3 μm to perform the SHG measurements. Wurtzite CdS has mirror symmetry along its a-axis with a uniform distribution of its electron wave function without a net nonlinear dipole moment  and drain (D) electrodes  at a FW wavelength of 1018 nm. The external normalized conversion efficiency of d11-SHG signal (−9 W−1) is 3.76 times stronger than d33-SHG at zero bias, an intrinsic and largest second-order nonlinear coefficient of CdS , is significantly larger than any other intrinsic second-order nonlinear coefficients of CdS and also other commonly used nonlinear crystals . The SHG signal ON/OFF ratio is estimated to be >104, with the ON state corresponding to the saturation SHG (SHGmax)  at ~22 V, where SHG starts to appear and can be reliably recorded above the noise floor (see Supplementary Note max−SHGmin)/(SHGmax+SHGmin) is ~1 due to the large ON/OFF ratio.To study dynamically controlled SHG, we fabricated two-terminal devices on a single-CdS nanobelt with a d.c. electric field applied along the CdS ent Fig. , hence cnal Fig. . Howeverdes Fig. , inset, CdS Fig. . The strion Fig.  and indud11-SHG, we measured the d11-SHG signal with respect to applied voltage at different fundamental wavelengths   behavior , then increases dramatically when Vt < VDS < Vs (where Vs ~ 40 V is the saturation SHG voltage) and eventually saturates at VDS > Vs  signal compared to 1043 nm and is expected since the third-order nonlinear coefficient  and eventually becomes zero   and saturation current (Js). Although, higher saturation SHG  and higher saturation current (Js) are observed at 1018 nm compared to 1043 nm   observed in our case is several orders of magnitude lower in comparison to other reports where current density > 106 A cm−2 was observed to modify SHG signals15. Therefore, the saturation of SHG signals at VDS > Vs indicates that a constant electric field is built in the optical excitation region  Fig.  and simuior Fig.  of the d Vs Fig. . d11-SHGage Fig. . The sloge) Fig. . Similar nm Fig. , the ele nm Fig.  while thVt) Fig. , and theVt) Fig.  while th Vs Fig. . Moreoveion Fig. .Fig. 2VoI–V behavior can be understood to originate from the Schottky junction  requiring very-high applied voltage (~kV)21, it still remains unclear if the high-field domain can be achieved in nanostructures (~100 nm thickness), and thus its dynamics  and unique attributes, such as their control by external stimuli remain unknown. Additionally, the nonlinear optical properties of high-field domains remain unknown along with their interactions with excitons that can influence bulk nonlinear optical processes.This ion Fig. , since o20. Once the high-field domain is initiated, the current also starts to saturate19 due to carrier depletion. Due to the similar features , our observations (SHG and I–V) are likely related to a stationary high-field domain formation in the CdS nanobelt. In order to validate this hypothesis, we studied the underlying mechanism of the high-field domain in our system and present a model based on field-induced ionization of deep-level acceptor traps in a Schottky barrier  and cadmium (VCd) vacancies and interstitials (Cd1 and S1)28. CdS is intrinsically n-type due to the presence of VS as donor traps (ionized donor density: ND)24. VCd in CdS have also been studied27 and demonstrated to function as acceptor traps28, which are initially neutral and become ionized or negatively charged after accepting an electron29. An increase in the reverse bias voltage can expand the Schottky barrier along the device and increase the internal electric field. As the internal field approaches a critical value (Fc) at Vc, the strong electric field can excite electrons from the valence to the acceptor states in the forbidden band-gap region27, which then become ionized and negatively charged (ionized acceptor density: NA)29. We refer to this process as field-induced ionization, which reduces the net space charge density (ρ) in the Schottky region from ρ = qND  to ρ = q(ND−NA) at Fc  and the internal field remains constant at Fc  Fig. . The region Fig. . The dom13,Iω is the FW intensity, L is the total sample length between the two contacts, f  is the spatial profile of the internal field  stands for the Gaussian beam profile of FW, centered at x0 with a width, w . Therefore, the SHG signal is d11-SHG , A is constant and the average field equals Fc, upon which the SHG signal saturates  is distinct from the saturation current–voltage (Vc), describing when the high-field domain is initiated (at xc = 0) with Vs = Vc + Fcx0.Based on the Schottky high-field domain model, we can quantitatively understand the experimental results by calculating the SHG field , where the Schottky barrier is initially formed, as predicted by the Schottky high-field domain model (Eq. 1). To understand the spatial dependence of the high-field domain, we measured SHG at different excitation positions (X1–X5) on the device with the same FW intensity , but greatly differs in threshold (Vt) and saturation voltage (Vs) values. For example, at X1 (close to the S-electrode at x = 0) where the high-field domain is initiated, the SHG signal saturates at Vs = 38 V, while at position X2 and X3, Vs = 45 and 60 V, respectively, indicating the high-field domain expands from position X1 to position X3, and eventually to position X4 and X5 with a continued increase of applied voltage. This is consistent with our model, thus demonstrating that the high-field domain due to field-induced ionization enables the swtiching of otherwise zero second-order nonlinear coefficients and contributes to the unique SHG behavior. The slight differences in the saturation SHG at some positions is likely due to inhomogeneity in the CdS