Fast activation of cardiac Ca++ channel gating charge by the dihydropyridine agonist, BAY K 8644.

Nonlinear charge movement (gating current) was studied by the whole-cell patch clamp method using cultured 17-d-old embryonic chick heart cells. Na+ and Ca++ currents were blocked by the addition of 10 microM TTX and 3 mM CoCl2; Cs+ replaced K+ both intra- and extracellularly. Linear capacitive and leakage currents were subtracted by a P/5 procedure. The small size (15 microns in diameter) and the lack of an organized internal membrane system in these myocytes permits a rapid voltage clamp of the surface membrane. Ca++ channel gating currents were activated positive to -60 mV; the rising phase was not distorted due to the system response time. The addition of BAY K 8644 (10(-6) M) caused a shortening of the time to peak of the Ca++ gating current, and a negative shift in the isochronal Qon vs. Vm curve. Qmax was unchanged by BAY K 8644. The voltage-dependent shift produced by BAY K 8644 is similar to that produced by isoproterenol (Josephson, I.R., and N. Sperelakis. 1990. Biophys. J. 57:305a. [Abstr.]). The results suggest that the binding of BAY K 8466 to one or more of the Ca++ channel subunits alters the kinetics and shifts the voltage dependence of gating. These changes in the gating currents can explain the parallel changes in the macroscopic Ca++ currents.     

Simple shifts in the voltage dependence of sodium channel gating caused by divalent cations

The effect of elevated divalent cation concentration on the kinetics of sodium ionic and gating currents was studied in voltage-clamped frog skeletal muscle fibers. Raising the Ca concentration from 2 to 40 mM resulted in nearly identical 30-mV shifts in the time courses of activation, inactivation, tail current decay, and ON and OFF gating currents, and in the steady state levels of inactivation, charge immobilization, and charge vs. voltage. Adding 38 mM Mg to the 2 mM Ca bathing a fiber produced a smaller shift of approximately 20 mV in gating current kinetics and the charge vs. voltage relationship. The results with both Ca and Mg are consistent with the hypothesis that elevated concentrations of these alkali earth cations alter Na channel gating by changing the membrane surface potential. The different shifts produced by Ca and Mg are consistent with the hypothesis that the two ions bind to fixed membrane surface charges with different affinities, in addition to possible screening.     

A study of the "outward potassium channel" (OPC) in the frog oocyte. II. A study of the "inside-out" configuration

The single K-channel current reported in a previous note was also studied in "outside-out" conditions. The electrode filling solutions used for the "cell-attached" experiments faced in this case the intracellular side of the membrane patches, the extracellular side facing the bath saline, i.e. Ringer standard. The most significant observations were obtained with filling solutions with varying proportions in K/Na concentrations solutions. In the absence of Na+ ([K+] = 110 mM), the elementary conductance was still around 90 pS and the I/V diagram was again somewhat bell shaped, though the distinctive reduction of the elementary conductance began at more positive potentials (+110 mV). No inward current could be detected upon membrane repolarization also in this case. The rectification became less evident and conductance increased with increasing Na+ concentration in the filling solution, until the I/V curve became a linear one and conductance was 270 pS with standard Ringer. Distinct inward elementary currents were evident upon repolarization in these conditions. Thus a complex interaction between Na+ and K+ takes place for conduction through the outward K channel in the frog oocyte, both cations probably competing for at least one active site inside. Another interesting observation concerns the process of gating of the OPC: the open times of the elementary currents were in fact much greater in outside out experiments as compared to cell-attached experiments, probably due to the presence of Ca++ in contact with the inner membrane side. Even increasing Na+ concentration prolonged the open time duration. The gating of the OPC in the membrane was not only voltage dependent, but also Ca++ and Na+ dependent.     

