Cation and voltage dependence of lidocaine inhibition of the hyperpolarization-activated cyclic nucleotide-gated HCN1 channel

Lidocaine is known to inhibit the hyperpolarization-activated mixed cation current (Ih) in cardiac myocytes and neurons, as well in cells transfected with cloned Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels. However, the molecular mechanism of Ih inhibition by this drug has been limitedly explored. Here, we show that inhibition of Ih by lidocaine, recorded from Chinese hamster ovary (CHO) cells expressing the HCN1 channel, reached a steady state within one minute and was reversible. Lidocaine inhibition of Ih was greater at less negative voltages and smaller current amplitudes whereas the voltage-dependence of Ih activation was unchanged. Lidocaine inhibition of Ih measured at −130 mV (a voltage at which Ih is fully activated) was reduced, and Ih amplitude was increased, when the concentration of extracellular potassium was raised to 60 mM from 5.4 mM. By contrast, neither Ih inhibition by the drug nor Ih amplitude at +30 mV (following a test voltage-pulse to −130 mV) were affected by this rise in extracellular potassium. Together, these data indicate that lidocaine inhibition of Ih involves a mechanism which is antagonized by hyperpolarizing voltages and current flow.

Lidocaine is a drug that depresses both sinus node activity and cardiac conduction, acts as a local anesthetic and topical analgesic, and, when applied systemically, produces central analgesia and sedation 1,2 . In addition to its well-known action on voltage-gated sodium channels [3][4][5] , lidocaine may produce its therapeutic actions, as well its known adverse effects, by acting on other target proteins, including Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels. For example, lidocaine has been shown to inhibit native HCN channels in cardiac Purkinje fibres 6,7 , sinoatrial node pacemaker cells 8 , dorsal root ganglion neurons 9 , and thalamocortical neurons 10 , with an IC 50 ranging from 38-99 µM and maximum inhibition varying from 70 to 100%. Two studies have examined the inhibition of heterologously expressed cloned HCN channels by lidocaine, with IC 50 and maximum inhibition values of 67-276 µM and 42-44%, respectively 11,12 . The molecular mechanism underlying the action of lidocaine on HCN channels and the basis for the variation in lidocaine's effect among studies of native and cloned HCN channels remain unknown.
In this study, we examine the mechanistic basis of the inhibition of the HCN channel and associated hyperpolarization-activated current, I h , by lidocaine. Four HCN genes have been identified in mammals (HCN1-HCN4) and they are similar in primary sequence to the Shaker-related voltage-gated potassium (Kv) channels [13][14][15] . However, HCN channels have recently been shown to have a structure that is similar but not identical to those channels 16,17 . Like Kv channels, HCN channels are tetrameric and each subunit is multi-membrane spanning and possesses an intracellular N and C-terminus 16 . However, the recently solved human HCN1 channel structure 16 shows that the selectivity filter (the portion of the pore closest to the outside of the cell) of the HCN channel contains fewer binding sites for cations, which may help to explain the permeability to both sodium and potassium ions [13][14][15]18 . The fourth S4 transmembrane segment, the voltage sensor, is long and appears to hold the pore (transmembrane S6 helices) in a compact and potentially closed conformation. Such a structural medium (Life Technologies, Burlington, ON, Canada) supplemented with 50 µg/ml penicillin/streptomycin (Life Technologies) and 10% fetal bovine serum (Life Technologies). Cells were grown on glass coverslips (22 × 22 mm) in 35 mm dishes at 37 °C with 5% CO 2 . Transfection of a mammalian expression vector encoding mouse HCN1 (pcDNA3, which was a kind gift from Martin Biel, Ludwig-Maximilians-Universität München, Germany) (2 µg per dish) was performed using the FuGene6 transfection reagent (Roche Diagnostics, Laval, QC, Canada) according to the manufacturer's protocol. The DNA plasmid encoding Green Fluorescence Protein (GFP) was co-transfected for visualization of transfected cells.

Electrophysiological Recordings.
