Distribution and kinetics of membrane dielectric polarization. 1. Long- term inactivation of gating currents

Gating currents were measured by subtracting the linear component of the capacitative current recorded at very positive or very negative potentials. When the membrane is depolarized for a few minutes, repolarized to the usual holding potential (HP) of --70 mV for 1 ms, and then pulsed to 0 mV, the charge transferred in 2--4 ms is approximately 50% of that which was transferred during the same pulse holding at --70 mV. This charge decrease, called slow inactivation of the gating current, was found to be consistent with a shift of the charge vs. potential (Q-V) curve to more hyperpolarized potentials. When the HP is 0 mV, the total charge available to move is the same as the total charge available when the HP is --70 mV. The time constants of the fast component of the ON gating current are smaller at depolarized holding potentials than at --70 mV. When the HP is --70 mV and a prepulse of 50 ms duration is given to 0 mV, the Q-V curve is also shifted to more hyperpolarized potentials (charge immobilization), but the effect is not as pronounced as the one obtained by holding at 0 mV. When the HP is 0 mV, a prepulse to --70 mV for 50 ms partially shifts back the Q-V curve, indicating that fast inactivation of the gating charge may be recovered in the presence of slow inactivation. A physical model consisting of a gating particle that interacts with a fast inactivating particle, and a slow inactivating particle, reproduces most of the experimental results.     

Sodium channel gating currents in frog skeletal muscle

Charge movements similar to those attributed to the sodium channel gating mechanism in nerve have been measured in frog skeletal muscle using the vaseline-gap voltage-clamp technique. The time course of gating currents elicited by moderate to strong depolarizations could be well fitted by the sum of two exponentials. The gating charge exhibits immobilization: at a holding potential of -90 mV the proportion of charge that returns after a depolarizing prepulse (OFF charge) decreases with the duration of the prepulse with a time course similar to inactivation of sodium currents measured in the same fiber at the same potential. OFF charge movements elicited by a return to more negative holding potentials of -120 or -150 mV show distinct fast and slow phases. At these holding potentials the total charge moved during both phases of the gating current is equal to the ON charge moved during the preceding prepulse. It is suggested that the slow component of OFF charge movement represents the slower return of charge "immobilized" during the prepulse. A slow mechanism of charge immobilization is also evident: the maximum charge moved for a strong depolarization is approximately doubled by changing the holding potential from -90 to -150 mV. Although they are larger in magnitude for a -150-mV holding potential, the gating currents elicited by steps to a given potential have similar kinetics whether the holding potential is -90 or -150 mV.     

Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation

The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.     

Isoform-specific lidocaine block of sodium channels explained by differences in gating

When depolarized from typical resting membrane potentials (V(rest) approximately -90 mV), cardiac sodium (Na) currents are more sensitive to local anesthetics than brain or skeletal muscle Na currents. When expressed in Xenopus oocytes, lidocaine block of hH1 (human cardiac) Na current greatly exceeded that of mu1 (rat skeletal muscle) at membrane potentials near V(rest), whereas hyperpolarization to -140 mV equalized block of the two isoforms. Because the isoform-specific tonic block roughly parallels the drug-free voltage dependence of channel availability, isoform differences in the voltage dependence of fast inactivation could underlie the differences in block. However, after a brief (50 ms) depolarizing pulse, recovery from lidocaine block is similar for the two isoforms despite marked kinetic differences in drug-free recovery, suggesting that differences in fast inactivation cannot entirely explain the isoform difference in lidocaine action. Given the strong coupling between fast inactivation and other gating processes linked to depolarization (activation, slow inactivation), we considered the possibility that isoform differences in lidocaine block are explained by differences in these other gating processes. In whole-cell recordings from HEK-293 cells, the voltage dependence of hH1 current activation was approximately 20 mV more negative than that of mu1. Because activation and closed-state inactivation are positively coupled, these differences in activation were sufficient to shift hH1 availability to more negative membrane potentials. A mutant channel with enhanced closed-state inactivation gating (mu1-R1441C) exhibited increased lidocaine sensitivity, emphasizing the importance of closed-state inactivation in lidocaine action. Moreover, when the depolarization was prolonged to 1 s, recovery from a "slow" inactivated state with intermediate kinetics (I(M)) was fourfold longer in hH1 than in mu1, and recovery from lidocaine block in hH1 was similarly delayed relative to mu1. We propose that gating processes coupled to fast inactivation (activation and slow inactivation) are the key determinants of isoform-specific local anesthetic action.     