Anion and cation permeability of a chloride channel in rat hippocampal neurons

The ionic permeability of a voltage-dependent Cl channel of rat hippocampal neurons was studied with the patch-clamp method. The unitary conductance of this channel was approximately 30 pS in symmetrical 150 mM NaCl saline. Reversal potentials interpreted in terms of the Goldman-Hodgkin-Katz voltage equation indicate a Cl:Na permeability ratio of approximately 5:1 for conditions where there is a salt gradient. Many anions are permeant; permeability generally follows a lyotropic sequence. Permeant cations include Li, Na, K, and Cs. The unitary conductance does not saturate for NaCl concentrations up to 1 M. No Na current is observed when the anion Cl is replaced by the impermeant anion SO4. Unitary conductance depends on the cation species present. The channel is reversibly blocked by extracellular Zn or 9-anthracene carboxylic acid. Physiological concentrations of Ca or Mg do not affect the Na:Cl permeability ratio. The permeability properties of the channel are consistent with a permeation mechanism that involves an activated complex of an anionic site, an extrinsic cation, and an extrinsic anion.     

Characterization of ion channels on the surface membrane of adult rat skeletal muscle.

The channels present on the surface membrane of isolated rat flexor digitorum brevis muscle fibers were surveyed using the patch clamp technique. 85 out of 139 fibers had a novel channel which excluded the anions chloride, sulfate, and isethionate with a permeability ratio of chloride to sodium of less than 0.05. The selectivity sequence for cations was Na+ = K+ = Cs+ greater than Ca++ = Mg++ greater than N-Methyl-D-Glucamine. The channel remained closed for long periods, and had a large conductance of approximately 320 pS with several subconductance states at approximately 34 pS levels. Channel activity was not voltage dependent and the reversal potential for cations in muscle fibers of approximately 0 mV results in the channel's behaving as a physiological leakage conductance. Voltage activated potassium channels were present in 65 of the cell attached patches and had conductances of mostly 6, 12, and 25 pS. The voltage sensitivity of the potassium channels was consistent with that of the delayed rectifier current. Only three patches contained chloride channels. The scarcity of chloride channels despite the known high chloride conductance of skeletal muscle suggests that most of the chloride channels must be located in the transverse tubular system.     

Chromatin aggregation depends on the anion species of the salts

The effects of anions on chromatin aggregation may be classified into three categories. First, monovalent anions, glutamate, acetate, chloride, and thiocyante, follow the lyotropic series in their effects on both H1 histone displacement and chromatin aggregation. Second, alkyl carboxylates and dicarboxylates differ in their ability to induce chromatin aggregation depending on charge density, suggesting possible interference by bulky alkyl chains with neutralization (screening) of closely spaced positive protein charges. Third, the multivalent anions, citrate3- and SO4(2-), bind tightly to histone and disrupt nucleosomes and thus interfere with chromatin aggregation. Substantial differences in chromatin aggregation were observed with different species of anions. At salt concentrations of 0-500 mN and pH 7.0, as much as 70% of the chromatin could be induced to aggregate by monosodium glutamate and sodium acetate, whereas only 10% or less was precipitated by NaSCN, Na2SO4, and Na3citrate. The physiological anion composition of the nucleus is not known; however, the anion effects discussed in the present work suggest a potential for regulation of chromatin condensation in higher eukaryotes.     

Aconitine modification of cell membrane sodium channels of a neuroblastoma

Currents through normal and aconitine-modified sodium channels in the perfused neuroblastoma cell are measured under voltage clamp conditions. Aconitine shifts the voltage range of activation of the sodium channels towards more negative potentials by about 20 mV, and changes the selectivity, so that channels become more permeable to NH4+ than to Na+ ions. The currents through aconitine--modified channels are inactivated almost completely like those through the normal ones. Aconitine is effective when applied to both sides of the cell membrane. Steady-state characteristics of gating are discussed in terms of the model assuming three main states of the gate machinery: closed, open and inactivated.     

Phosphorylation shifts the time-dependence of cardiac Ca++ channel gating currents.

A general mechanism for the physiological regulation of the activity of voltage-dependent Na+, Ca++, K+, and Cl channels by neurotransmitters in a variety of excitable cell types may involve a final common pathway of a cyclic AMP-dependent phosphorylation of the channel protein. The functional correlates of channel phosphorylation are known to involve a change in the probability of opening, and a negative or positive shift in the voltage dependence for activation of the conductance. The voltage dependence for activation appears to be governed by the properties of the charge movement of the voltage-sensing moiety of the channel. This study of the gating charge movement of cardiac Ca++ channels has revealed that isoproterenol or cAMP (via a presumed phosphorylation of the channel) speeds the kinetics of the Ca++ channel gating charge movement. These results suggest that the changes in the kinetics and voltage dependence of the cardiac calcium currents produced by beta-adrenergic stimulation are initiated, in part, by parallel changes in the gating charge movement.     