Approximately 24 to 48 hours post-transfection, a small piece of coverslip was paced into a recording chamber (900 µl) on the stage of an inverted microscope. Cells were perfused (0.5-1 ml/min) with a low potassium extracellular solution containing (mM): KCl, 5.4; NaCl, 135; MgCl 2 , 0.5; CaCl 2 , 1.8; HEPES, 5, and titrated to pH 7.4 with NaOH. It should be noted that "low" refers only to the other solutions used in this study (see below) whereas a concentration of 5.4 mM potassium in the extracellular solution is usually considered to be a normal concentration in vivo. Cells expressing GFP were selected for patch clamp recording. After attaining the whole-cell configuration, cells were perfused by using a Fast Step SF77B perfusion system (Warner Instruments, Hamden, CT), with either the same, low potassium extracellular solution or solutions containing different proportions of KCl and NaCl. The 30 and 60 mM potassium extracellular solutions contained 30 mM KCl and 110 mM NaCl and 60 mM KCl and 80 mM NaCl, respectively, and were titrated to pH 7.4 with either NaOH or KOH. The calculated osmolarities for the internal and external solutions used were ~290 mOsm. Patch pipettes were pulled from borosilicate glass (World Precision Instruments, Inc., Sarasota, FL) using a P-100 electrode puller (Sutter Instruments, Novato, CA) and filled with an intracellular solution containing (mM): KCl, 130; NaCl, 10; MgCl 2 , 0.5; EGTA, 1; HEPES, 5, adjusted to pH 7.4 with KOH, and had a resistance of 4-5 MΩ. Whole cell patch-clamp recordings were performed with an Axopatch 200B amplifier (Molecular Devices, LLC, Sunnyvale, CA) and a Digidata 1320A data acquisition system (Molecular Devices, LLC) using pCLAMP software (version 10, Molecular Devices, LLC). The membrane currents were low-pass filtered at a frequency of 2 kHz and digitized at 5 kHz. Data were collected more than 5-10 min after whole cell access to allow the internal pipette solution to equilibrate with the cell. Experiments were carried out at room temperature (21-23 °C).
In previous studies, we characterized aspects of HCN1 channel expression in CHO cells 28,29 . By Western blotting and patch clamp electrophysiology, we showed that untransfected CHO cells do not express HCN channel protein or HCN-mediated currents. Upon transfection, robust I h currents were elicited from GFP-expressing cells and HCN1 channel protein is abundant in Western blots. As we found previously, robust hyperpolarization-activated currents were observed in the current study when recording from cells that expressed GFP.
Lidocaine HCl was purchased from Sigma-Aldrich Canada Ltd. (Mississauga, ON, Canada), diluted in corresponding extracellular solution and applied by using the Fast Step SF77B perfusion system. Although the transfection efficiency was quite good, we recorded from only one cell per dish to avoid carrying out voltage protocols on cells that had already been exposed to the drug. New dishes were also employed if measurements were unfinished because of issues such as cells becoming unresponsive to voltage or large and irreversible alterations in access resistance or leak current. Lidocaine was perfused for 1-2 minutes in order to obtain steady state inhibition.
Data Analysis. Data were analyzed using ORIGIN 8 (OriginLab Corporation, Northampton, MA). To determine the IC 50 and Hill coefficient, concentration-response curves were normalized and fitted using the Hill equation as follows: where I represents the current measured in the presence of a given drug concentration; I max is the control current measured in the absence of the drug; [C] is the drug concentration; IC 50 is the half-maximal inhibitory concentration; and h is the Hill coefficient. The normalized conductance-voltage relationship for steady-state activation of HCN1 current was fitted by the Boltzmann equation: where G is the conductance estimated after the current activation at a hyperpolarizing step [V]; G max is the maximum conductance obtained after the most hyperpolarizing step; V 50 is the half-maximal activation potential; and k is the slope factor.
To model the effect of extracellular potassium on inward steady-state I h , the relationship between concentration and current obtained from individual cells was fitted to the Michaelis-Menten model: where I represents the current measured at a given potassium concentration, I max is the maximum current, [K + ] o is the extracellular potassium concentration, and K app is the apparent dissociation constant. Reversal potentials in different concentrations of extracellular potassium were determined from leak-subtracted steady state current obtained from a hyperpolarizing pulse to −90 mV and current obtained from a subsequent pulse to −60 mV. For each cell, values for the steady state at −90 mV and for the subsequent tail current at −60 mV was plotted versus voltage and joined by a straight line, which was extrapolated to cross the x-axis to yield an estimate of reversal potential.