nanobelt   is independent of the excitation intensity30. Moreover, higher near-IR intensity also induces higher saturation currents (Js) and saturation current–voltage (Vc) 25, or from mid-gap states into the conduction band 27. Typically, IR quenching would dominate, although because of the presence of the high-field domain, most of the low-level acceptor traps are already filled, allowing for more electrons to be excited from trap states (unionized donor and ionized acceptor) into the conduction band, and thereby increase the current , a higher electric field is required to induce additional NA  to maintain the condition (ND = NA) for space charge neutrality (Supplementary Equation Vs), current (Js), and field (Fc), which are consistent with our observations  Fig. . This prVc) Fig. . We can ons Fig. .Fig. 4NeVDS = −30 V, the SHG signal does not saturate yet and the near-IR excitation region is partially or completely overlapping with the high-field domain boundary  decreases /Fc = X 0 + (V−Vs)/Fc, Fig. x 0), generating weaker SHG  of ionized donor traps and consequently change the condition for initializing the high-field domain (ND = NA). We observed that when a negative (positive) gate was applied, the current (JDS) decreased (increased), whereas SHG within the high-field domain , a large number of electrons are injected from the contact, which can increase the current  to satisfy ND = NA, which require lower applied electric fields and hence induce weaker SHG signals  can remove electrons from the traps, causing a decrease in the current . This behavior is in contrast to previously reported gate-controlled SHG owing to gate-tuned charged excitons31 or gate-induced accumulation of carriers32, and demonstrates a completely different mechanism through gate voltage-engineered density of ionized donor traps to control the electric field strength and profile inside the material to generate very-strongly modulated SHG signals that may find applications in high-contrast optical modulator and transistor devices.The dynamic control over the SHG signal can be further demonstrated with the application of a gate voltage, which can change the occupation density  through a range of control measurements. The second-order nonlinear coefficient, d11, is intrinsically zero due to the mirror symmetry and can be tuned up to 151 pm V−1. This value was obtained by comparing d11-SHG with d33-SHG signals, which are produced from the same sample and with the same experimental conditions . In CdS, d33 = 78 pm V−1 is the largest intrinsic second-order nonlinear coefficient13 and cannot be tuned when the electric field is applied along the x-axis. Analogous to a conventional electrical transistor, the signal ON/OFF ratio of our SHG device is estimated to be >104. The modulation strength or subthreshold slope is around 200% V−1, which is obtained near the threshold voltage (see Supplementary Note NA), the thermal velocity of the electrons, and the field-enhanced cross-section for capturing electrons27. However, the response time can become much faster due to strong applied fields that modify the carrier trapping/de-trapping rates. The response time could also be improved via improved device geometries, such as smaller cross-section and smaller interaction region with the fundamental beam, and also by operating the device close to the threshold region. At high current densities, the sample heating may also influence SHG signals; however, in comparison to the strong field-induced SHG signals, heating does not have a significant impact on d11-SHG signals in our case (see Supplementary Note We observed d11), which is otherwise zero in CdS nanobelts. The field-induced d11-SHG signal rapidly increases with the applied voltage and eventually saturates, exhibiting a large ON/OFF ratio of ~104 and a steep subthreshold slope of ~200% V−1 and is much larger than previously reported values11. This unusual SHG behavior is attributed to the high-field domain generated in the CdS nanobelts, which is initiated at the Schottky contact region owing to the field-induced ionization of acceptor traps and can also be controlled by near-IR excitation and source–drain and gate voltage. Further improvement in device properties may be obtained by engineering defects in these materials and one can also envision utilizing high-field domains to induce large optical nonlinearities in materials-like GaAs for similar functionalities. Our study demonstrates a new way to dynamically control nonlinear optical signals in nanoscale materials with ultrahigh signal contrast and signal saturation, which can enable the development of nonlinear optical transistors and modulators for on-chip photonic devices with high-performance metrics and small-form factors, which can be further enhanced by integrating with nanoscale optical cavities28.In conclusion, by applying an external voltage, which breaks certain mirror symmetries, we are able to turn on and modulate the second-order nonlinear coefficient  followed by electron beam evaporation (Kurt Leskter PVD 75) of metal. In order to fabricate Schottky diode devices, lithography and metal deposition were performed for each metal contact in separate steps, with either (200/100/200) Ti/Au/Al or (200/100) Au/Al. No current leakage was observed in the device via the SiO2 layer (source–drain or gate) and also no measurable SHG was observed without the presence of a CdS nanobelt. If the laser was moved away from CdS device, no optical SHG signals were observed. Furthermore, SHG polarimetry data  and then dry-transferred onto a silicon substrate with a 300 nm SiO−1 500 nm blaze grating with a CCD detector (Princeton instruments) with a spectral resolution of 0.1 nm.The CdS