Voltage-dependent membrane capacitance in rat pituitary nerve terminals due to gating currents

We investigated the voltage dependence of membrane capacitance of pituitary nerve terminals in the whole-terminal patch-clamp configuration using a lock-in amplifier. Under conditions where secretion was abolished and voltage-gated channels were blocked or completely inactivated, changes in membrane potential still produced capacitance changes. In terminals with significant sodium currents, the membrane capacitance showed a bell-shaped dependence on membrane potential with a peak at approximately -40 mV as expected for sodium channel gating currents. The voltage-dependent part of the capacitance showed a strong correlation with the amplitude of voltage-gated Na+ currents and was markedly reduced by dibucaine, which blocks sodium channel current and gating charge movement. The frequency dependence of the voltage-dependent capacitance was consistent with sodium channel kinetics. This is the first demonstration of sodium channel gating currents in single pituitary nerve terminals. The gating currents lead to a voltage- and frequency-dependent capacitance, which can be well resolved by measurements with a lock-in amplifier. The properties of the gating currents are in excellent agreement with the properties of ionic Na+ currents of pituitary nerve terminals.     

Influence of anions and cations on the dipole potential of phosphatidylcholine vesicles: a basis for the Hofmeister effect

Anions and cations have long been recognized to be capable of modifying the functioning of various membrane-related physiological processes. Here, a fluorescent ratio method using the styrylpyridinium dyes, RH421 and di-8-ANEPPS, was applied to determine the effect of a range of anions and cations on the intramembrane dipole potential of dimyristoylphosphatidylcholine vesicles. It was found that certain anions cause a decrease in the dipole potential. This could be explained by binding within the membrane, in support of a hypothesis originally put forward by A. L. Hodgkin and P. Horowicz [1960, J. Physiol. (Lond.) 153:404-412.] The effectiveness of the anions in reducing the dipole potential was found to be ClO4- > SCN- > I- > NO3- > Br- > Cl- > F- > SO42-. This order could be modeled by a partitioning of ions between the membrane and the aqueous phase, which is controlled predominantly by the Gibbs free energy of hydration. Cations were also found to be capable of reducing the dipole potential, although much less efficiently than can anions. The effects of the cations was found to be trivalent > divalent > monovalent. The cation effects were attributed to binding to a specific polar site on the surface of the membrane. The results presented provide a molecular basis for the interpretation of the Hofmeister effect of lyotropic anions on ion transport proteins.     

Depolarizing response of rat parathyroid cells to divalent cations

Membrane potentials were recorded from rat parathyroid glands continuously perfused in vitro. At 1.5 mM external Ca++, the resting potential averages -73 +/- 5 mV (mean +/- SD, n = 66). On exposure to 2.5 mM Ca++, the cells depolarize reversibly to a potential of -34 +/- 8 mV (mean +/- SD). Depolarization to this value is complete in approximately 2-4 min, and repolarization on return to 1.5 mM Ca++ takes about the same time. The depolarizing action of high Ca++ is mimicked by all divalent cations tested, with the following order of effectiveness: Ca++ greater than Sr++ greater than Mg++ greater than Ba++ for alkali-earth metals, and Ca++ greater than Cd++ greater than Mn++ greater than Co++ greater than Zn++ for transition metals. Input resistance in 1.5 mM Ca++ was 24.35 +/- 14 M omega (mean +/- SD) and increased by an average factor of 2.43 +/- 0.8 after switching to 2.5 mM Ca++. The low value of input resistance suggests that cells are coupled by low-resistance junctions. The resting potential in low Ca++ is quite insensitive to removal of external Na+ or Cl-, but very sensitive to changes in external K+. Cells depolarize by 61 mV for a 10- fold increase in external K+. In high Ca++, membrane potential is less sensitive to an increase in external K+ and is unchanged by increasing K+ from 5 to 25 mM. Depolarization evoked by high Ca++ may be slowed, but is unchanged in amplitude by removal of external Na+ or Cl-. Organic (D600) and inorganic (Co++, Cd++, and Mn++) blockers of the Ca++ channels do not interfere with the electrical response to Ca++ changes. Our results show remarkable parallels to previous observations on the control of parathormone (PTH) release by Ca++. They suggest an association between membrane voltage and secretion that is very unusual: parathyroid cells secrete when fully polarized, and secrete less when depolarized. The extraordinary sensitivity of parathyroid cells to divalent cations leads us to hypothesize the existence in their membranes of a divalent cation receptor that controls membrane permeability (possibly to K+) and PTH secretion.     