Comparison of the endogenous IK currents in rat hippocampal neurons and cloned Kv2.1 channels in CHO cells

The Kv2.1 potassium channel is a principal component of the delayed rectifier I(K) current in the pyramidal neurons of cortex and hippocampus. We used whole-cell patch-clamp recording techniques to systemically compare the electrophysiological properties between the native neuronal I(K) current of cultured rat hippocampal neurons and the cloned Kv2.1 channel currents in the CHO cells. The slope factors for the activation curves of both currents obtained at different prepulse holding potentials and holding times were similar, suggesting similar voltage-dependent gating. However, the half-maximal activation voltage for I(K) was approximately 20 mV more negative than the Kv2.1 channel in CHO cells at a given prepulse condition, indicating that the neuronal I(K) current had a lower threshold for activation than that of the Kv2.1 channel. In addition, the neuronal I(K) showed a stronger holding membrane potential and holding time-dependence than Kv2.1. The Kv2.1 channel gave a U-shaped inactivation, while the I(K) current did not. The I(K) current also had much stronger voltage-dependent inactivation than Kv2.1. These results imply that the neuronal factors could make Kv2.1 channels easier to activate. The information obtained from these comparative studies help elucidate the mechanism of molecular regulation of the native neuronal I(K) current in neurons.     

Specific interaction of the potassium channel beta-subunit minK with the sarcomeric protein T-cap suggests a T-tubule-myofibril linking system.

Ion-channel beta-subunits are ancillary proteins that co-assemble with alpha-subunits to modulate gating kinetics and enhance stability of multimeric channel complexes. They provide binding sites for other regulatory proteins and are medically important as the targets of many pharmacological compounds. MinK is the beta-subunit of the slow activating component of the delayed rectifier potassium current (I(Ks)) channel, and associates with the alpha-subunit, KvLQT1. We report here that minK specifically interacts with the sarcomeric Z-line component, T-cap (also called telethonin). In vitro interaction studies indicated that the cytoplasmic domain of minK specifically binds to the sixteen C-terminal residues of T-cap; these residues are sufficient for its interaction with minK.Consistent with our in vitro studies, immunofluorescence staining followed by confocal analysis revealed that both minK and T-cap are localized within the Z-line region in cardiac muscle. Striated staining of minK was observed in non-washed, membrane-intact cardiac myofibrils, but not in well-washed, membrane-removed cardiac myofibrils, suggesting that minK localizes on T-tubular membranes surrounding the Z-line in the inner ventricular myocardium.Together with our previous data on the colocalization and interaction of T-cap with the N-terminus of the giant protein titin in the periphery of the Z-line, these data suggest that T-cap functions as an adapter protein to link together myofibrillar components with the membranous beta-subunit of the I(Ks) channel. We speculate that this interaction may contribute to a stretch-dependent regulation of potassium flux in cardiac muscle, providing a "mechano-electrical feedback" system.     

Rate-limiting Reactions Determining Different Activation Kinetics of Kv1.2and Kv2.1 Channels

To identify the mechanisms underlying the faster activation kinetics in Kv1.2 channels compared to Kv2.1 channels, ionic and gating currents were studied in rat Kv1.2 and human Kv2.1 channels heterologously expressed in mammalian cells. At all voltages the time course of the ionic currents could be described by an initial sigmoidal and a subsequent exponential component and both components were faster in Kv1.2 than in Kv2.1 channels. In Kv1.2 channels, the activation time course was more sigmoid at more depolarized potentials, whereas in Kv2.1 channels it was somewhat less sigmoid at more depolarized potentials. In contrast to the ionic currents, the ON gating currents were similarly fast for both channels. The main portion of the measured ON gating charge moved before the ionic currents were activated. The equivalent gating charge of Kv1.2 ionic currents was twice that of Kv2.1 ionic currents, whereas that of Kv1.2 ON gating currents was smaller than that of Kv2.1 ON gating currents. In conclusion, the different activation kinetics of Kv1.2 and Kv2.1 channels are caused by rate-limiting reactions that follow the charge movement recorded from the gating currents. In Kv1.2 channels, the reaction coupling the voltage-sensor movement to the pore opening contributes to rate limitation in a voltage-dependent fashion, whereas in Kv2.1 channels, activation is additionally rate-limited by a slow reaction in the subunit gating.     