nanobelt devices were mounted in a continuous flow optical microscopy cryostat  with electrical feedthroughs connected to an electrical measurement system, for room-temperature and low-temperature measurements. The voltage bias was sourced (0–200 V) and the output current signal was converted to an amplified voltage signal by a current preamplifier (DL instruments model 1211) and recorded continuously (~10 data points per second) by PCI card . A femtosecond pulsed Ti: sapphire laser (Chameleon), tuned from 680 to 1080 nm with ~140 fs pulse width and 80 MHz repetition rate, was used to perform SHG measurements. The laser polarization was controlled by a half-wave plate (HWP) and then focused (spot size ~3 μm) onto individual nanobelts by a home-built microscope equipped with a ×60, 0.7 NA objective (Nikon). The back-scattered SHG signals were imaged by a cooled charge-coupled device (CCD) and detected by a spectrometer (Acton) with a 300 groove mmThe data that support the findings of this study are available from the authors upon reasonable request.Supplementary MaterialsPeer Review File


In this study, the possible site(s) of action of AA in round spermatids, the signalling pathways associated and the intracellular Ca2+ stores targeted by AA-induced signalling were pharmacologically characterized by measuring intracellular Ca2+ using fluorescent Ca2+ probes. Our results suggest that AA acts by interacting with a fatty acid G protein coupled receptor, initiating a G protein signalling cascade that may involve PLA2 and ERK activation, which in turn opens intracellular ryanodine-sensitive channels as well as NAADP-sensitive channels in acidic intracellular Ca2+ stores. The results presented here also suggest that AMPK and PKA modulate this AA-induced Ca2+ release from intracellular Ca2+ stores in round spermatids. We propose that unsaturated free fatty acid lipid signalling in the seminiferous tubule is a novel regulatory component of rat spermatogenesis.Arachidonic acid (AA), a compound secreted by Sertoli cells (SC) in a FSH-dependent manner, is able to induce the release of Ca The functional relationship between germ and Sertoli cells (SCs) in the mammalian seminiferous tubules takes place through adhesion molecules and molecules secreted to the extracellular space in the adluminal compartment –3. The p2+ from intracellular Ca2+ stores (ICaS) in a dose-dependent manner in pachytene spermatocytes and round spermatids [2+ concentration ([Ca2+]i) in spermatogenic cells in the nominal absence of external Ca2+. This Ca2+ was released from ICaS, similarly to the effects of AA. Immunohistochemistry showed that GPR120 was present in spermatogenic cells. Using pharmacological effectors of downstream signalling pathways in these cells, we determined that the AA effect of releasing Ca2+ from ICaS was sensitive to ERK and PLA2 inhibition. Furthermore, modulators of AMPK and PKA affect this AA-induced intracellular Ca2+ release in round spermatids. Both the cADPribose-sensitive channel inhibitor ryanodine and the NAADP-sensitive channel inhibitor NED19 were able to slow the release of Ca2+ elicited by AA. We also showed that round spermatids presented NAADP-sensitive ICaS, which is consistent with the inhibitory action of NED19 on AA-induced Ca2+ release from ICaS. Thus, we provide evidences to support the hypothesis that UFAs can be part of the cell-cell signalling mechanisms in seminiferous tubules, leading to the control of spermatogenic cell differentiation or death by SCs in the testes.In a previous work, we showed that AA and other unsaturated fatty acids (UFA) were able to release Caermatids . In thisad libitum. Adult rats were maintained in groups of four animals per cage. Two rats were chosen at random and their testicles were used to isolate round spermatids as described below.Adult (40–60 days old) male Sprague–Dawley rats were acquired from the Animal Facility in the Faculty of Sciences University of Valparaiso. The rats were housed under a 12L:12D cycle with water and rat chow All the experiments were conducted in accordance with the guidelines outlined by the Consortium for Development in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching and by the National Research Council. All experimental protocols were reviewed and approved by the Chilean National Fund for Science and Technology (FONDECYT) and the Ethics Committee of the Pontificia Universidad Católica de Valparaíso (EC-PUCV-10/2013). None of the authors served on this committee.2 and then killed by cervical dislocation. Rat spermatogenic cell populations were isolated using velocity sedimentation separation in a 2–4% BSA gradient, as described by Romrell et al. (1976) -benzoic acid), PT1 -2-furanyl]methylene]-4,5-dihydro-4-oxo-2-thiazolyl]amino]benzoic acid), H89 (N-[2-[[3-(4-Bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride) and transNED19 -1-[3-[[4-(2-Fluorophenyl)piperazin-1-yl]methyl]-4-methoxyphenyl]-2,3,4,9-tetrahydro-1H-pyridoindole-3-carboxylic acid) were obtained from Tocris Bioscience . All the agonists, activators or inhibitors used in this work are listed in Fura-2 acetoxy methyl ester, 2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxy methyl ester (BCECF) and Hoescht 33342 were purchased from Molecular Probes . Ryanodine, GPR40 agonist phenyl)methyl)amino)phenyl) propanoic acid), ERK Activation Inhibitor Peptide I, ERK Activation Inhibitor Peptide II -1H-pyrazolopyridazin-3-ylamine), GPR120 agonist III -2-yl)methoxy)-phenyl)propanoic acid), GPR40 agonist II  ethynyl)phenyl)propanoic acid), okadaic acid -Hexahydro-3a,7a-dimethyl-4,7-epoxyisobenzofuran-1,3-dione), cyclosporin A and RU360 were purchased from Calbiochem . Arachidonic acid, dantrolene, caffeine, 