Selectivity characteristics of cation channels in Nitella flexilis plasmalemma

Ionic selectivity of Nitella flexilis plasmalemma cation channels is studied by voltage-clamp method with consecutive replacing of cations in the bathing medium. The selectivity sequence received by measuring the ionic current reversal potentials, psi alpha is: Ba++ approximately equal to Sr++ approximately equal to Ca++ greater than Mg++ greater than Cs+ approximately equal to K+ greater than Na+ greater than Li+. An analysis of results based on the three-barrier channel model suggests that when ions of the same valency are compared, the channel selectivity is determined by specific interactions between the ion and the nearest water molecules, which is possible both in a narrow and wide pore. On the other hand, when monovalent and divalent ions are compared the effects of ions binding in the channel or near the membrane surface prevail, thus causing the channel preference for divalent cations.     

Hypoxia and persistent sodium current

During prolonged depolarization of excitable cells, some voltage-activated, tetrodotoxin-sensitive sodium channels are resistant to inactivation and can continue to open for long periods of time, generating a "persistent" sodium current ( I(NaP)). The amplitude of I(NaP) is small [generally less than 1% of the peak amplitude of the transient sodium current ( I(NaT))], activates at potentials close to the resting membrane potential, and is more sensitive to Na channel blocking drugs than I(NaT). It is thought that persistent Na channels are generated by a change in gating of transient Na channels, possibly because of a change in phosphorylation or protein structure, e.g. loss of the inactivation gate. Drugs that block Na channels can prevent the increase in [Ca(2+)](i) in cardiac cells during hypoxia. Hypoxia increases the amplitude of I(NaP). Paradoxically, NO causes a similar increase in I(NaP) and the effects of both can be inhibited by reducing agents such as dithiothreitol and reduced glutathione. It is proposed that an increased inflow of Na(+) during hypoxia increases [Na(+)](i), which in turn reverses the Na/Ca exchanger so that [Ca(2+)](i) rises. An increase in I(NaP) and [Ca(2+)](i) could cause arrhythmias and irreversible cell damage.     

Interaction of internal anions with potassium channels of the squid giant axon

The interaction of internal anions with the delayed rectifier potassium channel was studied in perfused squid axons. Changing the internal potassium salt from K+ glutamate- to KF produced a reversible decline of outward K currents and a marked slowing of the activation of K channels at all voltages. Fluoride ions exert a differential effect upon K channel gating kinetics whereby activation of IK during depolarizing steps is slowed dramatically, but the rate of closing after the step is not much altered. These effects develop with a slow time course (30-60 min) and are specific for K channels over Na channels. Both the amplitude and activation rate of IK were restored within seconds upon return to internal glutamate solutions. The fluoride effect is independent of the external K+ concentration and test membrane potential, and does not recover with repetitive application of depolarizing voltage steps. Of 11 different anions tested, all inorganic species induced similar decreases and slowing of IK, while K currents were maintained during extended perfusion with several organic anions. Anions do not alter the reversal potential or shape of the instantaneous current-voltage relation of open K channels. The effect of prolonged exposure to internal fluoride could be partially reversed by the addition of cationic K channel blocking agents such as TEA+, 4-AP+, and Cs+. The competitive antagonism between inorganic anions and internal cationic K channel blockers suggests that they may interact at a related site(s). These results indicate that inorganic anions modify part of the K channel gating mechanism (activation) at a locus near the inner channel surface.     

