Voltage-activated ionic channels and conductances in embryonic chick osteoblast cultures

Summary Patch-clamp measurements were made on osteoblast-like cells isolated from embryonic chick calvaria. Cell-attachedpatch measurements revealed two types of high conductance (100–250 pS) channels, which rapidly activated upon 50–100 mV depolarization. One type showed sustained and the other transient activation over a 10-sec period of depolarization. The single-channel conductances of these channel types were about 100 or 250 pS, depending on whether the pipettes were filled with a low K⁺ (3mm) or high K⁺ (143mm) saline, respectively. The different reversal potentials under these conditions were consistent with at least K⁺ conduction. Whole-cell measurements revealed the existence of two types of outward rectifying conductances. The first type conducts K⁺ ions and activates within 20–200 msec (depending on the stimulus) upon depolarizing voltage steps from <–60 mV to >–30 mV. It inactivates almost completely with a time constant of 2–3 sec. Recovery from inactivation is biphasic with an initial rapid phase (1–2 sec) followed by a slow phase (>20 sec). The second whole-cell conductance activates at positive membrane potentials of >+50 mV. It also rapidly turns on upon depolarizing voltage steps. Activation may partly disappear at the higher voltages. Its single channels of 140 pS conductance were identified in the whole cell and did conduct K⁺ ions but were not highly Cl^– or Na⁺ selective. The results show that osteoblasts may express various types of voltage controlled ionic channels. We predict a role for such channels in mineral metabolism of bone tissue and its control by osteoblasts.     

Anion channel forming activity from the plant pathogenic bacteriumClavibacter michiganense ssp. nebraskense

Summary The plant pathogenic bacteriumClavibacter michiganense ssp. nebraskense secretes an anion channel forming activity (CFA) into the culture fluid. The CFA inserts spontaneously into planar lipid membranes when culture fluid of this species is added to the aqueous phase of the bilayer chamber. The channels formed are highly anion selective. The conductance decreases for larger anions (Cl^–>SCN^–>SO ₄ ^2– ) and is practically zero for gluconate. The channels show a unique voltage dependence : (i) The single-channel conductance increases linearly with voltage up to 200 mV saturating at 250 mV with 25±1 pS (300mm KCl). The channel is closed at negative voltage relative to the side of insertion (diode-typeI–V curve). (ii) The average number of open channels also increases with voltage. The Poisson distribution of channel numbers indicates independent opening of the channels.Channel activity can be abolished by protease treatment of the planar bilayer. The channels can be blocked by indanyloxyacetic acid (IAA-94) and by pH>10. The CFA was purified yielding one major band on the SDS gel with a relative molecular mass of 65,000. The putative involvement of the CFA in the toxicity of this plant pathogen is discussed and compared to other toxins like colicins and to the diphtheria toxin group.     

Ca2+‐dependent activation of guard cell anion channels,triggered by hyperpolarization,is promoted by prolonged depolarization

Rapid stomatal closure is driven by the activation of S‐type anion channels in the plasma membrane of guard cells. This response has been linked to Ca²⁺ signalling, but the impact of transient Ca²⁺ signals on S‐type anion channel activity remains unknown. In this study, transient elevation of the cytosolic Ca²⁺ level was provoked by voltage steps in guard cells of intact Nicotiana tabacum plants. Changes in the activity of S‐type anion channels were monitored using intracellular triple‐barrelled micro‐electrodes. In cells kept at a holding potential of ?100 mV, voltage steps to ?180 mV triggered elevation of the cytosolic free Ca²⁺ concentration. The increase in the cytosolic Ca²⁺ level was accompanied by activation of S‐type anion channels. Guard cell anion channels were activated by Ca²⁺ with a half maximum concentration of 515 nm (SE = 235) and a mean saturation value of ?349 pA (SE = 107) at ?100 mV. Ca²⁺ signals could also be evoked by prolonged (100 sec) depolarization of the plasma membrane to 0 mV. Upon returning to ?100 mV, a transient increase in the cytosolic Ca²⁺ level was observed, activating S‐type channels without measurable delay. These data show that cytosolic Ca²⁺ elevation can activate S‐type anion channels in intact guard cells through a fast signalling pathway. Furthermore, prolonged depolarization to 0 mV alters the activity of Ca²⁺ transport proteins, resulting in an overshoot of the cytosolic Ca²⁺ level after returning the membrane potential to ?100 mV.     