2-APB (2-aminoethyl diphenyl borate), enzymes, salts and buffers were obtained from Sigma-Aldrich . Grifolic acid  were loaded with 5 μM Fura-2 AM by incubation for 1 h at room temperature under an O2 atmosphere. Thereafter, the cells were washed three times in KH-lactate medium  and maintained in this media unless stated otherwise. The measurements were performed by adding concentrated cells in suspension  to a temperature-regulated and stirred spectro-fluorimeter cuvette that contained 2.5 mL of a KH-no Ca2+-EGTA-lactate  solution in a Fluoromax 2 fluorimeter . [Ca2+]i was calculated using a ratiometric method as described by i vs time, recorded between 30 and 200 s after addition of the compounds. Statistical differences between treatment and control rates of [Ca2+]i changes were analyzed by applying ANOVA followed by Welch’s t test. When the data was expressed as the percentage increase in [Ca2+]i by AA in the absence or presence of different effectors of [Ca2+]i homeostatic mechanisms or signalling pathways, or when the F test indicated significant differences in group variances, the comparison between treatment and control was performed using non-parametrical Mann-Whitney analysis.The initial rate of effector-induced Ca2+]i increase caused by the addition of arachidonic acid (AA), grifolic acid , GPR120 agonist 3, PPMAPP (GPR40 agonist) and GPR40 agonist 2 in round spermatids incubated in KH lactate-EGTA media . Fittin2+ release in round spermatids is shown in 2+]i was diminished as the GA concentration increased, and at 10 μM GA, it was completely abolished. These results strongly suggest that GA and AA release Ca2+ from similar ICaS in round spermatids.The kinetics of grifolic acid-induced intracellular Ca2+]i increase, we utilized an antiGPR120 antibody to perform immunohistochemistry in rat testes. To explore the presence of GPR120 in round spermatids as suggested by the pharmacological data and the AA-induced [Ca2+]i progressively increased at an average rate of 0.04 nM/s. As controls, but also to gain information on their actions on ICaS, the pharmacological agents used in this work were first tested for their basal effects on [Ca2+]i in round spermatids  and Ari2+]i increase in round spermatids. Thus, our data are consistent with involvement of G proteins in the signalling associated to the AA-induced changes in [Ca2+]i in round spermatids. In rat round spermatids, the commercially available inhibitor for GPR120  induced by itself an increase in [Ca2+]i in the absence of AA, discarding its use as an inhibitor of the AA-induced [Ca2+] increases shown in these cells. No further testing on the mechanisms of AH7614-induced changes in round spermatids [Ca2+]i was performed in this work.In order to explore the possible involvement of G protein-associated pathways activated by AA, we tested the effects of the following compounds: 1) SP 4–11, a Substance P-like hydrophobic peptide claimed to compete with its receptor binding to G proteins used here as a control peptide ; 2) mast2+ release from ICaS, we utilized ERK inhibitory peptide I, i in these cells.In order to explore the signalling pathways associated to AA-induced Captide I,  and ERK ptide I, ,58 and 5ptide I,  and wortptide I,  , calmidhibitor; ), KN-62 hibitor; , bisindohibitor;  and forshibitor; ; see alshibitor;  for non-agonist, ) but notagonist, ). Hence, kinases  signific2+ from ICaS in round spermatids, we used okadaic acid , canthaA, e.g., ) and cycn . S3C Fi2+ from ICaS could be via the activation of PLC and the generation of IP3. To test this hypothesis we used as pharmacological agents, U73122, an inhibitor of PLC  and U73M; e.g., ). Neithetids see . Aristol2+ form ICaS. Currently, ICaS are thought to comprise essentially three intracellular compartments: endoplasmic reticulum/nuclear envelope , the inhibitor of two-pore Ca2+ channels from acidic ICaS, produced an average of 50% inhibition of the AA-induced initial rate of Ca2+ release from ICaS.As shown by Paillamanque et al.  and the s; e.g., ,63), thes; e.g.,  and the s; e.g., ). As pre+-ATPase –67. Here+-ATPase ); of cAD+-ATPase ); of mit  and of  . Of the2+]i  did not have an effect on basal i in round spermatids in a dose-dependent fashion with a K0.5 and Vmax of 2.23±0.75 μM and 0.23±0.02 nM/s, respectively. Since the acethoxymethyl group in NAADP-AM is cleaved by intracellular esterases, the intracellular NAADP concentration is expected to be higher than the external concentration. As a reference, we show in the same graph the AA (4 μM) induced rate of [Ca2+]i changes. At 4 μM, NAADP-AM was able to induce an ICaS Ca2+ release rate approximately 35% of that induced by AA at the same concentration. Pre-incubation of the cells for 30 min with 25 μM NED19, an inhibitor of NAADP activated intracellular Ca2+ channels, produced on average a 95% inhibition of the NAADP-AM (1 μM)-induced Ca2+ release from ICaS in round spermatids.Since NED19 induced significant inhibition of the AA-induced Caof NAADP  that hasan sperm . As show2+ from intracellular Ca2+ stores (ICaS) in pachytene spermatocytes and round spermatids. The results presented in this work strongly suggest that arachidonic acid (AA), a PUFA that can be released to the external media by SCs [2+]i had a similar K0.5 to grifolic acid, a GPR120 agonist. However, this GPR120 agonist showed a stronger ability to induce intracellular Ca2+ release compared to AA. An antibody against rat C-terminal domain of GPR120 resulted in a marked labelling of interstitial cells and spermatid cells close to the lumen of the seminiferous tubules, which is consistent with a likely GPR120-mediated effect of AA on [Ca2+]i in round spermatids as shown here and in i was sensitive