Gating characteristics of a steeply voltage-dependent gap junction channel in rat Schwann cells

The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, Gss was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary junctional currents was detected corresponding to an unitary channel conductance of approximately 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic Gss voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states.     

The effects of external pH on calcium channel currents in bullfrog sympathetic neurons.

We have investigated the effects of external pH (pHo) on whole-cell calcium channel currents in bullfrog sympathetic neurons. The peak inward current increased at alkaline pHo and decreased at acidic pHo. We used tail currents to distinguish effects of pHo on channel gating and permeation. There were large shifts in the voltage dependence of channel activation (approximately 40 mV between pHo and 9.0 and pHo 5.6), which could be explained by binding of H+ to surface charge according to Gouy-Chapman theory. To examine the effects of pHo on permeation, we measured tail currents at 0 mV, following steps to + 120 mV to maximally activate the channels. Unlike most previous studies, we found only a approximately 10% reduction in channel conductance from pHo 9.0 to pHo 6.4, despite a approximately 25 mV shift of channel activation. At lower pHo the channel conductance did decrease, which could be described by binding of H+ to a site with pKa = 5.1. In some cells, there was a separate slow decrease in conductance at low pHo, possibly because of changes in internal pH. These results suggest that changes in current at pHo > 6.4 result primarily from a shift in the voltage dependence of channel activation. A H(+)-binding site can explain a rapid decrease in channel conductance at lower pHo. The surface charge affecting gating has little effect on the local ion concentration near the pore, or on the channel conductance.     

Sequence of Gating Charge Movement and Pore Gating in hERG Activation and Deactivation Pathways

K_V11.1 voltage-gated K⁺ channels are noted for unusually slow activation, fast inactivation, and slow deactivation kinetics, which tune channel activity to provide vital repolarizing current during later stages of the cardiac action potential. The bulk of charge movement in human ether-a-go-go-related gene (hERG) is slow, as is return of charge upon repolarization, suggesting that the rates of hERG channel opening and, critically, that of deactivation might be determined by slow voltage sensor movement, and also by a mode-shift after activation. To test these ideas, we compared the kinetics and voltage dependence of ionic activation and deactivation with gating charge movement. At 0 mV, gating charge moved ∼threefold faster than ionic current, which suggests the presence of additional slow transitions downstream of charge movement in the physiological activation pathway. A significant voltage sensor mode-shift was apparent by 24 ms at +60 mV in gating currents, and return of charge closely tracked pore closure after pulses of 100 and 300 ms duration. A deletion of the N-terminus PAS domain, mutation R4AR5A or the LQT2-causing mutation R56Q gave faster-deactivating channels that displayed an attenuated mode-shift of charge. This indicates that charge movement is perturbed by N- and C-terminus interactions, and that these domain interactions stabilize the open state and limit the rate of charge return. We conclude that slow on-gating charge movement can only partly account for slow hERG ionic activation, and that the rate of pore closure has a limiting role in the slow return of gating charges.     

Computing transient gating charge movement of voltage-dependent ion channels

The opening of voltage-gated sodium, potassium, and calcium ion channels has a steep relationship with voltage. In response to changes in the transmembrane voltage, structural movements of an ion channel that precede channel opening generate a capacitative gating current. The net gating charge displacement due to membrane depolarization is an index of the voltage sensitivity of the ion channel activation process. Understanding the molecular basis of voltage-dependent gating of ion channels requires the measurement and computation of the gating charge, Q. We derive a simple and accurate semianalytic approach to computing the voltage dependence of transient gating charge movement (Q–V relationship) of discrete Markov state models of ion channels using matrix methods. This approach allows rapid computation of Q–V curves for finite and infinite length step depolarizations and is consistent with experimentally measured transient gating charge. This computational approach was applied to Shaker potassium channel gating, including the impact of inactivating particles on potassium channel gating currents.     