Calcium and voltage dependent inactivation of sodium and calcium currents limits calcium influx in Helisoma neurons

The control of free intracellular calcium concentration ([Ca2+]i) is necessary for cell survival because of the ubiquitous and essential role this second messenger plays in regulating numerous intracellular processes. Calcium regulation in neurons is especially vigorous because of the large calcium influx that occurs through voltage-gated channels during membrane depolarization. In this study we examined changes in ionic currents that can limit calcium influx into neurons during electrical activity. We found that the [Ca2+]i in electrically stimulated Helisoma B4 neurons initially increased to a peak and then relaxed to lower concentrations in tandem with a decline in the action potential peak voltage. The decline in [Ca2+]i and the peak action potential voltage in this sodium and calcium driven neuron was found to be a dual manifestation of I(Na) and I(Ca) inactivation. I(Na) and I(Ca) both displayed voltage dependent inactivation. Additionally, I(Na) and I(Ca) progressively inactivated at [Ca2+]i above 200 nM, concentrations readily attained in electrically stimulated B4 neurons. Calcium and voltage dependent I(Na) and I(Ca) inactivation were found to reduce calcium influx during continuous electrical stimulation by decreasing both the magnitude of I(Ca) that could be activated and the percent of the available I(Ca) that would be activated due to the diminished peak action potential voltage. Calculations based on data herein suggest that the voltage and calcium dependent I(Na) and I(Ca) inactivation that occurs during continuous electrical stimulation dramatically reduces calcium influx in this sodium and calcium driven neuron and thus limits the increase in [Ca2+]i.     

Modification of sodium channel gating by lanthanum. Some effects that cannot be explained by surface charge theory

In clonal pituitary (GH3) cells we studied the changes in sodium channel gating caused by substitution of La3+ for Ca2+ ion. Gating of sodium channels was simplified by using intracellular papain to remove inactivation. To quantify La effects, we empirically fitted closing and the late phase of opening of the channels with single exponentials, determined the opening (a) and closing (b) rate, and plotted these rates as a function of Vm (membrane voltage). The midpoint of the fraction open-Vm curve was also determined. Changing from Ca to La shifted the curves for these three measures of Na channel gating along the voltage axis and changed their shape somewhat. Surface charge theory, in the form usually presented, predicts equal shifts of all three curves, with no change in shape. We found, however, that the shift for each of the measurements was different. 2 mM La, for example, shifted opening kinetics by +52 mV (i.e., 52 mV must be added to the depolarization to make activation in 2 mM La as fast as in 2 mM Ca), the fraction open voltage curve by +42.5 mV, and the closing rate curve by +28 mV. The shift was an almost linear function of log [La] for each of the measures. The main finding is that changing from 2 mM Ca to 10 microM La causes a positive shift of the opening rate and fraction open curves, but a negative shift of the closing rate curve. The opposite signs of the two effects cannot be explained in terms of surface charge theory. We briefly discuss some alternatives to this theory.     

Calcium activates and inactivates a photoreceptor soma potassium current.

Light-induced currents were measured with a two-microelectrode voltage clamp of type B photoreceptor somata, which had been isolated by axotomy from all synaptic interactions as well as from all membranes capable of generating impulse activity. In artificial seawater (ASW), light elicited a transient early inward current, INa+, which depended on Na+o and had a linear current-voltage relation and an extrapolated reversal potential of 30-40 mV (absolute). In 0-Na+ ASW, light elicited a transient short-latency outward current that dependent on K+o, increased exponentially with more positive voltages (greater than or equal to -40 mV), and reversed at -70 to -75 mV. This outward current was not blocked by Ca++ channel blockers (e.g., Cd++, Co++) or substitution of Ba++o, for Ca++o, but was reduced by iontophoretic injection of EGTA. In both ASW and 0-Na+ ASW, light also elicited a delayed, apparently inward current, which was associated with a decreased conductance, depended on K+o, increased exponentially with more positive voltages (greater than or equal to -40 mV), reversed at the equilibrium potential for K+ flux in elevated K+o was eliminated by substitution of Ba++o for Ca++o, and was greatly reduced by Cd++o or Co++o. Thus, light elicited an early Ca++-dependent K+ current, IC, and a prolonged decrease of IC. Iontophoretic injection of Ca++ through a third microelectrode caused prolonged reduction of both IC and the light-induced decrease of IC, but did not alter ICa++ or the current-voltage relation of IC. Ruthenium red (1 microM) in the external medium caused a prolongation of the light-induced decrease of IC. Iontophoretic injection of EGTA often eliminated the light-induced IC decrease while decreasing peak IC (during depolarizing steps to -5 or 0 mV) by less than one-half. EGTA injection, on the average, did not affect steady state IC but reduced the light-induced decrease of steady state IC to approximately one-third of its original magnitude. The prolonged IC decrease, elicited by dim light in the absence of light-induced IC or INa+, was more completely eliminated by EGTA injection. It was concluded that light, in addition to inducing a transient inward Na+ current, causes both a transient increase and a prolonged decrease of IC via elevation of Ca++i.     