Data are presented as mean ± SEM. We used one-way ANOVA to test for comparisons of more than two groups. Comparisons between two groups were conducted with the use of a paired or unpaired Student t test; Statistical tests were two-tailed and results were considered significant at α = 0.05.

Results
Inhibition of HCN1-mediated I h conductance by lidocaine is greater at less negative potentials. We examined the inhibition by lidocaine (600 µM) of steady-state I h at different voltages and of G h , which was estimated as the magnitude of the tail I h at a constant potential (−65 mV) in 30 mM extracellular potassium ( Fig. 1A-D). This concentration of lidocaine was used because it is in the mid-range of concentrations for inhibition of I h at 30 mM potassium (see below). The fraction of steady state I h inhibited by lidocaine increased from ~44% at −135 mV to ~62% at −90 mV ( Fig. 1B and D). Inhibition of G h by lidocaine was constant at ~70% and, in contrast to steady state I h , changed little over the same range of voltages ( Fig. 1C and D). The lack of change in G h and increase in inhibition of steady state I h at progressively less negative voltages in the same range suggest that lidocaine does not modify the voltage-dependence of I h activation whereas its inhibition of I h conductance increases at less negative potentials.
Raising extracellular potassium increases I h in HCN1-expressing CHO cells without altering the voltage-dependence of activation. We next examined the effect of increasing current flow on lidocaine inhibition of I h . In order to do this, we raised the proportion of extracellular potassium in order to increase current flow, which is one of two approaches used previously to examine how current flow impacts ivabradine inhibition of I h ; inhibition of I h by that drug is less at more negative potentials and, for I h in the sinoatrial node (I f ) and I h associated with the HCN4 isoform, inhibition is reduced by an increase in current flow 30,31 .
We first determined how raising extracellular potassium impacts I h conductance and the voltage-dependence of I h activation when measured in CHO cells expressing the HCN1 isoform. In addition to a depolarizing shift in shown below them from the same cell exposed to extracellular potassium concentrations of 5.4 and 60 mM. The magnitude of I h (for both inward current determined at −130 mV and outward current determined at +30 mV) was calculated as the difference between the instantaneous current at the beginning of each pulse (I inst ) and the steady-state current (I ss ) at the end of the pulse (arrows). Note the large difference in inward current measured at −130 mV and very little difference in current measured at +30 mV between the two extracellular solutions. (B) Amplitudes of I h density (nA/pF) measured at −130 mV (inward current) and +30 mV (outward current), plotted as a function of the extracellular potassium concentration. Increasing extracellular K + from 5.4 mM to 30 mM and to 60 mM produced a concentration-dependent increase in the average amplitude inward I h , which was measured at the end of the voltage pulse to −130 mV (filled circles; n = 22-32; ANOVA, P < 0.0001). The curved line represents a fit of the current density from individual cells to a Michaelis-Menten equation, yielding an apparent dissociation constant of 13.8 ± 7.1 mM. Maximum outward "tail" I h , which was measured 1 ms after the voltage step to +30 mV to minimize the contribution of capacitance current, was not significantly affected by raising the concentration of extracellular potassium (filled squares; P = 0.12). The reversal potentials for I h in different concentrations of extracellular potassium and sodium were −26 ± 5 mV (5.4 mM Na, 135 mM K), −20 ± 4 mV (30 mM K, 110 mM Na), and −5 ± 9 mV (60 mM K, 80 mM Na); extracellular sodium concentrations were reduced proportionally to maintain osmolarity (see Materials and Methods). reversal potential, raising extracellular potassium is known to increase maximum I h conductance in both native tissue 18, 32-37 as well as in cells that express cloned HCN channels 13,15,28,[38][39][40] . We previously examined this unusual potassium-induced increase in conductance of I h in CHO cells expressing the HCN1 isoform by substituting potassium for sodium in the extracellular solution and thereby leaving unchanged the concentration of both the total and permeant cations 28 . We used the same approach here to first characterize the effect of extracellular cations on I h conductance and voltage dependence of I h activation, and then to examine the effects of extracellular cations on inhibition of I h by lidocaine.