Sodium ionic and gating currents in mammalian cells

Ionic and gating currents from voltage-gated sodium channels were recorded in mouse neuroblastoma cells using the path-clamp technique. Displacement currents were measured from whole-cell recordings. The gating charge displaced during step depolarizations increased with the applied membrane potential and reached saturating levels above 20 mV Prolonged large depolarizations produced partial immobilization of the gating charge, and only about one third of the displaced charge was quickly reversed upon return to negative holding potentials. The activation and inactivation properties of macroscopic sodium currents were characterized by voltage-clamp analysis of large outside-out patches and the single-channel conductance was estimated from nonstationary noise analysis. The general properties of the sodium channels in mouse neuroblastoma cells are very similar to those previously reported for various preparations of invertebrate and vertebrate nerve cells. Offprint requests to: O. Moran     

Separation of sodium and calcium channels in the surface membrane of molluscan nerve cells

By intracellular dialysis of isolated neurons of the mollusksHelix pomatia andLimnaea stagnalis and by a voltage clamp technique the characteristics of transmembrane ionic currents were studied during controlled changes in the ionic composition of the extracellular and intracellular medium. By replacing the intracellular potassium ions by Tris ions, functional blocking of the outward potassium currents was achieved and the inward current distinguished in a pure form. Replacement of Ringer's solution in the extracellular medium with sodium-free or calcium-free solution enabled the inward current to be separated into two additive components, one carried by sodium ions, the other by calcium ions. Sodium and calcium inward currents were found to have different kinetics and different potential-dependence: _mNa=1±0.5 msec, _mCa=3±1 msec, _hNa=8±2 msec, _hCa=115±10 msec (V_m=0), G_Na=0.5 (V_m=–21±2 mV), G_Ca=0.5 (V_m=–8±2 mV). Both currents remained unchanged by tetrodotoxin, but the calcium current was specifically blocked by cadmium ions (2·10^–3 M), verapamil, and D=600, and also by fluorine ions if injected intracellularly. All these results are regarded as evidence that the soma membrane of the neurons tested possesses separate systems of sodium and calcium ion-conducting channels. Quantitative differences are observed in the relative importance of the systems of sodium and calcium channels in different species of mollusks.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 8, No. 2, pp. 183–191, March–April, 1976.     

Voltage-dependent calcium and potassium conductances in striated muscle fibers from the scorpion,Centruroides sculpturatus

Ionic currents responsible for the action potential in scorpion muscle fibers were characterized using a three-intracellular microelectrode voltage clamp applied at the fiber ends (8–12°C). Large calcium currents (I _Ca) trigger contractile activation in physiological saline (5 mm Ca) but can be studied in the absence of contractile activation in a low Ca saline (2.5 mm). Barium (Ba) ions (1.5–3 mm) support inward current but not contractile activation.Ca conductance kinetics are fast (time constant of 3 msec at 0 mV) and very voltage dependent, with steady-state conductance increasing e-fold in approximately 4 mV. Half-activation occurs at –25 mV. Neither I _Ca nor I _Ba show rapid inactivation, but a slow, voltage-dependent inactivation eliminates I _Ca at voltages positive to –40 mV. Kinetically, scorpion channels are more similar to L-type Ca channels in vertebrate cardiac muscle than to those in skeletal muscle.Outward K currents turn on more slowly and with a longer delay than do Ca currents, and K conductance rises less steeply with voltage (e-fold change in 10 mV; half-maximal level at 0 mV). K channels are blocked by externally applied tetraethylammonium and 3,4 diaminopyridine.This work was supported by a grant from the NIH (NS-17510) to W.F.G. and a NRSA award to T.S. (GM-09921).     

Alteration of Ion Channels in the Plasmalemma of Nitella flexilisCells during Long-Term Hyperthermia