TTX-sensitive Na(+) and nifedipine-sensitive Ca(2+) channels in rat vas deferens smooth muscle cells.

The inward currents in single smooth muscle cells (SMC) isolated from epididymal part of rat vas deferens have been studied using whole-cell patch-clamp method. Depolarising steps from holding potential -90 mV evoked inward current with fast and slow components. The component with slow activation possessed voltage-dependent and pharmacological properties characteristic for Ca(2+) current carried through L-type calcium channels (I(Ca)). The fast component of inward current was activated at around -40 mV, reached its peak at 0 mV, and disappeared upon removal of Na ions from bath solution. This current was blocked in dose-dependent manner by tetrodotoxin (TTX) with an apparent dissociation constant of 6.7 nM. On the basis of voltage-dependent characteristics, TTX sensitivity of fast component of inward current and its disappearance in Na-free solution it is suggested that this current is TTX-sensitive depolarisation activated sodium current (I(Na)). Cell dialysis with a pipette solution containing no macroergic compounds resulted in significant inhibition of I(Ca) (depression of peak I(Ca) by about 81% was observed by 13 min of dialysis), while I(Na) remained unaffected during 50 min of dialysis. These data draw first evidence for the existence of TTX-sensitive Na(+) current in single SMC isolated from rat vas deferens. These Na(+) channels do not appear to be regulated by a phosphorylation process under resting conditions.     

Voltage-gated transient currents in bovine adrenal fasciculata cells. II. A-type K+ current

In whole cell patch clamp recordings on enzymatically dissociated adrenal zona fasciculata (AZF) cells, a rapidly inactivating A-type K+ current was observed in each of more than 150 cells. Activation of IA was steeply voltage dependent and could be described by a Boltzmann function raised to an integer power of 4, with a midpoint of -28.3 mV. Using the "limiting logarithmic potential sensitivity," the single channel gating charge was estimated to be 7.2 e. Voltage-dependent inactivation could also be described by a Boltzmann function with a midpoint of -58.7 mV and a slope factor of 5.92 mV. Gating kinetics of IA included both voltage-dependent and -independent transitions in pathways between closed, open, and inactivated states. IA activated with voltage-dependent sigmoidal kinetics that could be fit with an n4h formalism. The activation time constant, tau a, reached a voltage- independent minimum at potentials positive to 0 mV. IA currents inactivated with two time constants that were voltage independent at potentials ranging from -30 to +45 mV. At +20 mV, tau i(fast) and tau i(slow) were 13.16 +/- 0.64 and 62.26 +/- 5.35 ms (n = 34), respectively. In some cells, IA inactivation kinetics slowed dramatically after many minutes of whole cell recording. Once activated by depolarization, IA channels returned to the closed state along pathways with two voltage-dependent time constants which were 0.208 s, tau rec-f and 10.02 s, tau rec-s at -80 mV. Approximately 90% of IA current recovered with slow kinetics at potentials between -60 and -100 mV. IA was blocked by 4-aminopyridine (IC50 = 629 microM) through a mechanism that was strongly promoted by channel activation. Divalent and trivalent cations including Ni2+ and La3+ also blocked IA with IC50's of 467 and 26.4 microM, respectively. With respect to biophysical properties and pharmacology, IA in AZF cells resembles to some extent transient K+ currents in neurons and muscle, where they function to regulate action potential frequency and duration. The function of this prominent current in steroid hormone secretion by endocrine cells that may not generate action potentials is not yet clear.     

The steady-state distribution of gating charge in crayfish giant axons.

