Capsaicin as an amphipathic modulator of NaV1.5 mechanosensitivity

ABSTRACT SCN5A-encoded NaV1.5 is a voltage-gated Na+ channel that drives the electrical excitability of cardiac myocytes and contributes to slow waves of the human gastrointestinal smooth muscle cells. NaV1.5 is mechanosensitive: mechanical force modulates several facets of NaV1.5’s voltage-gated function, and some NaV1.5 channelopathies are associated with abnormal NaV1.5 mechanosensitivity (MS). A class of membrane-active drugs, known as amphiphiles, therapeutically target NaV1.5’s voltage-gated function and produce off-target effects including alteration of MS. Amphiphiles may provide a novel option for therapeutic modulation of NaV1.5’s mechanosensitive operation. To more selectively target NaV1.5 MS, we searched for a membrane-partitioning amphipathic agent that would inhibit MS with minimal closed-state inhibition of voltage-gated currents. Among the amphiphiles tested, we selected capsaicin for further study. We used two methods to assess the effects of capsaicin on NaV1.5 MS: (1) membrane suction in cell-attached macroscopic patches and (2) fluid shear stress on whole cells. We tested the effect of capsaicin on NaV1.5 MS by examining macro-patch and whole-cell Na+ current parameters with and without force. Capsaicin abolished the pressure- and shear-mediated peak current increase and acceleration; and the mechanosensitive shifts in the voltage-dependence of activation (shear) and inactivation (pressure and shear). Exploring the recovery from inactivation and use-dependent entry into inactivation, we found divergent stimulus-dependent effects that could potentiate or mitigate the effect of capsaicin, suggesting that mechanical stimuli may differentially modulate NaV1.5 MS. We conclude that selective modulation of NaV1.5 MS makes capsaicin a promising candidate for therapeutic interventions targeting MS.


Introduction
The SCN5A-encoded voltage-gated sodium channel, Na V 1.5, is an ion channel gated by the electrical transmembrane potential present in excitable cells [1][2][3][4]. In addition to being voltage-gated, Na V 1.5 is also mechanosensitive: mechanical force modulates Na V 1.5's voltagedependent operation [5,6]. This mechanosensitivity (MS) contributes to a coupled mechanoelectrical feedback mechanism that drives contractile response in the mechanically active tissues of the heart and human gastrointestinal (GI) tract. Na V 1.5 is responsible for the action potential upstroke in cardiac myocytes [3]; and electrical slow waves in human interstitial cells of Cajal (ICC) and intestinal smooth muscle cells (SMC) [7][8][9]. Na V 1.5 channelopathies with abnormal MS are found in human cardiac and GI diseases [10][11][12][13][14][15][16]. Some SCN5A mutations responsible for cardiac arrhythmias result in impaired stretch modulation [14,17]; other mutations associated with altered MS in Na V 1.5 are found in patients with irritable bowel syndrome (IBS) [16,18]. Some IBS-associated SCN5A mutations have relatively unchanged voltage-dependent gating but a loss of MS [17], suggesting that the two mechanisms may be distinct processes that can be targeted separately. However, channelopathies associated with Na V 1.5 MS dysfunction are poorly studied, and pharmacological treatments targeting MS remain unexplored [14,18,19].
Ion channels are prime pharmacological targets because they are involved in many diseases and being embedded in the cells' plasma membrane-are highly accessible [20,21]. In patchclamp studies, mechanical stimuli modulate Na V 1.5's voltage-dependent function by increasing whole-cell conductance, shifting the voltagedependence to hyperpolarized potentials, and accelerating kinetics [5,22]. Na V 1.5 and other mechanosensitive channels detect mechanical stimuli through lipid-bilayer (membrane) tension and cytoskeletal deformation [23][24][25]. A class of membrane-active drugs, known as amphiphiles, have unique MS modulating properties and are frequently used to alter channel function [21,26,27]. The principal mechanism of action with amphiphiles may be associated with a lipid-bilayer modulation mechanism [28].
Interestingly, some amphipathic drugs modulate both Na V 1.5's voltage-dependent function and MS [21,26,29]. Ranolazine is a piperazine derivative and a therapeutic amphipath used in the treatment of chronic angina [30]. Ranolazine inhibits Na V 1.5 late current and MS in primary human GI SMCs, in addition to stretchdependent function in GI smooth muscle [21,26]. These modulations may contribute to therapeutic outcomes [27] and help explain commonly reported side effects [7,30]. Other amphiphiles frequently used to treat cardiac conditions, such as the antiarrhythmic, amiodarone [31]; and the β-blocker, propranolol [32]; also alter GI motility. The effects observed with these and other amphiphiles relate to their ability to modulate the voltage-gated activation and MS of Na V 1.5 and other channels.
Inhibiting Na V 1.5's voltage-dependent opening is usually undesired. Yet, selectively targeting Na V 1.5 MS while sparing voltage-gated activation could have novel therapeutic applications: this would allow mechano-electrical feedback modulation without direct inhibition of electrical activity. Amphiphilic drugs, including some antiarrhythmics, alter the membrane bilayer within the therapeutic range, and their efficacy has been correlated with membrane-modifying capacity [28,33]. Nonspecific membrane modulation may produce desirable effects, including changes in MS seen with some amphiphiles [28]. Therefore, we screened amongst membrane modifying and therapeutic amphiphiles for an amphipathic agent with minimal inhibition of Na V 1.5's voltage activation. Among the candidates, capsaicin shows promise; accordingly, we characterized its ability to modulate Na V 1.5 MS.