The conventional microelectrode technique was applied to study changes in conductance and activation characteristics of potassium and chloride channels in the plasmalemma of characean alga Nitella flexilis(L.) Agardz. during long-term heat treatment. Measurements were conducted at 18–20°C after preliminary exposure of cells to 33°C for 1–25 days. The conductance of outward- and inward-rectifying potassium channels, as well as the currents of excitable chloride channels, decreased after 2–3 days of heat treatment. By the 15th–17th days, the conductance of potassium channels was reduced by a factor of 3–5, whereas the peak values of the chloride current, associated with the action potential, was reduced by a factor of 8–10. These heat-induced changes were long lasting: the restoration of the initial parameters of transport systems after transferring cells to chilling or room temperature occurred within several days. Moreover, the recovery at chilling temperatures (8–10°C) proceeded nearly two times longer than at room temperature. Prolonged hyperthermia accelerated activation and deactivation of outward-rectifying potassium channels and caused the shift of their activation curve towards positive potentials by 35–40 mV. Analysis of current–voltage relations showed that the inward current in inward- and outward-rectifying potassium channels was reduced to a greater extent than the outward current. At the same time, both inward and outward currents of chloride channels were reduced to an equal extent. It is assumed that the changes observed are involved in thermal adaptation and account for the decrease in the intracellular concentrations of potassium and other cations and anions, which represents a nonspecific response of plant cells to stress.     

A gating charge interaction required for late slow inactivation of the bacterial sodium channel NavAb

Voltage-gated sodium channels undergo slow inactivation during repetitive depolarizations, which controls the frequency and duration of bursts of action potentials and prevents excitotoxic cell death. Although homotetrameric bacterial sodium channels lack the intracellular linker-connecting homologous domains III and IV that causes fast inactivation of eukaryotic sodium channels, they retain the molecular mechanism for slow inactivation. Here, we examine the functional properties and slow inactivation of the bacterial sodium channel NavAb expressed in insect cells under conditions used for structural studies. NavAb activates at very negative membrane potentials (V_1/2 of approximately −98 mV), and it has both an early phase of slow inactivation that arises during single depolarizations and reverses rapidly, and a late use-dependent phase of slow inactivation that reverses very slowly. Mutation of Asn49 to Lys in the S2 segment in the extracellular negative cluster of the voltage sensor shifts the activation curve ∼75 mV to more positive potentials and abolishes the late phase of slow inactivation. The gating charge R3 interacts with Asn49 in the crystal structure of NavAb, and mutation of this residue to Cys causes a similar positive shift in the voltage dependence of activation and block of the late phase of slow inactivation as mutation N49K. Prolonged depolarizations that induce slow inactivation also cause hysteresis of gating charge movement, which results in a requirement for very negative membrane potentials to return gating charges to their resting state. Unexpectedly, the mutation N49K does not alter hysteresis of gating charge movement, even though it prevents the late phase of slow inactivation. Our results reveal an important molecular interaction between R3 in S4 and Asn49 in S2 that is crucial for voltage-dependent activation and for late slow inactivation of NavAb, and they introduce a NavAb mutant that enables detailed functional studies in parallel with structural analysis.     

Sodium and calcium channels in the somatic membrane of artificially differentiated neuroblastoma cells

Using an intracellular dialysis technique a study was made on calcium and sodium inward currents at the neuroblastoma somatic cell membrane in suspension and during the course of artificial morphological differentiation produced by raising the pH of the culture medium to 8.0–8.2. The density of sodium currents in the somata of cells cultured in the suspension averaged 7.3±0.8 µA/µF, while this value varied from 37±5.2 to 54.7±3.6 µA/µF at various stages of culture. These values equalled 1.4±0.2 and 1.1±0.2 to 2.8±0.4 µA/µF in the case of calcium currents. Reciprocal changes were produced in the density of sodium and calcium channels by altering the culture medium.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 207–214, March–April, 1986.     