to G protein-receptor interaction, and appeared to involve ERK activation, and PLA2 activation. The possible participation of PLA2 and ERK and associated mechanisms of activation in the AA-induced release of Ca2+ from ICaS were not further explored in this work. Similarly, the mechanisms associated to the potentiation by activation of AMPK or inhibition of PKA on the effects of AA on ICaS release in round spermatids were not explored in this work. The effects of AA on ICaS release and the association with ERK in round spermatids are similar to those elicited by ω3 and ω6 PUFAs in Caco-2 cells, which express GPR120 but not GPR40  [2+]i and activated ERK1/2 in Caco-2 cells. Similarly, in HEK cells expressing GPR120 [2+]i with K0.5 values close to those found for grifolic acid and AA in our work. As for the possible involvement of a PLA2 in the AA-induced release of Ca2+ from intracellular stores suggested by our results, no literature has linked AA-Ca2+ and PLA2 in spermatogenic cells to our knowledge. However, Ca2+-dependent and Ca2+-independent PLA2 have been described in the testes and specifically in spermatogenic cells i increase in round spermatids. At the times indicated by arrows, grifolic acid or arachidonic acid (4 μM) were added to round spermatids previously loaded with fura-2 and incubated in KH-lactate-EGTA media without added Ca2+ at 33°C.Kinetics of grifolic acid-induced [Ca(TIF)Click here for additional data file.S2 Fig2+]i changes in round spermatids. Each bar and error bar represents the average and standard deviation of data obtained for at least 3 cell preparations (N = 3).Basal effects of different pharmacological molecules used in this work on the [Ca(TIF)Click here for additional data file.S3 FigA. Effects of ERK, Akt and PI3K inhibitors on the AA-induced (4 μM) release of Ca2+ from intracellular stores. ERK inhibitor I (10 μM), ERK inhibitor II (5 μM), PI3K inhibitor: wortmannin (1 μM), Akt inhibitor IV (1 μM) and Akt Inhibitor V (1 μM) were added to the cells in suspension and incubated for 5 min at 33°C before AA addition. Significance: **, p<0.01. N = 3. B. Effects of CaM, PKC, PKA, AMPK inhibitors or activators on the AA-induced (4 μM) release of Ca2+ from intracellular stores. CaM inhibitors: trifluoperazine , calmidazolium , KN-62 (5 μM). PKC inhibitor: bisindoleylmaleimide . AC activator: forskolin . AMPK activator: PT1 (2 μM). PKA inhibitor: H89 (2 μM) were added to the cells in suspension and incubated for 5 min at 33°C before AA addition. Significance: *, p<0.05. N = 3. C. Effects of protein phosphatase (PPase) inhibitors on the AA-induced (4 μM) release of Ca2+ from intracellular stores. Okadaic acid , cantharidin  and cyclosporine A  were added to the cells in suspension and incubated for 5 min at 33°C before AA addition. N = 3. D. Effects of phospholipases inhibitors on the AA-induced (4 μM) release of Ca2+ from intracellular stores. Aristolochic acid . U73122  and U73343  were added to the cells in suspension and incubated for 5 min at 33°C before AA addition. Significance: *, p<0.05. N = 3. Each bar and error bar in S3 Fig  represents the average and standard deviation of data obtained for at least 3 cell preparations (N = 3).(TIF)Click here for additional data file.


However, the role(s) of STIM2, a homolog of STIM1, in skeletal muscle has not been well addressed. The present study, first, was focused on searching for STIM2-binding proteins from among proteins mediating skeletal muscle functions. This study used a binding assay, quadrupole time-of-flight mass spectrometry, and co-immunoprecipitation assay with bona-fide STIM2- and SERCA1a-expressing rabbit skeletal muscle. The region for amino acids from 453 to 729 of STIM2 binds to sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a). Next, oxalate-supported 45Ca2+-uptake experiments and various single-myotube Ca2+ imaging experiments using STIM2-knockdown mouse primary skeletal myotubes have suggested that STIM2 attenuates SERCA1a activity during skeletal muscle contraction, which contributes to the intracellular Ca2+ distribution between the cytosol and the SR at rest. In addition, STIM2 regulates Ca2+ movement through RyR1 during skeletal muscle contraction as well as SOCE. Therefore, via regulation of SERCA1a activity, STIM2 regulates both intracellular Ca2+ distribution and Ca2+ movement in skeletal muscle, which makes it both similar to, yet different from, STIM1.Stromal interaction molecule 1 (STIM1) along with Orai1 mediates extracellular Ca Skeletal muscle contraction occurs via excitation-contraction (EC) coupling. During EC coupling, the action potential in skeletal muscle cells  by neural stimulation spreads along the surface of the plasma membrane and to the interior of skeletal muscle cells via transverse (t)-tubules, which activate dihydropyridine receptors  in the t-tubule membrane. Subsequently, RyR1  in the sarcoplasmic reticulum (SR) membrane is activated by the active DHPRs via physical interactions, which allows Ca2+ release from the SR to the cytosol via the RyR1 for skeletal muscle contraction (up to sub-micromolar concentrations of Ca2+ in the cytosol). Ca2+-uptake from the cytosol to the SR is a critical step for skeletal muscle relaxation6. Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a  is a Ca2+ pump in the SR membrane and uptakes Ca2+ from the cytosol into the SR to reduce cytosolic Ca2+ levels to that at rest (nanomolar range) and to refill the SR with Ca2+ during skeletal muscle relaxation9. Therefore, Ca2+ in the SR is the major source of Ca2+ for skeletal muscle contraction, and spatial and temporal distribution of the intracellular Ca2+ between the SR and the cytosol via RyR1, and SERCA1 is a key factor for the cycling of skeletal muscle contraction and relaxation6. Patients with Brody syndrome suffer from exercise-induced muscle stiffness and delayed muscle relaxation due to a reduction in SERCA1a activity, however, the reduced SERCA1a activity does not involve a mutation in the SERCA1a gene12. This suggests that SERCA1 is not the only cause of Brody syndrome, and regulatory proteins of SERCA1a activity could be candidates for the diagnosis or treatment of patients with Brody syndrome. The DHPR, RyR1, and SERCA1a have a functionally efficient arrangement in the t-tubule and the SR membranes that are closely juxtaposed 15. Junctophilin (JP) is required for the proper formation of the junctional membrane complex16.Contraction and relaxation are the main tasks of skeletal muscle2+ entry (SOCE) is a ubiquitous Ca2+ entry way in various cells such as skeletal muscle cells, and stromal interaction molecule 1 , and Orai1 (a Ca2+ entry channel in t-tubule/plasma membranes) are the main proteins responsible for SOCE18. SOCE is required for replenishing the ER/SR with Ca2+ and sustaining the presence of a certain concentration of Ca2+ in the cytosol. In general, during SOCE, the depletion of Ca2+ in the ER induces the dissociation of Ca2+ from STIM1, which allows the relocation of STIM1 to the ER membranes near plasma membranes, the interaction of STIM1 with Orai1 , and extracellular Ca2+ entry through Orai123. The formation of puncta in skeletal muscle occurs as a part of the terminal differentiation of myoblasts  to form mature myotubes in an SR depletion-independent manner, which results in SOCE that is more rapid in skeletal muscle than in other cells, as it occurs in a matter of seconds26.Store-operated Ca2+ release-activated Ca2+ (CRAC)-activating domain) domain, an ezrin-radixin-moesin domain, a proline/serine-rich domain, and a lysine-rich domain30. The EF-hand domain is a Ca2+-sensing region32. The EF-SAM domain is responsible for the self-oligomerization and relocalization of STIM1s to form puncta21. The first coiled-coil domain participates in the self-oligomerization of STIM1 at rest33. The CAD/SOAR domain physically interacts with, and activates, Orai135. The CAD also participates in the self-oligomerization of STIM133. The lys-rich domain is responsible for the Orai1-independent plasma membrane targeting of STIM135. STIM2 is a homolog of STIM1 and is widely expressed in many types of tissues, although the expression level of STIM2 is usually lower than that of STIM129. The role of STIM2 in SOCE is less clear because initial studies on the role of STIM2 in SOCE using smooth muscle cells or heterologous expression systems have produced contradictory results: a positive role in SOCE36, or no effect on SOCE38. STIM2 has an EF-hand region that is more sensitive to Ca2+ than STIM140.STIM1 has a short intraluminal N-terminus that contains an actual EF-hand domain and a sterile α-motif (SAM) domain, a single-transmembrane domain, and a cytosolic C-terminus that contains three coiled-coil domains, SOAR (STIM1-Orai1-activating region)/CAD  show muscular atrophy, tubular aggregate myopathy, and/or progressive muscle weakness47. Patients with a loss-of-function mutation of STIM1, E136X, show congenital myopathies as well as severe combined immunodeficiency49. Patients with another STIM1 mutation, R429C, also show muscular hypotonia50. Muscle fibers from mdx mice, an animal model for Duchenne muscular dystrophy that is characterized by progressive muscle weakness, show increases in both STIM1 expression and SOCE52. It is possible that STIM2 could be also related to the skeletal muscle diseases because STIM2 shares high homologies with STIM1 in their amino acid sequences and domains although there has yet been no report on the direct relevance of STIM2 to skeletal muscle diseases. In case of the smooth muscle, STIM2 contributes to the proliferation of pulmonary arterial smooth muscle cells in patients with pulmonary arterial hypertension54.The conditional deletion of STIM1 in skeletal muscle is prenatally lethal in mice due to myopathy and defective muscle differentiation52, it is possible that STIM2 could also participate in unique functions of skeletal muscle such as terminal differentiation, Ca2+ movements during contraction and relaxation, and more. However, until now, the role of STIM2 in skeletal muscle has not been well addressed. Therefore, in the present study, biochemical approaches were used to identify STIM2-binding proteins from among the proteins that mediate skeletal muscle function. We found that STIM2 binds to SERCA1a, and the functional contribution of the binding between STIM2 and SERCA1a in skeletal muscle was examined using mouse primary skeletal myotubes (instead of a heterologous expression system involving variations in expression) and single-myotube Ca2+-imaging experiments.Considering that STIM1 plays important and various roles in skeletal muscle functions and diseasesTo find unique STIM2-binding proteins among proteins mediating or regulating the contraction and relaxation of skeletal muscle, first, cDNA of the variable region between STIM1 and STIM2 . Supplementary Fig. IM2 Fig. . TherefoIt is possible that, according to the expected protein size, band 8 could be responsible for an oligomeric STIM2-UI (approximately 224 kDa in the case of a tetramer). However, there was no matching signal to the band 8 in the known databases, suggesting that the STIM2-UI region is irrelevant to the self-oligomerization of STIM2. It is possible that the signal from the band 8 could be obtained from experimental shortcomings, for example, an incomplete digestion of the band 8 by trypsin, which could result in a mixed signal that does not match