Heterologous expression and cell culture
We used the wild-type SCN5A variant, Q1077del Na V 1.5 [1], which makes up 65% of the mRNA transcripts for Na V 1.5 in the heart [1]. SCN5A was co-transfected with pEGFP-C1 into HEK-293 cells using Lipofectamine 3000 (Thermo Fisher Scientific, Massachusetts, USA).

Pipette fabrication
For whole-cell experiments, electrodes were pulled on a P-97 puller (Sutter Instruments, CA) from KG12 glass to a resistance of 2-5 MΩ. For cell-attached patch experiments, electrodes were pulled from 8250 glass (King Precision Glass, California, USA) then firepolished to wide-bore, bullet-shaped tips with a final resistance of 1-2 MΩ. Electrodes were coated with R6101 elastomer (Dow Corning, MI) and then cured with a heat gun to reduce capacitive transients.

Data acquisition
Whole-cell and cell-attached patch data from HEK-293 cells were recorded at 20 kHz with an Axopatch 200B patch-clamp amplifier, Digidata 1550, and pClamp11 software (Molecular Devices, CA).

Cell-attached patch
Solutions. The pipette solution contained (in mM): 149 NaCl, 4.7 KCl, 2.5 CaCl 2 , 10 HEPES, and 5.5 glucose; with an osmolality of 290 mmol/kg. GdCl 3 (10 µM) was included in the pipette solution to inhibit endogenous stretch-activated channels [39]. The bath solution contained (in mM): 139 CsCl, 15 NaCl, 4.7 KCl, 2.5 CaCl 2 , 10 HEPES, and 5.5 glucose; with an osmolality of 305 mmol/kg. Both solutions were made to a pH of 7.35. Where applicable, capsaicin was diluted 1000-fold in bath solution from a 20 mM ethanol stock then added to the recording chamber. Seal pressures were digitally controlled and monitored by High-Speed Pressure Clamp (HSPC-2, ALA Scientific, NY). Suction ≤10 mmHg was applied to establish giga-seals.
Episodic protocol and mechanical stimulation by pressure. Na + currents in macroscopic patches were elicited by an identical pair of voltage ladders with 31-ms pressure steps up to −50 mmHg encompassing the second voltage ladder. Patches were held at +100 mV, stepped briefly for 10 ms to +190 mV to close Na V channels, then stepped through a 10-step voltage ladder from +100 to 0 mV in 21-ms long, 10-mV increments with a total duration of 280 ms per sweep. Recordings were an average of 5 runs. Capsaicin (20 µM) was added to the chamber 5 min before testing the effects of the drug.