Progressive shifts of holding potential (Vh) in crayfish giant axons, from -140 to -70 mV, reduce gating currents seen in depolarizing steps (to 0 mV test potential) while proportionately increasing gating currents in hyperpolarizing steps (to -240 mV). The resulting sigmoid equilibrium charge distribution (Q-Vh curve) shows an effective valence of 1.9e and a midpoint of -100 mV. By contrast, Q-V curves obtained using hyperpolarizing and/or depolarizing steps from a single holding potential, change their "shape" depending on the chosen holding potential. For holding potentials at the negative end of the Q-Vh distribution (e.g., -140 mV), negligible charge moves in hyperpolarizing pulses and the Q-V curve can be characterized entirely from depolarizing voltage steps. The slope of the resulting simple sigmoid Q-V curve also indicates an effective valence of 1.9e. When the axon is held at less negative potentials significant charge moves in hyperpolarizing voltage steps. The component of the Q-V curve collected using hyperpolarizing pulses shows a significantly reduced slope (approximately 0.75e) by comparison with the 1.9e slope found using depolarizing pulses or from the Q-Vh curve. As holding potential is shifted in the depolarizing direction along the Q-Vh curve, an increasing fraction of total charge movement must be assessed in hyperpolarizing voltage steps. Thus charge moving in the low slope component of the Q-V curve increases as holding potential is depolarized, while charge moving with high apparent valence decreases proportionately. Additional results, together with simulations based on a simple kinetic model, suggest that the reduced apparent valence of the low slope component of the Q-V curve results from gating charge immobilization occurring at holding potential. Immobilization selectively retards that fraction of total charge moving in hyperpolarizing pulses. Misleading conclusions, as to the number and valence of the gating particles, may therefore be derived from Q-V curves obtained by other than depolarizing pulses from negative saturated holding potentials.     

Long QT syndrome-associated mutations in the S4-S5 linker of KvLQT1 potassium channels modify gating and interaction with minK subunits.

Long QT syndrome is an inherited disorder of cardiac repolarization caused by mutations in cardiac ion channel genes, including KVLQT1. In this study, the functional consequences of three long QT-associated missense mutations in KvLQT1 (R243C, W248R, E261K) were characterized using the Xenopus oocyte heterologous expression system and two-microelectrode voltage clamp techniques. These mutations are located in or near the intracellular linker between the S4 and S5 transmembrane domains, a region implicated in activation gating of potassium channels. The E261K mutation caused loss of function and did not interact with wild-type KvLQT1 subunits. R243C or W248R KvLQT1 subunits formed functional channels, but compared with wild-type KvLQT1 current, the rate of activation was slower, and the voltage dependence of activation and inactivation was shifted to more positive potentials. Co expression of minK and KvLQT1 channel subunits induces a slow delayed rectifier K(+) current, I(Ks), characterized by slow activation and a markedly increased magnitude compared with current induced by KvLQT1 subunits alone. Coexpression of minK with R243C or W248R KvLQT1 subunits suppressed current, suggesting that coassembly of mutant subunits with minK prevented normal channel gating. The decrease in I(Ks) caused by loss of function or altered gating properties explains the prolonged QT interval and increased risk of arrhythmia and sudden death associated with these mutations in KVLQT1.     

Steady-state availability of sodium channels. Interactions between activation and slow inactivation.

Changes in holding potential (Vh), affect both gating charge (the Q(Vh) curve) and peak ionic current (the F(Vh) curve) seen at positive test potentials. Careful comparison of the Q(Vh) and F(Vh) distributions indicates that these curves are similar, having two slopes (approximately 2.5e for Vh from -115 to -90 mV and approximately 4e for Vh from -90 to -65 mV) and very negative midpoints (approximately -86 mV). Thus, gating charge movement and channel availability appear closely coupled under fully-equilibrated conditions. The time course by which channels approach equilibration was explored using depolarizing prepulses of increasing duration. The high slope component seen in the F(Vh) and Q(Vh) curves is not evident following short depolarizing prepulses in which the prepulse duration approximately corresponds to the settling time for fast inactivation. Increasing the prepulse duration to 10 ms or longer reveals the high slope, and left-shifts the midpoint to more negative voltages, towards the F(Vh) and Q(Vh) distributions. These results indicate that a separate slow-moving voltage sensor affects the channels at prepulse durations greater than 10 ms. Charge movement and channel availability remain closely coupled as equilibrium is approached using depolarizing pulses of increasing durations. Both measures are 50% complete by 50 ms at a prepulse potential of -70 mV, with proportionately faster onset rates when the prepulse potential is more depolarized. By contrast, charge movement and channel availability dissociate during recovery from prolonged depolarizations. Recovery of gating charge is considerably faster than recovery of sodium ionic current after equilibration at depolarized potentials. Recovery of gating charge at -140 mV, is 65% complete within approximately 100 ms, whereas less than 30% of ionic current has recovered by this time. Thus, charge movement and channel availability appear to be uncoupled during recovery, although both rates remain voltage sensitive. These data suggest that channels remain inactivated due to a separate process operating in parallel with the fast gating charge. We demonstrate that this behavior can be simulated by a model in which the fast charge movement associated with channel activation is electrostatically-coupled to a separate slow voltage sensor responsible for the slow inactivation of channel conductance.     