Using patch clamp electrophysiology, we investigated inward I h activated by a hyperpolarization to −130 mV and outward I h recorded as the tail current at +30 mV (deactivation) from prior hyperpolarization to −130 mV. Increasing extracellular potassium from 5.4 mM to 60 mM produced a reversible increase in the magnitude of the inward I h , which reached a maximum in ~1 minute (Fig. 2). The dependence of the inward I h on the extracellular potassium concentration was fitted to the Michaelis-Menten equation, yielding an apparent dissociation constant of ~14 mM. Our data are consistent with previous experiments on native and cloned HCNs showing that extracellular potassium has an activating effect on I h , which is separate from the changes in driving force. Outward I h was minimally affected because raising extracellular potassium also depolarizes the reversal potential as shown in the original studies of the hyperpolarization-activated current 18,33 . We have shown previously that raising potassium produces both a depolarizing shift in the reversal potential and an increase in slope conductance of the instantaneous I-V curve of I h measured in CHO cells expressing the HCN1 isoform 28 .
To examine the effect of extracellular potassium on the voltage dependence of I h activation, we measured tail I h amplitude at −65 mV (Fig. 3A) after a series of hyperpolarizing test voltages from a holding potential of −35 mV. Normalized tail current amplitude versus test voltage was plotted and fit with a Boltzmann equation to yield values for half-activation voltage (V 1/2 ) and slope factor (k), as described in the Methods. Raising extracellular potassium did not significantly modify the range of I h activation, although the slope factor was slightly increased (Fig. 3B). Thus, raising extracellular potassium increases the maximal conductance of I h without altering the voltage-dependence of activation; its effect is the opposite of the effect of lidocaine on I h .
We next investigated the effect of extracellular potassium on the I h activation rate, which was estimated by fitting the activation phase of the current with a double-exponential function. Neither the fast nor the slow time constant was affected by raising extracellular potassium (Fig. 3C). The time constant of I h deactivation, which was determined by fitting the deactivating current with a single-exponential function (Fig. 3D), was significantly decreased by raising extracellular potassium.

Elevation of extracellular potassium concentration opposes the effects of lidocaine on HCN1-mediated I h amplitude measured at −130 mV.
We subsequently examined the influence of extracellular potassium on lidocaine inhibition of I h . In order to also assess the time it takes for inhibition by lidocaine, a hyperpolarizing voltage pulse to −130 mV (1 s) to activate I h , followed by a repolarizing pulse to +30 mV to deactivate the current (0.5 s), was applied every 15 seconds. Bath application of 100 µM lidocaine at 5.4 mM extracellular potassium reversibly inhibited inward I h (at −130 mV) by ~48% (Fig. 4A,B). We noted close to complete inhibition of I h by lidocaine after the first sweep of the protocol, within 15 s of application of the drug. The percentage of inward I h (−130 mV) inhibited by 100 µM lidocaine was less at 30 and 60 mM extracellular potassium (Fig. 4C).
Next, we investigated how lidocaine impacts the concentration-response relationship for the effect of extracellular potassium on I h at −130 mV. We plotted current amplitude remaining after exposure to a given concentration of lidocaine as a function of the extracellular potassium concentration (Fig. 5A). Fitting these plots with the Michaelis-Menten equation showed that an increase in the concentration of lidocaine from 100 to 600 µM resulted in an increase in the apparent dissociation constant of potassium from ~5.3 to ~27 mM. We also sought to determine the effects of raising extracellular potassium on the concentration-response relationship for lidocaine inhibition of I h at −130 mV. Fitting plots of percentage of inhibition versus lidocaine concentration with the Hill equation showed that raising the concentration of extracellular potassium reduced the potency of the drug at 30 mM potassium and both the potency and maximum inhibition of the drug at 60 mM potassium (Fig. 5B).
Elevation of extracellular potassium concentration does not modify inhibition of HCN1-mediated I h measured at +30 mV. Bath application of 100 µM lidocaine also reversibly inhibited outward I h (measured as deactivating tail currents at +30 mV) (Fig. 4A). When I h is compared near the beginning of the test voltage (determined at 1 ms, after the capacitive transient), the degree of inhibition is greater at lower levels of potassium (See Fig. 4C). However, inhibition of I h at this voltage was complex and appeared to vary over the time as the current deactivated and the channel closed.