The permeability of the lysosomal membrane to small ions.

The permeability of the lysosomal membrane to small anions and cations was studied at 37 degrees C and pH 7.0 in a lysosomal-mitochondrial fraction isolated from the liver of untreated rats. The extent of osmotic lysis following ion influx was used as a measure of ion permeancy. In order to preserve electroneutrality, anion influx was coupled to an influx of K+ in the presence of valinomycin, and cation influx was coupled to an efflux of H+ using the protonophore 3-tert-butyl-5,2'-dichloro-4'-nitrosalicilylanilide. Lysosomal lysis was monitored by observing the loss of latency of two lysosomal hydrolases. The order of permeability of the lysosomal membrane to anions was found to be SCN- greater than I- greater than CH3COO- greater than Cl- approximately Pi greater than SO24- and that to cations Cs+ greater than K+ greater than Na+ greater than H+. These orders are largely in agreement with the lyotropic series of anions and cations. The implications of these findings for the mechanism by means of which a low intralysosomal pH is produced and maintained are discussed.     

Asymmetric electrostatic effects on the gating of rat brain sodium channels in planar lipid membranes.

The effects of ionic strength (10-1,000 mM) on the gating of batrachotoxin-activated rat brain sodium channels were studied in neutral and in negatively charged lipid bilayers. In neutral bilayers, increasing the ionic strength of the extracellular solution, shifted the voltage dependence of the open probability (gating curve) of the sodium channel to more positive membrane potentials. On the other hand, increasing the intracellular ionic strength shifted the gating curve to more negative membrane potentials. Ionic strength shifted the voltage dependence of both opening and closing rate constants of the channel in analogous ways to its effects on gating curves. The voltage sensitivities of the rate constants were not affected by ionic strength. The effects of ionic strength on the gating of sodium channels reconstituted in negatively charged bilayers were qualitatively the same as in neutral bilayers. However, important quantitative differences were noticed: in low ionic strength conditions (10-150 mM), the presence of negative charges on the membrane surface induced an extra voltage shift on the gating curve of sodium channels in relation to neutral bilayers. It is concluded that: (a) asymmetric negative surface charge densities in the extracellular (1e-/533A2) and intracellular (1e-/1,231A2) sides of the sodium channel could explain the voltage shifts caused by ionic strength on the gating curve of the channel in neutral bilayers. These surface charges create negative electric fields in both the extracellular and intracellular sides of the channel. Said electric fields interfere with gating charge movements that occur during the opening and closing of sodium channels; (b) the voltage shifts caused by ionic strength on the gating curve of sodium channels can be accounted by voltage shifts in both the opening and closing rate constants; (c) net negative surface charges on the channel's molecule do not affect the intrinsic gating properties of sodium channels but are essential in determining the relative position of the channel's gating curve; (d) provided the ionic strength is below 150 mM, the gating machinery of the sodium channel molecule is able to sense the electric field created by surface changes on the lipid membrane. I propose that during the opening and closing of sodium channels, the gating charges involved in this process are asymmetrically displaced in relation to the plane of the bilayer. Simple electrostatic calculations suggest that gating charge movements are influenced by membrane electrostatic potentials at distances of 48 and 28 A away from the plane of the membrane in the extracellular sides of the channel, respectively.