Recovery from inactivation.
To test the effect of pressure on the recovery of Na V 1.5 from inactivation, cells were held at 120 mV and stepped to (1) 20 mV for 30 ms, next to (2) 120 mV for a variable duration to recover, then to (2) 20 mV for 30 ms. The time between the beginning of each sweep was 5 s. The duration of the recovery time in stage (2) was varied between 1 and 300 ms in half-log unit increments. The pressure step per sweep was 400 ms regardless of recovery time.

Use-dependent inactivation.
To test the effect of pressure on the onset (use dependence) of Na V 1.5 inactivation, cells were held at 120 mV and stepped 20 times to 20 mV, with the frequency between steps varying between 33.33 and 3.33 Hz. The duration of the pressure step per sweep was 30 ms.

Peak current, voltage dependence of activation, and kinetics of activation and inactivation.
To measure peak Na + current density, cells transfected with Na V 1.5 were held at −120 mV then stepped through a 2-stage, 19-step voltage ladder (1) from −110 to −30 mV in 5 mV intervals for 2.9 s each and (2) to −30 mV for 100 ms. The time from the start of each sweep to the next was 5 s. Peak currents at each voltage step were normalized to the cell capacitance (pF) to quantify current densities (Figure 1 and 2) or to the maximum peak inward current without shear to quantify the change in current over baseline (Figures 3-4).

Recovery from inactivation.
Recovery from inactivation was measured by holding cells at −130 mV and stepping through a 3-stage, 10step protocol to (1) −30 mV for 100 ms, next to (2) −130 mV for a variable duration to recover, then to (2) −30 mV for 100 ms. The time between each sweep start was 2.5 s. The length of the recovery time in stage (2) of sweep n was 4*2 n ms for a total of n = 10 sweeps.

Use-dependent inactivation.
To measure the onset of Na V 1.5 inactivation, cells were held at −130 mV and depolarized 10 times to −40 mV, in which the frequency of steps recorded ranged between 0.3 and 50 Hz. Mechanical stimulation by shear stress. When testing the effect of shear stress, the extracellular (bath) solution was perfused by gravity drip (at 10 mL/min) for the duration of the voltage protocol.

Data analysis
The maximum peak Na + current and voltage dependence of activation were determined by fitting the Na V 1.5 current-voltage (I-V) plots with where G MAX is the maximum Na + conductance in whole cells (I MAX substitutes G MAX for the maximum Na + current in patches), V is the voltage, E REV is the reversal potential, V 1/2A is the voltage of half-maximal activation, and δV is the slope. Conductance measurements were performed for the whole-cell configuration. Activation kinetics were determined by fitting currents with a twoterm weighted exponential function: I(t) = A 1 e (-t/ τA) +A 2 e (-t/τI) +C, where τ A and τ I are the time constants of activation and inactivation, respectively, and A 1 ,A 2 , and C are constants. Steady-state inactivation was obtained by fitting remaining peak Na + currents with a 3-parameter sigmoid curve: I = 1/(1 + e ((V-V1/2I)/δVI) ), where V is the voltage, V 1/2I is the half-point of steady-state inactivation (availability), and δV I the slope. For graphing, each IV curve was normalized to the peak effect without mechanical stimulation to demonstrate the increases in peak current with mechanical stimulation. To calculate recovery from inactivation, peak Na + currents were fit with the equation: I/I 0 = 1/(1 + t/t 1/2 ) b , in which I/I 0 is the ratio of Na + current recovered following inactivation from the control current, b is the rate of inactivation recovery, t is time, and t 1/2 is the midpoint in which half of the Na + current has recovered from inactivation. To calculate the plateau for use-dependent inactivation, peak Na + currents of successive pulses were fit with the 3-parameter exponential decay equation: I 10 /I 1 = I ƒ e b/(t+c) , in which I 10 /I 1 is the peak Na + current of step 10 normalized to the peak of step 1, and I ƒ is the maximally inactivated peak Na + current at frequency ƒ, t is time, and b or c is the rate or constant of use-dependent inhibition, respectively. To determine the voltage-step frequency at which peak Na + currents were inhibited by 50% (or half-frequency of use-dependent inhibition), I ƒ was plotted vs. step frequency ƒ and fit with I ƒ = (1-a)/(1 + e (ƒ1/2-ƒ)/δV ), where a is the limit of use-dependent inhibition, ƒ 1/2 the halffrequency of use-dependent inhibition, ƒ the frequency and δV the slope. Data are expressed as the mean ± standard error of the mean (SEM). Significance was assigned when P < 0.05 by a 2-way ANOVA and Tukey posttest when comparing force to rest or capsaicin to drug-free.