Single channel measurements demonstrate the voltage dependence of permeation through N-type and L-type CaV channels

The delivery of Ca²⁺ into cells by Ca_V channels provides the trigger for many cellular actions, such as cardiac muscle contraction and neurotransmitter release. Thus, a full understanding of Ca²⁺ permeation through these channels is critical. Using whole-cell voltage-clamp recordings, we recently demonstrated that voltage modulates the apparent affinity of N-type (Ca_V2.2) channels for permeating Ca²⁺ and Ba²⁺ ions. While we took many steps to ensure the high fidelity of our recordings, problems can occur when Ca_V currents become large and fast, or when currents run down. Thus, we use here single channel recordings to further test the hypothesis that permeating ions interact with N-type channels in a voltage-dependent manner. We also examined L-type (Ca_V1.2) channels to determine if these channels also exhibit voltage-dependent permeation. Like our whole-cell data, we find that voltage modulates N-channel affinity for Ba²⁺ at voltages > 0 mV, but has little or no effect at voltages < 0 mV. Furthermore, we demonstrate that permeation through L-channel is also modulated by voltage. Thus, voltage-dependence may be a common feature of divalent cation permeation through Ca_V1 and Ca_V2 channels (i.e. high-voltage activated Ca_V channels). The voltage dependence of Ca_V1 channel permeation is likely a mechanism mediating sustained Ca²⁺ influx during the plateau phase of the cardiac action potential.     

Effects of low concentrations of tetradotoxin on rat trigeminal ganglion neurons

Voltage clamping and intracellular perfusion methods were used to investigate ionic currents produced by depolarizing shifts of –120 mV from holding potential during experiments on neurons isolated from the trigeminial ganglion of one-month-old rats. It was found that tetradotoxin at low (external) concentrations of 10^–12–10¹⁰ M produced an increase in the amplitude and alternations in the kinetics of inward ionic currents. Similar effects were observed in 8 test cells out of 29. The extent to which the increase in the amplitude of inward ionic currents depended on concentration level could be described by Langmuir's isotherm, with a dissociation constant of the order of 5·10^–12 M. No such tetrodotoxin effects were observed when chloride ions were replaced by a non-penetrating anion in the intracellular solution. It is suggested that tetrodotoxin-sensitive channels exist in the neuronal membrane of the rat trigeminal ganglion, letting through chloride ions during depolarization of the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 6, pp. 723–729, November–December, 1986.     

Excitatory action of γ-aminobutyric acid (GABA) on crustacean neurosecretory cells

Summary 1. Intracellular and voltage-clamp recordings were obtained from a selected population of neuroscretory (ns) cells in the X organ of the crayfish isolated eyestalk. Pulses of -aminobutyric acid (GABA) elicited depolarizing responses and bursts of action potentials in a dose-dependent manner. These effects were blocked by picrotoxin (50 µM) but not by bicuculline. Picrotoxin also suppressed spontaneous synaptic activity.2. The responses to GABA were abolished by severing the neurite of X organ cells, at about 150 µm from the cell body. Responses were larger when the application was made at the neuropil level.3. Topical application of Cd²⁺ (2 mM), while suppressing synaptic activity, was incapable of affecting the responses to GABA.4. Under whole-cell voltage-clamp, GABA elicited an inward current with a reversal potential dependent on the chloride equilibrium potential. The GABA effect was accompanied by an input resistance reduction up to 33% at a –50 mV holding potential. No effect of GABA was detected on potassium, calcium, and sodium currents present in X organ cells.5. The effect of GABA on steady-state currents was dependent on the intracellular calcium concentration. At 10^–6 M [Ca²⁺]_i, GABA (50 µM) increased the membrane conductance more than threefold and shifted the zero-current potential from–25 to–10 mV. At 10^–9 M [Ca²⁺]_i, GABA induced only a 1.3-fold increase in membrane conductance, without shifting the zero-current potential.6. These results support the notion that in the population of X organ cells sampled in this study, GABA acts as an excitatory neurotransmitter, opening chloride channels.     