Nonlinear charge movement in mammalian cardiac ventricular cells. Components from Na and Ca channel gating [published erratum appears in J Gen Physiol 1989 Aug;94(2):401]

Intramembrane charge movement was recorded in rat and rabbit ventricular cells using the whole-cell voltage clamp technique. Na and K currents were eliminated by using tetraethylammonium as the main cation internally and externally, and Ca channel current was blocked by Cd and La. With steps in the range of -110 to -150 used to define linear capacitance, extra charge moves during steps positive to approximately -70 mV. With holding potentials near -100 mV, the extra charge moving outward on depolarization (ON charge) is roughly equal to the extra charge moving inward on repolarization (OFF charge) after 50-100 ms. Both ON and OFF charge saturate above approximately +20 mV; saturating charge movement is approximately 1,100 fC (approximately 11 nC/muF of linear capacitance). When the holding potential is depolarized to -50 mV, ON charge is reduced by approximately 40%, with little change in OFF charge. The reduction of ON charge by holding potential in this range matches inactivation of Na current measured in the same cells, suggesting that this component might arise from Na channel gating. The ON charge remaining at a holding potential of -50 mV has properties expected of Ca channel gating current: it is greatly reduced by application of 10 muM D600 when accompanied by long depolarizations and it is reduced at more positive holding potentials with a voltage dependence similar to that of Ca channel inactivation. However, the D600-sensitive charge movement is much larger than the Ca channel gating current that would be expected if the movement of channel gating charge were always accompanied by complete opening of the channel.     

Ionic currents in dispersed chemoreceptor cells of the mammalian carotid body

Ionic currents of enzymatically dispersed type I and type II cells of the carotid body have been studied using the whole cell variant of the patch-clamp technique. Type II cells only have a tiny, slowly activating outward potassium current. By contrast, in every type I chemoreceptor cell studied we found (a) sodium, (b) calcium, and (c) potassium currents. (a) The sodium current has a fast activation time course and an activation threshold at approximately -40 mV. At all voltages inactivation follows a single exponential time course. The time constant of inactivation is 0.67 ms at 0 mV. Half steady state inactivation occurs at a membrane potential of approximately -50 mV. (b) The calcium current is almost totally abolished when most of the external calcium is replaced by magnesium. The activation threshold of this current is at approximately -40 mV and at 0 mV it reaches a peak amplitude in 6-8 ms. The calcium current inactivates very slowly and only decreases to 27% of the maximal value at the end of 300-ms pulses to 40 mV. The calcium current was about two times larger when barium ions were used as charge carriers instead of calcium ions. Barium ions also shifted 15-20 mV toward negative voltages the conductance vs. voltage curve. Deactivation kinetics of the calcium current follows a biphasic time course well fitted by the sum of two exponentials. At -80 mV the slow component has a time constant of 1.3 +/- 0.4 ms whereas the fast component, with an amplitude about 20 times larger than the slow component, has a time constant of 0.16 +/- 0.03 ms. These results suggest that type I cells have predominantly fast deactivating calcium channels. The slow component of the tails may represent the activity of a small population of slowly deactivating calcium channels, although other possibilities are considered. (c) Potassium current seems to be mainly due to the activity of voltage-dependent potassium channels, but a small percentage of calcium-activated channels may also exist. This current activates slowly, reaches a peak amplitude in 5-10 ms, and thereafter slowly inactivates. Inactivation is almost complete in 250-300 ms. The potassium current is reversibly blocked by tetraethylammonium. Under current-clamp conditions type I cells can spontaneously fire large action potentials. These results indicate that type I cells are excitable and have a variety of ionic conductances. We suggest a possible participation of these conductances in chemoreception.     