Ca2+-activated K+ currents inNecturus choroid plexus

Summary The tight-seal whole-cell recording method has been used to studyNecturus choroid plexus epithelium. A cell potential of –59±2 mV and a whole cell resistance of 56±6 M were measured using this technique. Application of depolarizing step potentials activated voltage-dependent outward currents that developed with time. For example, when the cell was bathed in 110mm NaCl Ringer solution and the interior of the cell contained a solution of 110mm KCl and 5nm Ca²⁺, stepping the membrane potential from a holding value of –50 to –10 mV evoked outward currents which, after a delay of greater than 50 msec, increased to a steady state in 500 msec. The voltage dependence of the delayed currents suggests that they may be currents through Ca²⁺-activated K^_ channels. Based on the voltage dependence of the activation of Ca²⁺-activated K⁺ channels, we have devised a general method to isolate the delayed currents. The delayed currents were highly selective for K⁺ as their reversal potential at different K⁺ concentration gradients followed the Nernst potential for K⁺. These currents were reduced by the addition of TEA⁺ to the bath solution and were eliminated when Cs⁺ or Na⁺ replaced intracellular K⁺. Increasing the membrane potential to more positive values decreased both the delay and the half-times (t _1/2) to the steady value. Increasing the pipette Ca²⁺ also decreased the delay and decreasedt _1/2. For instance, when pipette Ca²⁺ was increased from 5 to 500nm, the delay andt _1/2 decreased from values greater than 50 and 150 msec to values less than 10 and 50 msec. We conclude that the delayed currents are K⁺ currents through Ca²⁺-activated K⁺ channels.At the resting membrane potential of –60 mV, Ca²⁺-activated K⁺ channels contribute between 13 to 25% of the total conductance of the cell. The contribution of these channels to cell conductance nearly doubles with membrane depolarization of 20–30 mV. Such depolarizations have been observed when cerebrospinal fluid (CSF) secretion is stimulated by cAMP and with intracellular Ca²⁺. Thus the Ca²⁺-activated K⁺ channels may play a specific role in maintaining intracellular K⁺ concentrations during CSF secretion.     

Gating of Shaker K+ channels: I. Ionic and gating currents.

Ionic and gating currents from noninactivating Shaker B K+ channels were studied with the cut-open oocyte voltage clamp technique and compared with the macropatch clamp technique. The performance of the cut-open oocyte voltage clamp technique was evaluated from the electrical properties of the clamped upper domus membrane, K+ tail current measurements, and the time course of K+ currents after partial blockade. It was concluded that membrane currents less than 20 microA were spatially clamped with a time resolution of at least 50 microseconds. Subtracted, unsubtracted gating currents with the cut-open oocyte voltage clamp technique and gating currents recorded in cell attached macropatches had similar properties and time course, and the charge movement properties directly obtained from capacity measurements agreed with measurements of charge movement from subtracted records. An accurate estimate of the normalized open probability Po(V) was obtained from tail current measurements as a function of the prepulse V in high external K+. The Po(V) was zero at potentials more negative than -40 mV and increased sharply at this potential, then increased continuously until -20 mV, and finally slowly increased with voltages more positive than 0 mV. Deactivation tail currents decayed with two time constants and external potassium slowed down the faster component without affecting the slower component that is probably associated with the return between two of the closed states near the open state. In correlating gating currents and channel opening, Cole-Moore type experiments showed that charge moving in the negative region of voltage (-100 to -40 mV) is involved in the delay of the conductance activation but not in channel opening. The charge moving in the more positive voltage range (-40 to -10 mV) has a similar voltage dependence to the open probability of the channel, but it does not show the gradual increase with voltage seen in the Po(V).     

Sodium currents in the giant axon of the crabCarcinus maenas

Summary Measurements were made of the kinetics and steady-state properties of the sodium conductance changes in the giant axon of the crabCarcinus maenas. The conductance measurements were made in the presence of small concentrations of tetrodotoxin and as much electrical compensation as possible in order to minimize errors caused by the series resistance. After an initial delay of 10–150 sec, the conductance increase during depolarizing voltage clamp pulses followed the Hodgkin-Huxley kinetics. Values of the time constant for the activation of the sodium conductance lay on a bell-shaped curve with a maximum under 180 sec at –40 mV (at 18°C). Values of the time constant for the inactivation of the sodium conductance were also fitted using a bell-shaped curve with a maximum under 7 msec at –70 mV. The effects of membrane potential on the fraction of Na channels available for activation studied using double pulse protocols suggest that hyperpolarizing potentials more negative than –100 mV lock a fraction of the Na channels in a closed conformation.     