Role of voltage-dependent ionic currents in coupling glucose stimulation to insulin secretion in canine pancreatic islet B-Cells

Summary Glucose-induced electrical activity in canine pancreatic islet B cells is distinct from that in rodent islets, though both display Ca²⁺-dependent insulin secretion. Rodent islet B cells undergo regular bursts of Ca²⁺-dependent action potentials, while canine islet B cells generate isolated Na⁺-dependent action potentials which often give way to a plateau depolarization. Here we present evidence to reconcile the species difference in electrical activity with the similarity of Ca²⁺ dependence of secretion. (i) In canine B cells increasing glucose concentrations produce membrane depolarization and increasing frequency of Na_o-dependent action potentials until a background membrane potential (-40mV) is reached where Na⁺ currents are inactivated. (ii) Voltage-dependent Ca²⁺ currents are present which are activated over the voltage excursion of the action potential (–50 to +20 mV) and inactivate slowly, (over seconds) in the range of the plateau depolarization (–40 to –25 mV). Hence, they are available to contribute to both phases of depolarization. (iii) Tetrodotoxin (TTX) reduces by half an early transient phase of glucosestimulated insulin secretion but not a subsequent prolonged plateau phase. The transient phase of secretion often corresponds well in time to the period of initial high frequency action potential activity. These latter results suggest that in canine B cells voltagedependent Na⁺ and Ca²⁺ currents mediate biphasic glucose-induced insulin secretion. The early train of Na⁺-dependent action potentials, by transiently activating Ca²⁺ channels and allowing pulsatile Ca²⁺ entry, may promote an early transient phase of insulin secretion. The subsequent sustained plateau depolarization, by allowing sustained Ca²⁺ entry, may permit steady insulin release.     

Expression and regulation of chloride channels in neonatal rat cardiomyocytes

Using an ¹²⁵I^– efflux assay, we have studied the expression of various types of chloride channels in isolated neonatal rat cardiomyocytes. Three different classes of anion conductances were distinguished: (1) a Cal²⁺-sensitive Cl^– conductance, triggered upon stimulation of the cells with endothelin-1 or Ca²⁺-ionophore; (2) a CAMP/protein kinase A-operated Cl^– conductance, activated by addition of forskolin. This anion channel could be identified as the Cystic Fibrosis Transmembrane conductance Regulator (CFTR-CI^– channel) by Western blotting as well as by its enhanced activity in cultures pretreated with the tyrosine kinase inhibitor genistein; (3) a distinct class of cell volume-regulated Cl^– channels, potentiated in the presence of endothelin-1 or the phosphotyrosine phosphatase inhibitor pervanadate. The potential role of each class of Cl^– channels in the generation and/or modulation of action potentials as well as in maintaining cell volume is discussed.     

Ion channels activated by swelling of Madin Darby Canine Kidney (MDCK) cells

Summary According to previous studies hyposmotic swelling of Madin Darby Canine Kidney (MDCK) cells leads to a marked decrease of cell membrane resistance. The present study has been performed to identify the underlying ion channels using the patchclamp technique: reduction of extracellular osmolarity to 230 mmol/liter leads to a transient activation of K⁺ channels and a sustained activation of anion channels. The K⁺ channels are inwardly rectifying with a single-channel slope conductance of 56 ± 3 pS at –50 mV (cell negative) and of 29 ± 2 pS at 0 mV PD across the patch 150 mmol/liter K⁺ in pipette). The same channels are activated by an increase of intracellular calcium activity, as shown previously. The anion channels display a single-channel slope conductance of 41 ± 4 pS at –50 mV (cell negative) and of 25 ± 3 pS at 0 mV PD across the patch (150 mmol/liter Cl^– in pipette). The channel is anion selective and conducts both bicarbonate and chloride with a preference for bicarbonate. Its open probability is not affected by changing intracellular calcium from 0.1–10 mol/liter. The channels observed explain the effects of cell swelling on PD, ion selectivity and resistance of the cell membrane in MDCK cells.The authors gratefully acknowledge the valuable discussion with Drs. P. Deetjen, E. Wöll and F. Friedrich, the skilled technical assistance of G. Siber and S. David, and the excellent mechanic and electronic support by K.-H. Streicher, Ing. M. Hirsch and M. Plank. This study was supported by the Fonds zur Förderung der wissenschaftlichen Forschung, Grant No. P5813 and P6792M.     