To determine how inhibition of current by lidocaine varied over time at +30 mV, the outward tail I h in the presence of lidocaine was normalized to that measured under control conditions and fitting the obtained kinetics with a double-exponential function (Fig. 6A,B). The extent of inhibition differed over the course of the tail current; inhibition at the beginning of the voltage pulse to +30 mV increased over the course of deactivation for each concentration of extracellular potassium. The initial value for inhibition was larger for the lower concentrations of extracellular potassium, and they reflected the mean values plotted in Fig. 4C. However, the maximum inhibition reached a plateau and was the same at all potassium concentrations (~70-80%). Raising extracellular potassium from 5.4 to 60 mM did not significantly alter either the fast and slow time constants of inhibition in the presence of 600 µM lidocaine.
Lastly, we determined the degree of lidocaine inhibition of outward I h at +30 mV by calculating the area under the current relaxation trace upon deactivation with the drug and normalizing this to the area under the relaxation curve in the absence of drug. We expected the degree of lidocaine inhibition of I h determined in this way to reflect mainly the maximum inhibition reached after ~10 ms (~70-80%). Indeed, plotting the total normalized degree of inhibition of tail current area versus lidocaine concentration and fitting with the Hill plot produced three curves with similar values for maximum inhibition. Fitting also produced Hill coefficients of ~1 and values for IC 50

Discussion
In this study, we demonstrate that lidocaine inhibition of I h in CHO cells that express the HCN1 channel depends upon the extracellular concentration of potassium and voltage. We show that inhibition of I h is increased at less negative potentials whereas the dependence of I h activation over the same range of voltages is not affected. We also show that the inhibition of I h at −130 mV is reduced, and I h conductance are increased, by raising potassium from 5.4 mM to 60 mM, whereas inhibition at +30 mV, and I h conductance, are unaffected by the same changes in potassium. Thus, the data show that lidocaine inhibition of I h and I h conductance are inversely correlated and,  50 of 78 ± 7, 495 ± 133, and 332 ± 151 µM (n = 4-6 cells; ANOVA, P < 0.0001) and estimated Hill coefficients of 1.6 ± 0.2, 0.84 ± 0.12, and 0.92 ± 0.3 at 5.4, 30, and 60 mM extracellular potassium, respectively. Lidocaine's estimated maximum inhibition was also lower at the higher concentrations of extracellular potassium, declining from 80.7 ± 3.5 to 71.1 ± 5.9% and to 26.6 ± 3.4% upon raising the extracellular potassium concentration from 5.4 to 30 mM, and to 60 mM, respectively. together with concentration response curves, suggest that inhibition of I h by lidocaine is antagonized by current flow and that the increase in current is antagonized by the drug. Finally, we show that lidocaine has no effect on the I h activation curve.
Lidocaine, a weak base with a pK a of approximately 7.9, exists in an uncharged basic form and a charged cationic form. As has been proposed for other channels 41 , the charged form may inhibit the HCN1 channel from the extracellular side or the uncharged form may diffuse through the plasma membrane, become re-protonated in the cytoplasm and inhibit the channel from the inside. Alternatively, the uncharged form of the drug may interact with the channel by traversing a lipophilic pathway in the plasma membrane towards a site on the channel which is embedded in the plasma membrane. Studies have shown that the application of QX-314, a quaternary derivative of lidocaine which is protonated and unable to cross the lipid bilayer, has no effect on I h in neurons when applied on the external side whereas it inhibits I h when applied from the intracellular side 9,42,43 . Thus, we currently favor the view that uncharged lidocaine diffuses through the plasma membrane, becomes re-protonated inside the cell and inhibits the HCN1 channel from the inside.