Voltage Gating of Shaker K+ Channels : The Effect of Temperature on Ionic and Gating Currents

Ionic (I_i) and gating currents (I_g) from noninactivating Shaker H4 K⁺ channels were recorded with the cut-open oocyte voltage clamp and macropatch techniques. Steady state and kinetic properties were studied in the temperature range 2–22°C. The time course of I_i elicited by large depolarizations consists of an initial delay followed by an exponential rise with two kinetic components. The main I_i component is highly temperature dependent (Q₁₀ > 4) and mildly voltage dependent, having a valence times the fraction of electric field (z) of 0.2–0.3 e_o. The I_g On response obtained between −60 and 20 mV consists of a rising phase followed by a decay with fast and slow kinetic components. The main I_g component of decay is highly temperature dependent (Q₁₀ > 4) and has a z between 1.6 and 2.8 e_o in the voltage range from −60 to −10 mV, and ∼0.45 e_o at more depolarized potentials. After a pulse to 0 mV, a variable recovery period at −50 mV reactivates the gating charge with a high temperature dependence (Q₁₀ > 4). In contrast, the reactivation occurring between −90 and −50 mV has a Q₁₀ = 1.2. Fluctuation analysis of ionic currents reveals that the open probability decreases 20% between 18 and 8°C and the unitary conductance has a low temperature dependence with a Q₁₀ of 1.44. Plots of conductance and gating charge displacement are displaced to the left along the voltage axis when the temperature is decreased. The temperature data suggests that activation consists of a series of early steps with low enthalpic and negative entropic changes, followed by at least one step with high enthalpic and positive entropic changes, leading to final transition to the open state, which has a negative entropic change.     

Gating of the bacterial sodium channel, NaChBac: voltage-dependent charge movement and gating currents

The bacterial sodium channel, NaChBac, from Bacillus halodurans provides an excellent model to study structure-function relationships of voltage-gated ion channels. It can be expressed in mammalian cells for functional studies as well as in bacterial cultures as starting material for protein purification for fine biochemical and biophysical studies. Macroscopic functional properties of NaChBac have been described previously (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001. Science. 294:2372-2375). In this study, we report gating current properties of NaChBac expressed in COS-1 cells. Upon depolarization of the membrane, gating currents appeared as upward inflections preceding the ionic currents. Gating currents were detectable at -90 mV while holding at -150 mV. Charge-voltage (Q-V) curves showed sigmoidal dependence on voltage with gating charge saturating at -10 mV. Charge movement was shifted by -22 mV relative to the conductance-voltage curve, indicating the presence of more than one closed state. Consistent with this was the Cole-Moore shift of 533 micros observed for a change in preconditioning voltage from -160 to -80 mV. The total gating charge was estimated to be 16 elementary charges per channel. Charge immobilization caused by prolonged depolarization was also observed; Q-V curves were shifted by approximately -60 mV to hyperpolarized potentials when cells were held at 0 mV. The kinetic properties of NaChBac were simulated by simultaneous fit of sodium currents at various voltages to a sequential kinetic model. Gating current kinetics predicted from ionic current experiments resembled the experimental data, indicating that gating currents are coupled to activation of NaChBac and confirming the assertion that this channel undergoes several transitions between closed states before channel opening. The results indicate that NaChBac has several closed states with voltage-dependent transitions between them realized by translocation of gating charge that causes activation of the channel.     

Cl− currents of unstimulated T84 intestinal epithelial cells studied by intracellular recording