Biphasic increase of apical Cl− conductance by muscarinic stimulation of ht-29cl.19a human colon carcinoma cell line: Evidence for activation of different cl− conductances by carbachol and forskolin

Summary The modulation of ion transport pathways in filtergrown monolayers of the Cl^–-secreting subclone (19A) of the human colon carcinoma cell line HT-29 by muscarinic stimulation was studied by combined Ussing chamber and microimpalement experiments.Basolateral addition of 10^–4 m carbachol induced a complex poly-phasic change of the cell potential consisting of (i) a fast and short (30-sec) depolarization of 15±1 mV from a resting value of –52±1 mV and an increase of the fractional resistance of the apical membrane (first phase), (ii) a repolarization of 22±1 mV leading to a hyperpolarization of the cell (second phase), (iii) a depolarization of 11±1 mV and a decrease of the fractional resistance of the apical membrane (the third phase), (iv) and sometimes, a hyperpolarization of 6±1 mV and an increase of the fractional resistance of the apical membrane (fourth phase). The transepithelial potential increased with a peak value of 2.4±0.3 mV (basolateral side positive). The transepithelial PD started to increase (serosa positive), coinciding with the start of the second phase of the intracellular potential change, and continued to increase during the third phase. Ion replacements and electrical circuit analyses indicate that the first phase is caused by increase of the Cl^– conductance in the apical and basolateral membrane, the second phase by increased K⁺ conductance of the basolateral membrane, and the third phase and the fourth phase by increase and decrease, respectively, of an apical Cl^– conductance. The first and second phase of the carbachol effect could be elicited also by ionomycin. They were strongly reduced by EGTA. Phorbol dibutyrate (PDB) induced a sustained depolarization of the cell and a decrease of the apical fractional resistance. The results suggest that two different types of Cl^– channels are involved in the carbachol response: one Ca²⁺ dependent and a second which may be PKC sensitive.In the presence of a supramaximal concentration of forskolin, carbachol evoked a further increase of the apical Cl^– conductance.It is concluded that the short-lasting carbachol/Ca²⁺-dependent Cl^– conductance is different from the forskolin-activated conductance. The increase of the Cl^– conductance in the presence of forskolin by carbachol may be due to activation of different Cl^– channels or to modulation of the PKA-activated Cl^– channels by activated PKC.The authors are grateful to Drs. Laboisse and Augeron for providing the cell clone, and we thank Prof. Dr. F.H. Lopes da Silva for his comments. This work was supported by a grant from the Dutch Organization for Scientific Research, NWO.     

Voltage-gated calcium and Ca2+-activated chloride channels and Ca2+ transients: voltage-clamp studies of perfused and intact cells of Chara

The voltage-clamp technique was used to study Ca²⁺ and Cl^– transient currents in the plasmalemma of tonoplast-free and intact Chara corallina cells. In tonoplast-free cells [perfused medium with ethylene glycol bis(2-aminoethyl ether)tetraacetic acid] long-term inward and outward currents through Ca channels consisted of two components: with and without time-dependent inactivation. The voltage dependence of the Ca channel activation ratio was found to be sigmoid-shaped, with about –140-mV activation threshold, reaching a plateau at V>50 mV. As the voltage increased, the characteristic activation time decreased from approximately 10³ ms in the threshold region to approximately 10 ms in the positive region. The positive pulse-activated channels can then be completely deactivated, which is recorded by the Ca²⁺ tail currents, at below-threshold negative voltages with millisecond-range time constants. This tail current is used for fast and brief Ca²⁺ injection into tonoplast-free and intact cells, to activate the chloride channels by Ca²⁺ . When cells are perfused with EDTA-containing medium in the presence of excess Mg²⁺, this method of injection allows the free submembrane Ca²⁺ concentration, [Ca²⁺]_c, to be raised rapidly to several tens of micromoles per liter. Then a chloride component is recorded in the inward tail current, with the amplitude proportional to . When Ca²⁺ is thus injected into an intact cell, it induces an inward current in the voltage-clamped plasmalemma, having activation–inactivation kinetics qualitatively resembling that in EDTA-perfused cells, but a considerably higher amplitude and duration (approximately 10 A m^–2 and _inact~0.5 s at –200 mV). Analysis of our data and theoretical considerations indicate that the [Ca²⁺]_c rise during cell excitation is caused mainly by Ca²⁺ entry through plasmalemma Ca channels rather than by Ca²⁺ release from intracellular stores.     