Our data on lidocaine are similar to previous data on the inhibition of I h by other drugs. For example, inhibition of I h by the drugs ZD7288, UL-FS49, DK-AH3, and ivabradine is reduced at more negative voltages and these molecules have only small or negligible effects on the voltage-dependence of I h activation [44][45][46][47][48] . Like the lidocaine analog, QX-314, the aforementioned drugs are thought inhibit I h in their cationic form by acting on a site which is accessible from the inside of the cell 31,44,45,49 . Like lidocaine inhibition of I h in our study, ivabradine inhibition of I h in rabbit sino-atrial node cells and I h in HEK cells heterologously expressing the HCN4 isoform is also increased when current is reduced; in those studies, current was reduced by reducing the proportion of extracellular potassium (as we did in our study) or by adding millimolar levels of extracellular cesium 30,31 . In these studies, it was proposed that the dependence of ivabradine inhibition on current was due to its interaction with permeant ions on the inner aspect of the pore. Thus, together with the intracellular action of QX-314 on I h in other systems, it seems possible that, in our study, the reduced inhibition of I h by lidocaine at larger current amplitudes is due to its greater interaction with permeant ions on the inner side of the HCN1 pore. Further studies will be required to understand the interaction between current amplitude and lidocaine, and why drug potency is reduced mainly in 30 mM potassium whereas both potency and maximum inhibition of I h by the drug are impacted by the drug in 60 mM potassium.
The present findings are important for understanding the myriad of effects of lidocaine in vivo. The greatest inhibition of I h by lidocaine in our study occurred at the lowest concentration of extracellular potassium utilized, 5.4 mM, and the highest concentration of sodium (130 mM); these are close to the usual physiological concentrations for these cations. Our data in Fig. 1 imply that the inhibition of I h by lidocaine increases progressively as the membrane potential becomes less negative suggesting that the effect of this drug might vary with membrane potential in vivo e.g. during an action potential. The concentration of potassium and sodium are also known to vary in vivo. In the heart, the concentration of extracellular potassium may range between 3 and 10 mM, and has been proposed to contribute to bradycardia that is mediated by HCN channels [50][51][52] . In the central nervous system, a range of potassium concentrations between 3 and 12 mM has been measured, while extracellular sodium may decrease by up to 7 mM during repetitive activity [53][54][55] . This variation in cation concentration encountered in vivo could influence the inhibition of I h by lidocaine.
The results from our study and others support the probability that lidocaine exerts some its effects in vivo by acting on the HCN1 isoform. For example, knockout of the HCN1 isoform from somatosensory neurons in the mouse has been shown to reduce cold allodynia and to contribute to action potential generation (Momin et al. 2008). Thus, inhibition of HCN1 by lidocaine likely contributes to its analgesic effects, and it will be important to determine the extent to which lidocaine inhibition of HCN1 contributes to this drug's in vivo actions and how inhibition is impacted by voltage and current. However, lidocaine inhibits three of the four mammalian HCN isoforms (HCN1 as well as HCN2 and HCN4), all of which are found in neurons and cardiomyocytes 11 . While it remains unknown if lidocaine inhibition of HCN2 and HCN4 is as sensitive to current and voltage as HCN1, there are noted differences in the inhibition of HCN1 and HCN4 isoforms by ivabradine, with only the latter being prominently current-dependent 31 . In brain slice whole-cell recordings of rat thalamocortical neurons, we previously found that lidocaine inhibition of I h (100 μM) was not dependent on voltage and was comparatively potent (IC 50 = 72 μM) and near complete; on the basis of the present findings, these differences could be explained by the relatively low extracellular potassium concentrations utilized (2.5 mM) 10 . Differences in lidocaine inhibition of I h in thalamocortical neurons versus I h in HCN1-expressing CHO cells could be due to the different HCN isoforms that are expressed in the former, thought to be mainly HCN2 and HCN4 56-60 , or to other factors such as other differences in recording conditions and cellular context.
In summary, the results of the present study show that lidocaine inhibition of I h in CHO cells expressing the HCN1 isoform is reduced at hyperpolarizing voltages and inversely correlated with increases in I h amplitude. Our data, together with data on lidocaine inhibition of I h in other systems, suggest that lidocaine may diffuse through the plasma membrane, becomes re-protonated inside the cell and inhibits the channel by binding to the inner side of the HCN1 pore, where it interacts with permeant ions. Future studies will be required test this idea and to determine if inhibition occurs in the same way in other members of the HCN channel family and other members of the lidocaine family of drugs.