The screen of amphipathic membrane-permeable drugs
As a preliminary screen, we examined select membrane-bilayer-modifying amphipathic agents with high partition coefficients (Table 1) as potential modulators of Na V 1.5 MS [34][35][36][37][38]. We selected amphiphiles with known therapeutic potential and membrane stiffness modifying properties as previously examined with a gA channel assay [31,38,[40][41][42]. Each compound was tested (10 −9 to 10 −4 M) for its ability to inhibit peak voltage-gated Na + currents ( Figure 1(a,d)). Triton X-100 was the most potent (logP OW 4.6, IC 50 5.3 µM, slope 1.00; Figure 1(c,d), Table 1) and capsaicin the least potent (logP OW 3.04, IC 50 60.2 µM, slope 0.64; Figure 1(c,d), Table 1). The antiarrhythmic amiodarone (logP OW Table 1. Partition coefficients and IC 50 values for amphipathic agents. Partition coefficients denoted logP OW for amiodarone, capsaicin, propranolol, and Triton-X100 were previously reported [34][35][36][37]. IC 50 , concentration at which an amphipathic agent inhibited half of the maximum peak whole cell Na + current from HEK293 cells transfected with Na V 1.5. Slope, the hill slope to mechanistically characterize drug behavior based on the slope of fit.  Figure 1(c,d)) and βblocker propranolol (logP OW 3.48, IC 50 7.6 µM, slope 1.01; Figure 1(c,d)) also inhibited Na V 1.5. Propranolol, which had a partition coefficient (logP OW ) similar to capsaicin, was an 8-fold more potent Na V 1.5 inhibitor than the latter, indicating that logP OW is not a good predictor of the drug's effect on Na V 1.5 voltage-gated function, consistent with the previous literature [43]. Similarly, we did not observe a discernable trend in the Hill slope for inhibitionamiodarone and capsaicin had the lowest Hill slopes (0.50 and 0.64, respectively). Nevertheless, amiodarone's potency (IC 50 8.4 µM) was comparable to the two most potent current inhibitors: Triton X-100 and propranolol (IC 50 5.3 and 7.6 µM, respectively). Overall, capsaicin, compared against the other amphiphiles tested, was an order of magnitude less potent for current inhibition (IC 50 60.2 µM for capsaicin vs. IC 50 5.3 to 8.4 µM for the other amphiphiles tested; Figure 1(c,d), Table 1). Because our goal was to find a candidate that would selectively modulate MS while minimizing Na V 1.5 voltage-dependent current inhibition, we chose capsaicin (20 µM) for further investigation, as this dose inhibited voltagedependent Na + current by ≤25% (Figure 1(b,d)).

Capsaicin inhibits mechanically induced hyperpolarizing shifts in the voltage dependence of activation and channel availability
Pressure [6,21,26,48], and shear [17,18,25] produce hyperpolarizing shifts in the voltage dependence of Na V 1.5 activation and inactivation. Membrane-permeable amphipathic drugs like lidocaine and ranolazine reduce these mechanosensitive shifts in voltage dependence [21,26]. Therefore, we explored whether capsaicin could reduce the pressure-or shear-induced shifts in voltage dependence. Comparable to our previous work without drug [21,26] in the absence of capsaicin (−6.0 ± 0.9 mV with pressure or −2.5 ± 0.9 mV with shear; P < 0.05 to no force), though the technical limitations of holding the voltage to more negative potentials prevented us from reaching the plateau in wholecell experiments (Tables 2-3, Figure 2(d-h)). Without force, capsaicin produced a hyperpolarizing shift in the whole-cell V 1/2I by −5.1 ± 0.7 mV, as previously observed [42].
In the presence of capsaicin, neither pressure nor shear significantly affected the V 1/2I (−1.4 ± 1.2 or −0.5 ± 0.6 mV change, respectively; P > 0.05 to drug with no force), suggesting loss of the MS of Na V 1.5 inactivation.
Overall, our results show that capsaicin inhibited the mechanosensitive shifts in Na V 1.5 voltage-gating.