K+ currents activated by depolarization in cardiac fibroblasts

K(+) currents expressed in freshly dispersed rat ventricular fibroblasts have been studied using whole-cell patch-clamp recordings. Depolarizing voltage steps from a holding potential of -90 mV activated time- and voltage-dependent outward currents at membrane potentials positive to approximately -30 mV. The relatively slow activation kinetics exhibited strong dependence on the membrane potential. Selected changes in extracellular K(+) concentration ([K(+)](o)) revealed that the reversal potentials of the tail currents changed as expected for a K(+) equilibrium potential. The activation and inactivation kinetics of this K(+) current, as well as its recovery from inactivation, were well-fitted by single exponential functions. The steady-state inactivation was well described by a Boltzmann function with a half-maximal inactivation potential (V(0.5)) of -24 mV. Increasing [K(+)](o) (from 5 to 100 mM) shifted this V(0.5) in the hyperpolarizing direction by -11 mV. Inactivation was slowed by increasing [K(+)](o) to 100 mM, and the rate of recovery from inactivation was decreased after increasing [K(+)](o). Block of this K(+) current by extracellular tetraethylammonium also slowed inactivation. These [K(+)](o)-induced changes and tetraethylammonium effects suggest an important role for a C-type inactivation mechanism. This K(+) current was sensitive to dendrotoxin-I (100 nM) and rTityustoxin Kalpha (50 nM).     

Electrical remodeling in a canine model of ischemic cardiomyopathy

The nature of electrical remodeling in a canine model of ischemic cardiomyopathy (ICM; induced by repetitive intracoronary microembolizations) that exhibits spontaneous ventricular tachycardia is not entirely clear. We used the patch-clamp technique to record action potentials and ionic currents of left ventricular myocytes isolated from the region affected by microembolizations. We also used the immunoblot technique to examine channel subunit expression in adjacent affected tissue. Ventricular myocytes and tissue isolated from the corresponding region of normal hearts served as control. ICM myocytes had prolonged action potential duration (APD) and more pronounced APD dispersion. Slow delayed rectifier current (I(Ks)) was reduced at voltages positive to 0 mV, along with a negative shift in its voltage dependence of activation. Immunoblots showed that there was no change in KCNQ1.1 (I(Ks) pore-forming or alpha-subunit), but KCNE1 (I(Ks) auxiliary or beta-subunit) was reduced, and KCNQ1.2 (a truncated KCNQ1 splice variant with a dominant-negative effect on I(Ks)) was increased. Transient outward current (I(to)) was reduced, along with an acceleration of the slow phase of recovery from inactivation. Immunoblots showed that there was no change in Kv4.3 (alpha-subunit of fast-recovering I(to) component), but KChIP2 (beta-subunit of fast-recovering component) and Kv1.4 (alpha-subunit of slow-recovering component) were reduced. Inward rectifier current was reduced. L-type Ca current was unaltered. The immunoblot data provide mechanistic insights into the observed changes in current amplitude and gating kinetics of I(Ks) and I(to). We suggest that these changes, along with the decrease in inward rectifier current, contribute to APD prolongation in ICM hearts.     

Inactivation kinetics and pharmacology distinguish two calcium currents in mouse pancreatic B-cells

Summary Voltage-dependent calcium currents were studied in cultured adult mouse pancreatic B-cells using the whole-cell voltage-clamp technique. When calcium currents were elicited with 10-sec depolarizing command pulses, the time course of inactivation was well fit by the sum of two exponentials. The more rapidlyinactivating component had a time constant of 75±5 msec at 0 mV and displayed both calcium influx- and voltage-dependent inactivation, while the more slowly-inanctivating component had a time constant of 2750±280 msec at 0 mV and inactivated primarily via voltage. The fast component was subject to greater steady-state inactivation at holding potentials between –100 and –40 mV and activated at a lower voltage threshold. This component was also significantly reduced by nimodipine (0.5 m) when a holding potential of –100 mV was used, whereas the slow component was unaffected. In contrast, the slow component was greatly increased by replacing external calcium with barium, while the fast component was unchanged. Cadmium (1–10 m) displayed a voltage-dependent block of calcium currents consistent with a greater effect on the high-threshold, more-slowly inactivating component. Taken together, the data suggest that cultured mouse B-cells, as with other insulin-secreting cells we have studied, possess at least two distinct calcium currents. The physiological significance of two calcium currents having distinct kinetic and steady-state inactivation characteristics for B-cell burst firing and insulin secretion is discussed.     