to the known databases.To examine how STIM2 is related to SERCA1a activity, STIM2 in mouse primary skeletal myotubes was knocked down using siRNA. Three different siRNAs for mouse STIM2 were used to knock down STIM2 Table . Therefo45Ca2+-uptake assay was conducted. The Ca2+-uptake activity of SERCA1a was not changed at a resting cytosolic Ca2+ concentration , the Ca2+-uptake activity of SERCA1a was significantly increased by the STIM2-knockdown .To examine SERCA1a activity in the STIM2-knockdown myotubes, an oxalate-supported 2+ level at rest and the amount of Ca2+ in the SR in the STIM2-knockdown myotubes were measured using single-myotube Ca2+-imaging experiments , the SR of the STIM2-knockdown myotubes was depleted with thapsigargin (TG) in the absence of extracellular Ca2+. Extracellular free Ca2+ allows the avoidance of extracellular Ca2+ entry and an assessment of the amount of Ca2+ exclusively in the SR . There was no significant change in the protein content levels of either Orai1 or STIM1 via STIM2-knockdown. Therefore, the decrease in SOCE via STIM2-knockdown was not due to a simple change in the expression levels of either Orai1 or STIM1.2+ level to a certain point at a given time is the key step in skeletal muscle contraction, and RyR1 is the only channel that is responsible for intracellular Ca2+ release from the SR to the cytosol for skeletal muscle contraction. Intracellular Ca2+ releases through RyR1 in response to caffeine, a direct agonist of RyR1 in skeletal muscle55, were examined in the STIM2-knockdown myotubes using Ca2+-imaging experiments  were also increased by the STIM2-knockdown to an approximately equal response to caffeine  participates in the interaction with a SERCA1a.During SOCE, three dimers of STIM1  are required to activate ‘a’ functional hexameric Orai1 that contains a central pore45. In the present study, we found another in-situ SERCA1a regulator in skeletal muscle, STIM2, but its regulation is the reverse of STIM1, which means STIM2 is a negative regulator of SERCA1a. The relationship between STIM1 and STIM2 in terms of regulating SERCA1a is akin to a tug-of war that results in SERCA1a that is neither too much nor too little.Previously, we reported that STIM1 binds to SERCA1a in skeletal muscle and is required for the full-activity of SERCA1a during skeletal muscle relaxation, which means STIM1 is a positive regulator of SERCA1a2+ levels at rest, an increase in the amount of Ca2+ in the SR, but no effect on terminal differentiation45. However, under the disrupted interaction of STIM1 or STIM2 with SERCA1a, the regulation of intercellular Ca2+ movement through RyR1 in response to either caffeine or KCl differed: Ca2+ movement through RyR1 was decreased by a disrupted STIM1-SERCA1a interaction45, but it was increased by a disrupted STIM2-SERCA1a interaction, as shown via the use of STIM2-knockdown in the present study. The binding region of STIM1 or STIM2 to SERCA1a belongs to the variable region between STIM1 and STIM2 , which possibly creates the differences in the roles of STIM1 and STIM2 in skeletal muscle. Different properties in the sensing of Ca2+ and in the self-oligomerization of STIM1 and STIM2 could also account for the different roles of STIM1 and STIM2 in skeletal muscle61. Considering that the regulation of SERCA2a activity by phospholamban depends on the phosphorylation status of phospholamban62, possible phosphorylation sites were searched in the SERCA1a-bindgin regions using NetPhos 3.1 (that predicts both generic- and kinase-specific phosphorylation sites), GPS 3.0 (that predicts kinase-specific phosphorylation sites in a proteome-wide level), or PhosphoSVM (that is a non-kinase-specific prediction tool)  that was used by others, siRNA for STIM2 was transfected to immature myotubes on differentiation day 3  exerts an inhibitory effect on RyR1 channel activity69. Less availability of Ca2+ due to decreases in SOCE by the STIM2-knockdown in the present study could induce a switch of Ca2+-CaM1 to aop-CaM1 that could enhance RyR1 activity. In addition, it is possible that the decreased protein content level of CaM1 by the STIM2-knockdown  have also been proposed as Ca2+ entry channels for SOCE in skeletal muscle74. Skeletal muscle expresses mainly four types of TRPCs: TRPC1, TRPC3, TRPC4, and TRPC675. TRPC1, TRPC3, and TRPC4 are known to mediate SOCE in skeletal muscle77. However, little is known about TRPC6 in skeletal muscle, although it is reported that myoblasts from mdx mice, a mouse model of Duchenne muscular dystrophy, shows a reduced expression of TRPC678. In the present study, TPRC6 protein content was significantly decreased by the STIM2-knockdown . To prepare the cDNA for the STIM2-UI region, oligonucleotide primers were designed on the basis of human STIM2 (GenBank accession number: NM_ AF328905.1) . The affinity beads were then incubated with 150 μg of the solubilized triad sample for 8 h at 4 °C. The proteins that were bound to the affinity beads were separated on a 10% SDS-PAGE gel, and the gel was stained with Coomassie Brilliant Blue in order to obtain the protein bands.The triad vesicles that are enriched portions with proteins mediating or regulating skeletal muscle functions, including SERCA1a81. The digested peptide solution was desalted and concentrated, and was eluted using a homemade C18 nano-column ) and 1.5 ul of 50% MeOH, 49% H2O, and 1% HCO2H. qTOF-MS of the eluted peptides was performed using a Hybrid Quadrupole-TOF LC/MS/MS Mass Spectrometer  equipped with an electrospray ionization (ESI) source. The quadrupole analyzer was used to select precursor ions for fragmentation in the hexapole collision cell. The produced ions were analyzed using an orthogonal TOF analyzer