Effects of capsaicin and mechanical stimuli on recovery from inactivation
Both capsaicin and pressure delay the recovery of Na V 1.5 from fast inactivation [42,48]. Therefore, we tested whether the presence of capsaicin affected the recovery from fast inactivation (1 to 1000 ms) in the absence or presence of mechanical stimuli (Tables 2-3, Figure 3(a,b)). Without force or drug, Na + currents recovered within ~100 ms in either configuration (Figure 3(c)); the half-time of Na V 1.5 inactivation recovery (t 1/2R ) at rest was 13.2 ± 2.5 ms in the patch and 18.8 ± 1.7 ms in whole-cell (Tables 2-3, Figure 3(c-f)). In addition, unlike the consistent responses to force regardless of stimulus or configuration described above, we observed consistent differences between the two approaches when assessing recovery. Shear accelerated Na V 1.5's t 1/2R by 2.2 ± 0.6 ms (P < 0.05, 0 to 10 mL/min), whereas pressure delayed the t 1/2R (+8.9 ± 3.9 ms; P < 0.05, 0 to −30 mmHg) (Tables 2-3, Figure 3(c-f)). In whole cells, without force, capsaicin delayed the recovery from inactivation; the t 1/2R increased from 18.8 ± 1.7 to 38.0 ± 4.5 ms (P < 0.05, 0 to 20 µM capsaicin). With capsaicin present, pressure increased the t 1/2R by 19.2 ± 8.2 ms (P < 0.05 to drug with no pressure), whereas shear reduced the t 1/2R in whole cells by 9.5 ± 0.9 ms (P < 0.05 to drug with no shear). In all, the recovery from inactivation was delayed by capsaicin across both approaches. In the presence of capsaicin, pressure further delayed recovery in pressurized patches, but shear accelerated recovery in whole cells.

Effects of capsaicin and mechanical stimuli on use-dependent inactivation
Capsaicin can stabilize the inactivated state of Na V 1.5 through use-dependent inhibition [42]. Therefore, we tested whether force could alter the use-dependent inactivation of Na V 1.5 in the absence of capsaicin and reexamined usedependent inactivation in the presence of capsaicin (Figure 4(a-f)). To measure the use-dependent Table 2. Effect of capsaicin on pressure-induced Na   Table 3. Effect of capsaicin on shear-induced Na   Figure 4(c-f)) and 64.6 ± 2.4% with a ƒ 1/2 of 26.1 ± 2.0 Hz in whole cells (Table 3, Figure 4(d-f)). In the absence of capsaicin, the use-dependence did not change with either pressure or shear (P > 0.05 to no force; Tables 2-3, Figure 4(c-f)). Without shear force, capsaicin increased the maximum use-dependent inhibition of Na V 1.5 to 89.0 ± 1.4% and decreased ƒ 1/2 to 18.9 ± 1.0 Hz (P < 0.05, 0 to 20 µM capsaicin) in the whole-cell configuration. In the presence of capsaicin, shear produced a modest decrease in the maximum use-dependent inhibition (5.1 ± 0.9%; P < 0.05 to drug with no shear), and ƒ 1/2 was unaffected, suggesting that shear partially reverses the use-dependent inhibition of Na V 1.5 promoted by capsaicin. In patches, capsaicin affected neither the use-dependent inhibition nor ƒ 1/2 at rest (P > 0.05, 0 to 20 µM capsaicin) but increased the pressure-sensitivity (ƒ 1/2 decreased by 2.4 ± 3.3 Hz; P < 0.05 to drug with no pressure). Together, our results suggest that, though capsaicin enhances use-dependent inhibition, its effect on force-dependent changes to Na V 1.5 use dependence may be specific to the type of force applied.