Outward-rectifying chloride channels in cultured adult and fetal human nasal epithelial cells

Summary The patch-clamp technique was used to characterize ion channels in the apical membranes of cultured human nasal epithelial cells, dissociated from fetal nasal mucosa and from adult nasal polyps. Outward-rectifying chloride channels were found in 4.3% of the cell-attached patches from fetal cells (n=258) and in 3.1% of the patches from adult cells (n=320). After exeision the number of patches containing active chloride channels increased threefold to 13% of the patches from the fetal cells and 10% from adult cells. The single-channel conductance at 0 mV in symmetrical 150mm NaCl solutions was 24.3 ±0.9 pS (n=28) and 26.0 ± 1.2 pS (n=30), respectively, in adult and fetal cells and showed outward rectification in the potential range from –80 to +80 mV. In fetal cells as well as in adult cells the channels were anion selective, and were almost impermeable for larger anions and monovalent cations. In cell-free patches the channels were Ca²⁺ independent. In most of the channels the open probability was voltage independent and high (±0.86); in 20% of the channels, however, the open probability increased with depolarization. In conclusion, fetal nasal epithelial cells contain chloride channels in their apical membranes with singlechannel properties and regulatory mechanisms similar to those found in cells from adults.     

Anion channels in giant liposomes made of endoplasmic reticulum vesicles from rat exocrine pancreas

Summary Using the method of dehydration and rehydration, rough endoplasmic reticulum (RER) vesicles, isolated by differential centrifugation, can be enlarged to giant liposomes with diameters ranging from 5 to 200 m. Patch-clamp studies on these giant RER liposomes revealed the existence of a channel with a mean conductance of 260±7 pS (n=23; 140 mmol/liter KCl on both sides). The channel is about four times more permeable for Cl^– than for K⁺. Its activity is strongly voltage regulated. At low potentials (±20 mV) the channel is predominantly in its open state with an open probability near 1.0, whereas it closes permanently at high positive and negative voltages (±70 mV). The channel activity is not influenced by changing the free Ca²⁺ concentration from 1 mmol/liter to less than 10^–9 mol/liter on either side, and is also not affected by typical Cl^–-channel blockers like diphenylamine-2-carboxylate (DPC, 1 mmol/liter) or 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS, 1 mmol/liter). Another chloride channel with a singlechannel conductance of 79±6 pS (n=4) was less frequently observed. In the potential range of –80 to +40 mV this channel displayed no voltage-dependent gating. We assume that these anion channels are involved in the maintenance of electroneutrality during Ca²⁺ uptake in the RER.     

A9C sensitive Cl? - accumulation in A. thaliana root cells during salt stress is controlled by internal and external calcium

The involvement of chloride in salt stress symptoms and salt tolerance mechanisms in plants has been less investigated in the past. Therefore, we studied the salt-induced chloride influx in Arabidopsis expressing the GFP-based anion indicator Clomeleon. High salt concentrations induce two phases of chloride influx. The fast kinetic phase is likely caused by membrane depolarization, and is assumed to be mediated by channels. This is followed by a slower "saturation" phase, where chloride is accumulated in the cytoplasm. Both phases of chloride uptake are dependent on the presence of external calcium. In general: with high [Ca²⁺] less chloride is accumulated in the cytoplasm. Surprisingly, also the internal calcium availability has an impact on chloride transport. A complete block of the second phase of chloride influx is achieved by the anion channel blocker A9C and trivalent cations (La³⁺, Gd³⁺, and Al³⁺). Other channel blockers and diuretics were found to inhibit the process partially. The results suggest that several transporter species are involved here, including electroneutral cation-chloride-cotransporters, and a part of chloride possibly enters the cells through cation channels after salt application.     