and fitted with a reflector, a micro-channel plate detector, and a time-to-digital converter.Protein bands obtained from the binding assay were subjected to in-gel digestion with trypsin87. For the terminal differentiation of the primary skeletal myoblasts to myotubes, myoblasts were re-plated either on 10-cm plates  or on 96-well plates (for the single-myotube Ca2+ imaging experiments) coated with Matrigel . All reagents for the cell cultures were obtained from Invitrogen (ThermoFisher Scientific).Mouse primary skeletal myoblasts were derived from mouse skeletal muscle using a single-cell cloning method, and these proliferated and differentiated to myotubes, as previously describedμl of X-tremeGENE siRNA Transfection Reagent , and 200 nM of the specific synthetic siRNAs, as previously described45. After the transfection, fully differentiated myotubes on differentiation day 5 were subjected to further experiments.Three different siRNAs for mouse STIM2 (GenBank accession number NM_001081103.2) were selected using two forms of siRNA design software from Ambion (ThermoFisher Scientific) and from Sigma-Aldrich , anti-SERCA1a , or anti-TRPC6  antibody was used for immunoblot assay. For the immunoblot assay, fully differentiated mouse primary skeletal myotubes on 10-cm plates on differentiation day 5 were solubilized, and the solubilized lysate  was subjected to SDS-PAGE  and immunoblot assay, as previously described87. Anti-RyR1, anti-CSQ, anti-CaM1, anti-JP1, and anti-JP2 antibodies were obtained from Thermo Scientific Inc. Anti-TRPC1, anti-TRPC3, and anti-TRPC4 antibodies were obtained from Alomone Laboratories. Anti-DHPR, anti-Orai1, and anti-α-actin antibodies were obtained from Abcam. Horseradish peroxidase-conjugated anti-goat, anti-mouse, or anti-rabbit secondary antibodies were obtained from Jackson ImmunoResearch Laboratories . The membranes were washed three times with PBS and developed using a SuperSignal Ultra Chemiluminescent substrate .For the co-immunoprecipitation assay, the solubilized triad sample  was incubated with anti-STIM2 antibody (Sigma-Aldrich) overnight at 4 °C, as previously described2PO4, 10 mM NaF, 1 mM EDTA, 0.3 M sucrose, protease inhibitor cocktail (Roche Applied Science), and 0.5 mM DTT at pH 7.0) with a homogenizer for 15 s at speed 5 80. Then, 250 μg of the myotube homogenate was subjected to an oxalate-supported 45Ca2+-uptake experiment. Briefly, the reaction buffer was composed of 40 mM imidazole, 100 mM KCl, 5 mM MgCl2, 5 mM NaN3, and 0.5 mM EGTA at pH 7.0. The washing buffer was composed of 100 mM KCl and 20 mM MOPS at pH 7.0. The uptake reaction was begun by the rapid sequential addition of 5 mM Mg-ATP, 5 mM K-oxalate, and either 70 nM or 1 μM of free 45Ca2+ . The rate of 45Ca2+ uptake was calculated from the linear regression of 45Ca2+ uptake at 0, 1, 2, 3, and 4 min.The STIM2-knockdown myotubes were homogenated in a buffer  equipped with a 40X oil-immersion objective (NA 1.30), a high-speed monochromator with a 75 W xenon lamp , and a 12-bit charge-coupled device camera , as previously described88. Fully differentiated mouse primary skeletal myotubes on 96-well plates were loaded with 5 μM fura-2-AM  or with 5 μM fluo-4-AM  in an imaging buffer  at 37 °C for 45 min. The data were displayed and analyzed using image acquisition and analysis software . Either KCl or caffeine was dissolved in the imaging buffer and was applied via an auto-perfusion system . To measure the amount of Ca2+ in the SR, TG  was applied to the myotubes in the absence of extracellular Ca2+ in order to avoid extracellular Ca2+ entry. DMSO (0.05%) alone had no effect on the release of Ca2+. For the measurement of SOCE, the SR Ca2+ was depleted with TG (2.5 μM) in the absence of extracellular Ca2+, and once the cytosolic Ca2+ level returned to the baseline, 2 mM Ca2+ was added to the myotubes to measure the SOCE. To analyze the Ca2+ release obtained from the Ca2+ imaging experiments, the peak amplitude, which exhibited similar increases or decreases in peak areas, was considered. For long-term Ca2+ releases such as SOCE and TG responses, the areas under the curves were analyzed. For the analysis of SOCE, the slope at the rising phase of the peaks  was also examined by a linear equation that was obtained from a linear fitting of the rising phase of the peaks.Single-myotube Ca89, GPS 3.090, or PhosphoSVM91. Under NetPhos 3.1, both generic- and kinase-specific predictions are performed, and the kinase-specific predictions by NetPhos 3.1 cover the prediction by NetPhosK 1.0. GPS 3.0 predicts kinase-specific phosphorylations with a large-scale prediction of >13,000 mammalian phosphorylation sites and a proteome-wide prediction of Aurora-B specific substrates including protein-protein interaction information. PhosphoSVM is a non-kinase-specific prediction tool which detects possible phosphorylation sites for which the associated kinase is unknown or the number of known substrate sequences of the associated kinase is few. For the prediction using NetPhos 3.1 or PhosphoSVM, predictions with more than 0.7 in score were considered. For the predictions using GPS 3.0, threshold was set at high and predictions with more than 20 in score were considered.The possible phosphorylation site was predicted using NetPhos 3.1t-test . The differences were considered to be significant at p < 0.05. The graphs were prepared using Origin v7 software.The results are presented as the mean ± S.E. for the number of myotubes shown in the parentheses of Tables or in the legends of either the Tables or Figures. The values were normalized to the mean value from the corresponding controls. The significant differences were analyzed using an unpaired Supplementary Figures