Discussion
The MS of voltage-gated ion channels contributes to a mechano-electrical feedback system that has important implications in organs with primarily mechanical functions [14,48,49]. Examples of such mechanically active organs include the heart and gut, where ion channels contribute to important physiologic functions [14,50]. In the human heart and gut, the voltagegated sodium channel, Na V 1.5 serves as a key electrically excitable component in both the cardiac action potential and GI smooth muscle contraction [17,50]. Besides being voltage-gated, Na V 1.5 is mechanosensitive [37]; this property contributes to mechanoelectrical feedback [49]. Membrane-permeable amphiphiles impact Na V 1.5 MS [7,21,51] and may be capable of modulating this feedback mechanism to exert therapeutic effects [21]. How membrane-permeable amphipathic drugs alter the MS of voltage-gated channels, like Na V 1.5, and whether they do so by a mechanism separate from voltage-dependent current inhibition remain critical points for understanding amphiphilemediated effects [21,26,29]. For example, the membrane-permeable amphiphile and local anestheticlidocaine-inhibits Na V 1.5's peak current, and at lower concentrations, inhibits MS [21,26]. Supporting a separate-mechanisms hypothesis for current inhibition and altered mechanosensation by amphiphiles, the anesthetic-binding site mutation, F1760A, eliminates the voltage-dependent inhibition by lidocaine without altering lidocaine's effect on MS [21]; while the membrane-impermeant lidocaine analog, QX-314, does not affect MS.
Amphiphiles often have significant effects on both voltage-gating and MS [21]. Thus, we searched for a membrane-permeable amphipathic agent that alters Na V 1.5 MS without significant inhibition of the voltage-gated channel opening. In principle, this would allow selective targeting Na V 1.5 MS in conditions where it is abnormal. We selected amphiphiles with high partition coefficients and tested each candidate's inhibition of peak voltage-gated currents in Na V 1.5. Our choices were motivated by these molecules' therapeutic use and ability to alter membrane stiffness, as demonstrated with the gA channel assay [31,38,[40][41][42]. Interestingly, the partition coefficients for these compounds do not predict the drug's effects on Na V 1.5's voltage-gated function. Our results, interpreted together with earlier studies with membrane-impermeable quaternary ammonium amphiphilic local anesthetics [52,53], suggest that amphiphile-mediated effects require membrane partitioning as a prerequisite for peak current inhibition. However, the level of partitioning estimated using logP ow does not determine the level of Na V 1.5 current inhibition.
The class of therapeutic amphiphiles, local anesthetics [52] have been shown to interact with the channel pore to exert current inhibition through intracellular access. Local anesthetics exert open-state block through the channel pore via a local anesthetic-binding site that is accessible during opening events. However, local anesthetics exert poor closed-state block [52,54], which may involve a different mechanism that is shared with other amphiphiles. The Hill slopes, ranging from 0.5 to 1 for the chosen amphiphiles, did not predict the closed-state current inhibition level, suggesting that amphiphile-mediated peak current inhibition does not have a precise binding modality [55]. This may indicate that mechanisms of current inhibition by amphiphiles depend on the identity of the amphiphile used. While propranolol (slope of 1.0) and Triton X-100 (slope of 1.0) could be interacting with a binding site, amiodarone (slope 0.5) and capsaicin (slope 0.6) appear to be acting by another nonspecific mechanism. This finding is supported by earlier work with the gA channel assay and KcsA K + channel. Using the KcsA channel as a model for the voltage-gated ion channel [56], amphiphiles were generally found to alter KcsA function, but amphiphiles altered different gating steps in an identity-specific manner.
Both amiodarone and propranolol are antiarrhythmics, yet amiodarone exerts membraneperturbing effects within the therapeutic range of use while propranolol-induced perturbations occur above therapeutic concentrations [28]. This may underlie amiodarone's therapeutic and offtarget effects, including changes in MS. In the case of thiazolidinediones, a class of insulinsensitizing drugs, greater lipid-bilayer altering potency is strongly correlated with efficacy and side effects [33]. This implies that some pharmacological agents alter the membrane to produce desired therapeutic outcomes. Similar to these drugs, capsaicin alters the membrane bilayer at concentrations required to exert effects [42]. These nonspecific membrane-modulatory behaviors may describe one mechanism by which some amphiphiles function therapeutically and alter MS. From our selection however, choosing among amphiphiles with bilayer-modulating effects and minimal closed-state current inhibition as selection criteria may indicate therapeutic potential. Capsaicin inhibited Na V 1.5 MS in a manner comparable to other amphipaths-such as lidocaine [21,26], and ranolazine [14]-yet with minimal inhibitory effects. Capsaicin consistently inhibited the MS effects of pressure and shear stress on Na V 1.5 in membrane patches and whole cells, respectively, by inhibiting the: (1) mechanosensitive increases in Na + current, (2) shifts in steady-state voltage-dependence, and (3) acceleration of Na V 1.5 gating kinetics.