Resting potential-dependent regulation of the voltage sensitivity of sodium channel gating in rat skeletal muscle in vivo

Normal muscle has a resting potential of -85 mV, but in a number of situations there is depolarization of the resting potential that alters excitability. To better understand the effect of resting potential on muscle excitability we attempted to accurately simulate excitability at both normal and depolarized resting potentials. To accurately simulate excitability we found that it was necessary to include a resting potential-dependent shift in the voltage dependence of sodium channel activation and fast inactivation. We recorded sodium currents from muscle fibers in vivo and found that prolonged changes in holding potential cause shifts in the voltage dependence of both activation and fast inactivation of sodium currents. We also found that altering the amplitude of the prepulse or test pulse produced differences in the voltage dependence of activation and inactivation respectively. Since only the Nav1.4 sodium channel isoform is present in significant quantity in adult skeletal muscle, this suggests that either there are multiple states of Nav1.4 that differ in their voltage dependence of gating or there is a distribution in the voltage dependence of gating of Nav1.4. Taken together, our data suggest that changes in resting potential toward more positive potentials favor states of Nav1.4 with depolarized voltage dependence of gating and thus shift voltage dependence of the sodium current. We propose that resting potential-induced shifts in the voltage dependence of sodium channel gating are essential to properly regulate muscle excitability in vivo.     

Hysteresis in the voltage dependence of HCN channels: conversion between two modes affects pacemaker properties

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels are important for rhythmic activity in the brain and in the heart. In this study, using ionic and gating current measurements, we show that cloned spHCN channels undergo a hysteresis in their voltage dependence during normal gating. For example, both the gating charge versus voltage curve, Q(V), and the conductance versus voltage curve, G(V), are shifted by about +60 mV when measured from a hyperpolarized holding potential compared with a depolarized holding potential. In addition, the kinetics of the tail current and the activation current change in parallel to the voltage shifts of the Q(V) and G(V) curves. Mammalian HCN1 channels display similar effects in their ionic currents, suggesting that the mammalian HCN channels also undergo voltage hysteresis. We propose a model in which HCN channels transit between two modes. The voltage dependence in the two modes is shifted relative to each other, and the occupancy of the two modes depends on the previous activation of the channel. The shifts in the voltage dependence are fast (tau approximately 100 ms) and are not accompanied by any apparent inactivation. In HCN1 channels, the shift in voltage dependence is slower in a 100 mM K extracellular solution compared with a 1 mM K solution. Based on these findings, we suggest that molecular conformations similar to slow (C-type) inactivation of K channels underlie voltage hysteresis in HCN channels. The voltage hysteresis results in HCN channels displaying different voltage dependences during different phases in the pacemaker cycle. Computer simulations suggest that voltage hysteresis in HCN channels decreases the risk of arrhythmia in pacemaker cells.     

Slow components of potassium tail currents in rat skeletal muscle

The kinetics of potassium tail currents have been studied in the omohyoid muscle of the rat using the three-microelectrode voltage-clamp technique. The currents were elicited by a two-pulse protocol in which a conditioning pulse to open channels was followed by a test step to varying levels. The tail currents reversed at a single well-defined potential (VK). At hyperpolarized test potentials (-100 mV and below), tail currents were inward and exhibited two clearly distinguishable phases of decay, a fast tail with a time constant of 2-3 ms and a slow tail with a time constant of approximately 150 ms. At depolarized potentials (-60 mV and above), tail currents were outward and did not show two such easily separable phases of decay, although a slow kinetic component was present. The slow kinetic phase of outward tail currents appeared to be functionally distinct from the slow inward tail since the channels responsible for the latter did not allow significant outward current. Substitution of Rb for extracellular K abolished current through the anomalous (inward-going) rectifier and at the same time eliminated the slow inward tail, which suggests that the slow inward tail current flows through anomalous rectifier channels. The amplitude of the slow inward tail was increased and VK was shifted in the depolarizing direction by longer conditioning pulses. The shift in VK implies that during outward currents potassium accumulates in a restricted extracellular space, and it is suggested that this excess K causes the slow inward tail by increasing the inward current through the anomalous rectifier. By this hypothesis, the tail current slowly decays as K diffuses from the restricted space. Consistent with such a hypothesis, the decay of the slow inward tail was not strongly affected by changing temperature. It is concluded that a single delayed K channel is present in the omohyoid. Substitution of Rb for K has little effect on the magnitude or time course of outward current tails, but reduces the magnitude and slows the decay of the fast component of inward tails. Both effects are consistent with a mechanism proposed for squid giant axon (Swenson and Armstrong, 1981): that (a) the delayed potassium channel cannot close while Rb is inside it, and (b) that Rb remains in the channel longer than K.