The ionic currents spontaneously present in T84 intestinal epithelial cells, a line of colonie carcinoma origin, have been studied using the whole-cell recording mode of the patch-clamp technique and the single-electrode voltage-clamp method. Patch-clamp experiments showed that nonstimulated T84 cells already possess large currents but that these tend to disappear during the course of the experiments, presumably through the dialysis of some essential cytoplasmic component against the micropipette solution. The main charge carrier in these experiments appears to be Cl^– as judged from ion replacement. Microelectrode impalement of T84 cells gave a membrane potential of around –30 mV, similar to the equilibrium potential for Cl^– estimated from previously published values for intracellular Cl^– concentration. Voltage-clamp experiments with a single microelectrode revealed three kinetically distinguishable current patterns; currents decaying during hyperpolarizing voltage pulses, currents slowly activating during hyperpolarizing pulses and time-independent currents. The appearance of these distinct kinetic patterns was not predictable from cell to cell, and was not dependent on extracellular Ca²⁺. Ionic replacement experiments suggest that the charge carrier was always Cl^–, regardless of the kinetic pattern observed. No K⁺ currents appear to be present in the nonstimulated T84 cells. Exposure of T84 cells to the muscarinic agonist carbachol induced a shift in the membrane potential towards more negative values, consistent with an activation of a K⁺ conductance. Thus, we suggest that the resting membrane potential in T84 cells is determined by the distribution of Cl^–. This might imply that activation of K⁺ conductance could by itself support secretion by T84 monolayers through tonically active Cl^– channels.G.M.V. and M.A.V. were supported by AFRC (UK) LRG 111 and DGICYT (Spain), respectively. We are grateful to John O'Brien for culturing the cells, to John Dempster (University of Strathclyde, Glasgow, UK) for providing the analysis software, and to Geoff Warhurst (Hope Hospital, Salford, UK) for generously providing the initial batch of T84 cells.     

Gating of Na channels. Inactivation modifiers discriminate among models

Macroscopic Na currents were recorded from N18 neuroblastoma cells by the whole-cell voltage-clamp technique. Inactivation of the Na currents was removed by intracellular application of proteolytic enzymes, trypsin, alpha-chymotrypsin, papain, or ficin, or bath application of N-bromoacetamide. Unlike what has been reported in squid giant axons and frog skeletal muscle fibers, these treatments often increased Na currents at all test pulse potentials. In addition, removal of inactivation gating shifted the midpoint of the peak Na conductance-voltage curve in the negative direction by 26 mV on average and greatly prolonged the rising phase of Na currents for small depolarizations. Polypeptide toxins from Leiurus quinquestriatus scorpion and Goniopora coral, which slow inactivation in adult nerve and muscle cells, also increase the peak Na conductance and shift the peak conductance curve in the negative direction by 7-10 mV in neuroblastoma cells. Control experiments argue against ascribing the shifts to series resistance artifacts or to spontaneous changes of the voltage dependence of Na channel kinetics. The negative shift of the peak conductance curve, the increase of peak Na currents, and the prolongation of the rise at small depolarization after removal of inactivation are consistent with gating kinetic models for neuroblastoma cell Na channels, where inactivation follows nearly irreversible activation with a relatively high, voltage-independent rate constant and Na channels open only once in a depolarization. As the same kind of experiment does not give apparent shifting of activation and prolongation of the rising phase of Na currents in adult axon and muscle membranes, the Na channels of these other membranes probably open more than once in a depolarization.     

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.     

FPL 64176 modification of Ca(V)1.2 L-type calcium channels: dissociation of effects on ionic current and gating current

FPL 64176 (FPL) is a nondihydropyridine compound that dramatically increases macroscopic inward current through L-type calcium channels and slows activation and deactivation. To understand the mechanism by which channel behavior is altered, we compared the effects of the drug on the kinetics and voltage dependence of ionic currents and gating currents. Currents from a homogeneous population of channels were obtained using cloned rabbit Ca(V)1.2 (alpha1C, cardiac L-type) channels stably expressed in baby hamster kidney cells together with beta1a and alpha2delta1 subunits. We found a striking dissociation between effects of FPL on ionic currents, which were modified strongly, and on gating currents, which were not detectably altered. Inward ionic currents were enhanced approximately 5-fold for a voltage step from -90 mV to +10 mV. Kinetics of activation and deactivation were slowed dramatically at most voltages. Curiously, however, at very hyperpolarized voltages (< -250 mV), deactivation was actually faster in FPL than in control. Gating currents were measured using a variety of inorganic ions to block ionic current and also without blockers, by recording gating current at the reversal potential for ionic current (+50 mV). Despite the slowed kinetics of ionic currents, FPL had no discernible effect on the fundamental movements of gating charge that drive channel gating. Instead, FPL somehow affects the coupling of charge movement to opening and closing of the pore. An intriguing possibility is that the drug causes an inactivated state to become conducting without otherwise affecting gating transitions.