Quantifying ion channel MS is challenging [57], and different approaches often yield different results [23,44]. Few studies have explored Na V 1.5 MS using both whole-cell and macroscopic patch modes in parallel [17,21,48]. To our knowledge, this is the first study to directly compare pressure and shear effects on the biophysics of Na V 1.5 MS in the absence or presence of a drug in detail. As with other amphiphiles studied [21,58], capsaicin hyperpolarized parameters of voltage-dependence. In the whole-cell mode, capsaicin has previously demonstrated the ability to shift the V 1/2A and V 1/2I to hyperpolarized potentials [42]. Comparably, we observed significant shifts in both the patch and whole-cell configuration. Impressively, despite the stimuli being two distinct modes of mechanical stimulation, with unique mechanisms, most of Na V 1.5's mechanosensitive responses and capsaicin's effects on Na V 1.5 MS were similar across techniques. Both produced an increase in peak Na + current, shifts in the V 1/2A and V 1/2I , and an acceleration in τ A . Capsaicin in both approaches inhibited most of these changes. These effects are important in the context of MS channel functionmechanical strain leads to faster and greater Na + influx, which increases Na V channel availability and further depolarizes the membrane. When Na V 1.5 is mechanically stimulated in the presence of capsaicin, there would be a reduction in Na + influx and consequently a slower membrane depolarization, which would reduce the effect of mechanical force on Na V channel availability.
Many membrane modulating amphiphilic drugs alter recovery from inactivation and exert usedependent block on Na V channels [59][60][61]: these responses are modulated by capsaicin among other amphiphiles [42]. Changes in channel properties due to mechanical stimuli can be considered mechanosensitive processes [23]. Therefore, it was important to understand how capsaicin could modulate these effects in addition to the commonly studied mechanosensitive parameters of activation and inactivation gating. We found opposite responses in the pressure-and shearsensitivity of Na V 1.5 inactivation recovery and use-dependent inactivation. Pressure applied to patches increased the time to recover from inactivation, whereas shear stress applied in the wholecell configuration decreased it. As previously reported [42], the addition of capsaicin delayed Na V 1.5's recovery from inactivation in patches and whole cells. Intriguingly, the opposing effects on recovery with mechanical stimuli were further amplified with capsaicin. Capsaicin and pressure cooperatively delayed recovery further, in contrast to whole cells, where shear stress accelerated inactivation recovery, and thus reduced capsaicin mediated recovery delays.
At the concentration we tested, capsaicin alone did not significantly alter the half-frequency for use-dependent inactivation in patches, but it did in whole cells. When capsaicin and pressure were applied together, they decreased the frequency of use-dependent inactivation in patches; while in whole cells, capsaicin alone lowered usedependence frequency, a process unaffected by shear stress. Pressure has previously been shown to prolong Na V 1.5 inactivation recovery time in patches [48], but the effect of shear stress on inactivation recovery or use-dependent inhibition of Na V 1.5 was previously unknown.
The opposing responses in use-dependence and recovery using the two approaches are independent of capsaicin, suggesting that these mechanical stimuli may act through different mechanisms [45,62]. Conceivably, the effect of pressure or shear stress on the membrane or cytoskeleton could be different. Shear can lead to uniaxial elastic tension along the membrane, yielding asymmetrical sliding of lipid membrane leaflets [29,30]. The associated effects of lipid bilayer thinning affect some functional states, such as inactivation, more than others [40,41]. Meanwhile, macroscopic patch suction can create unequal transmembrane surface tension [23,45,63], with most tension at the dome peak. Accordingly, from the perspective of the membrane, two different phenomena may be occurring with each respective stimulus that may translate to divergent effects on Na V 1.5 MS. Negative pressures in the patch may alter the properties of voltage-gating through a lipidstretch mechanism, whereas shear stress across the membrane may alter gating though a mechanism involving an asymmetric tension across on the entire cell that is attached to the electrode [23,62,64].