Halide permeation through three types of epithelial anion channels after reconstitution into giant liposomes

Anion-selective channels from apical membranes of cultured CFPAC-1 cells were isolated and incorporated into giant liposomes for patch clamp recording. Liposomes were formed from L--lecithin by a dehydration-hydration method. Ion channels were characterized using the excised inside-out patch clamp configuration. The most commonly observed anion channels were similar to those observed in native epithelial tissues. The linear 20 pS Cl channel had the halide permeability sequence Cl^– > I^– Br^– > F^–, and showed anomalous mole-fraction behavior in solutions containing different proportions of Cl^– and F^–, ions. The autwardly rectifying Cl^– channel had the halide permeability sequence I^– > Br^– > Cl^– > F^–, and also showed anomalous molefraction behavior, indicating that both these channels probably contain multi-ion pores. The third, voltage-dependent anion channel showed at least five different substrates, had a conductance of 390 pS in the main state, and showed two types of kinetics, fast (openings and closings < 1 ms), and slow (openings and closings > 1 s). The channel was seen more frequently after reconstitution into giant liposomes than in intact cells. It was not selective amongst the halides, and there was no deviation from a linear dependence of relative current on molar fractions, indicating relatively simple permeation through the pore. Differences in halide permeabilities suggest that different anion channels may be related to different membrane proteins. Comparison with the chloride channel proteins isolated biochemically from epithelial cell membranes is discussed. Correspondence to: M. Duszyk     

Single channel measurements demonstrate the voltage dependence of permeation through N-type and L-type CaV channels

The delivery of Ca²⁺ into cells by Ca_V channels provides the trigger for many cellular actions, such as cardiac muscle contraction and neurotransmitter release. Thus, a full understanding of Ca²⁺ permeation through these channels is critical. Using whole-cell voltage-clamp recordings, we recently demonstrated that voltage modulates the apparent affinity of N-type (Ca_V2.2) channels for permeating Ca²⁺ and Ba²⁺ ions. While we took many steps to ensure the high fidelity of our recordings, problems can occur when Ca_V currents become large and fast, or when currents run down. Thus, we use here single channel recordings to further test the hypothesis that permeating ions interact with N-type channels in a voltage-dependent manner. We also examined L-type (Ca_V1.2) channels to determine if these channels also exhibit voltage-dependent permeation. Like our whole-cell data, we find that voltage modulates N-channel affinity for Ba²⁺ at voltages > 0 mV, but has little or no effect at voltages < 0 mV. Furthermore, we demonstrate that permeation through L-channel is also modulated by voltage. Thus, voltage-dependence may be a common feature of divalent cation permeation through Ca_V1 and Ca_V2 channels (i.e. high-voltage activated Ca_V channels). The voltage dependence of Ca_V1 channel permeation is likely a mechanism mediating sustained Ca²⁺ influx during the plateau phase of the cardiac action potential.     

Electrical activity of an intestinal epithelial cell line: Hyperpolarizing responses to intestinal secretagogues

Summary Cultured epithelial cells (Intestine 407) derived from fetal human small intestine exhibited spontaneous oscillations of membrane potential between the resting level of about –20 mV and the activated level of about –75mV. The cells were hyperpolarized to the latter level in response to mechanical or electrical stimuli. The hyperpolarizing responses were also elicited by the application of intestinal secretagogues: acetylcholine, histamine, serotonin and vasoactive intestinal polypeptide (VIP). The spontaneous oscillation of membrane potential became prominent and long-lasting in the presence of acetylcholine, histamine, serotonin or VIP. These secretagogue-induced responses were mediated by individual independent receptors on the cell membrane. Muscarinic receptors were responsible for the acetylcholine response, and H₁-receptors for the histamine response. The cells also responded with a slow hyperpolarization to calcium ionophore A23187, which is known to induce intestinal secretion. The spontaneously occurring hyperpolarizing responses and those induced by stimuli were both due to an increase in the K⁺ conductance of the cell membrane. Since acetylcholine, histamine, serotonin and A23187 are known to promote mobilization of cellular Ca²⁺ ions in intestinal secretory cells, it is hypothesized that these electrical activities of the cell are closely related to the receptor stimulation which leads to the Ca²⁺-mediated intestinal secretion.     

Built for speed

Many of us were taught in high school biology that the action potential waveform in nerves and other excitable tissues was generated by an initial rapid influx of external Na⁺ ions across the plasma membrane, followed by an outward movement of intracellular K⁺ ions. The former event, mediated by voltage-gated Na⁺ channels, is responsible for the fast depolarizing upstroke of the action potential, while voltage-gated K+ channels are responsible for the subsequent repolarizing phase, which largely controls action potential duration. Although Hodgkin and Huxley described the fundamental importance of this sequential activation process more than 60 y ago, the molecular and structural details underlying the faster activation of voltage-gated Na⁺ (Nav) vs. K⁺ (Kv) channels have yet to be fully resolved.