Lipid compositions surrounding channels play critical roles in force transduction and gating mechanisms. Changing the lipid composition of synthetic bilayers alters mechano-gated MscL channel gating parameters-adding lysophosphatidylcholine to vesicles composed of phosphatidylcholine was sufficient to measurably open MscL channels [65]. In another study, charged amphiphiles of a like charge were capable of activating the MscL channel, and effects could be neutralized with the addition of amphiphiles of an opposing charge [66]. Furthermore, the gA channel reporter suggests that membrane elasticity may be essential in the Na V channel gating mechanism [42]. Two gA channel subunits, which function as molecular force transducers, join to form an open pore when the membrane elastic disjoining force is overcome [40]. Amphiphiles like capsaicin and capsazepine increased gA channel appearance rate by lowering the elastic disjoining force energy barrier for gA dimerization [40,42]. These findings suggest the presence of an important membranechannel force transduction mechanism and that the application of therapeutic amphiphiles may alter/modulate channel behavior by this mechanism. The decrease in membrane stiffness by amphiphiles, such as capsaicin, may be responsible for the loss of mechanosensitive effects observed in our study. Overall, the membrane bilayer likely plays a critical role in channel MS.
Mechanical stimuli modulate components of cardiac and intestinal contractility [24]. In the setting of mechanically active organs, such as the heart and GI, the mechanoelectric feedback loop has important system level regulatory functions [49,67]. This feedback loop could serve as point of therapeutic regulation in the treatment diseases with MS dysfunction [26]. Amphiphiles that blunt mechanosensitive effects, like capsaicin, may reduce MS when mechanoelectric feedback is disrupted in cases of cardiac and gastrointestinal disease [24,68]. Pharmacologic modulation via capsaicin could conceivably reduce channel activity through a combination of use-dependent block and recovery delay, while sparing voltage-gated operation.
Capsaicin joins a growing group of amphipaths that modulate Na V 1.5 voltage-gating and MS with therapeutic potential. The Na V 1.5 targeting amphiphilic drug, ranolazine is a common antiischemic and anginal medication. Ranolazine inhibits the increase in peak Na + current and the hyperpolarization of voltage-dependence of activation induced by pressure or shear stress in a manner comparable to capsaicin [26]. Abnormalities in gut transit are common side effects of ranolazine [30]. This could be explained by ranolazine's ability to inhibit both muscle contractility in human colon smooth muscle cells (SMCs), and Na V 1.5 peak current and MS [7]. Capsaicin activates its canonical target, TRPV1, in sensory neurons to improve GI dysfunction in IBS-D patients [69]; however, TRPV1 is not pressure-sensitive [70] and is minimally expressed in HEK cells [63,71]. Capsaicin has shown promise in targeting IBS pain [69]. Fascinatingly, it also affects gut motility [72][73][74], possibly through its effects on Na V 1.5 MS, since TRPV1 is only expressed in extrinsic sensory fibers, which are not primary regulators of motility [75]. Building on this study, there may be a possibility of using capsaicin to affect sensory (TRPV1) and motility (SCN5A/Na V 1.5) processes by different mechanisms in the GI tract. Amphipathic drugs are widely used in clinical practice to target ion channels but are rarely used for mechano-modulation [12,13]. The ability of capsaicin, lidocaine [21], and ranolazine [26] to inhibit Na V 1.5 MS demonstrates that membrane-partitioning amphipathic agents can effectively alter MS and may have pharmacologic potential. Such amphiphiles may be viable candidates for therapeutic modulation of Na V 1.5 MS and for targeting dysfunction in channelopathies with disordered MS. Channelopathies involving mechanosensitive dysfunction are an emerging area of study [12,16,17,19,76,77]. Voltage-sensitive mechanogated Piezo channels [78][79][80] and Na V 1.5 MS channelopathies currently lack targeted treatment options. While continued progress is required, this study suggests that therapeutically targeting the voltage-gated and mechanosensitive functions of Na V 1.5 separately in human diseases may hold promise for MS-associated disorders.