Effects of membrane potential on the capacitance of skeletal muscle fibers

A method for measuring muscle fiber capacitance using small test pulses applied with the three-microelectrode voltage clamp is presented. Using this method, three membrane potential-dependent changes in capacitance were observed: (a) Capacitance of polarized fibers increased by 5--15% with depolarization from V less then -100 mV to voltages slightly below the contraction threshold. (b) Capacitance of fibers depolarized to -30 mV by 100 mM Rb solution decreased by roughly 8% with further depolarization to about +50 mV and increased with repolarization, exhibiting a maximum increase of about 10% at -80 to -90 mV. (c) Capacitance of fibers depolarized to -15 mV by 100 mM K solution increased by about 19% with further depolarization to +43 mV and decreased by about 23% with repolarization to -62 mV. Effects a and b are attributed to changes in specific membrane capacitance due to voltage-dependent redistribution of mobile charged groups within surface of T-tubule membranes. Effect c is caused by changes in the T-system space constant lambdaT due to the voltage dependence of K conductance (inward rectification). Analysis of c showed that in 100 mM K solution lambdaT congruent to 30 mum when inward rectification was fully activated by hyperpolarization and that the density of inward rectifier channels is about the same in surface and tubular membranes. Fiber internal resistance was found to be independent of voltage, a necessary condition for the interpretation of the capacitance measurements.     

Properties of sodium pumps in internally perfused barnacle muscle fibers

To study the properties of the Na extrusion mechanism, giant muscle fibers from barnacle (Balanus nubilus) were internally perfused with solutions containing tracer 22Na. In fibers perfused with solutions containing adenosine 5'-triphosphate (ATP) and 30 mM Na, the Na efflux into 10 mM K seawater was approximately 25-30 pmol/cm2.s; 70% of this efflux was blocked by 50-100 microM ouabain, and approximately 30% was blocked by removal of external K. The ouabain-sensitive and K-dependent Na effluxes were abolished by depletion of internal ATP and were sigmoid-shaped functions of the internal Na concentration ([Na]i), with half-maxima at [Na]i approximately or equal to 20 mM. These sigmoid functions fit the Hill equation with Hill coefficients of approximately 3.5. Ouabain depolarized ATP-fueled fibers by 1.5-2 mV ([Na]i greater than or equal to 30 mM) but had very little effect on the membrane potential of ATP-depleted fibers; ATP depletion itself caused a 2-2.5- mV depolarization. When fueled fibers were treated with 3,4- diaminopyridine or Ba2+ (to reduce the K conductance and increase membrane resistance), application of ouabain produced a 4-5 mV depolarization. These results indicate that an electrogenic, ATP- dependent Na-K exchange pump is functional in internally perfused fibers; the internal perfusion technique provides a convenient method for performing transport studies that require good intracellular solute control.     

Effects of sulfhydryl inhibitors on nonlinear membrane currents in frog skeletal muscle fibers

The effect of sulhydryl reagents on nonlinear membrane currents of frog skeletal muscle fibers has been studied using the triple Vaseline gap voltage-clamp technique. These compounds, which are known to interfere with depolarization contraction coupling, also appear to diminish intramembranous charge movement recorded with fibers polarized to -100 mV (charge 1). This effect, however, is accompanied by changes in the fiber membrane conductance and in most cases by the appearance of an inwardly directed current in the potential range between -60 and +20 mV. This current is reduced by both cadmium and nifedipine and does not occur in Ca-free solution, suggesting that it is carried by calcium ions flowing through regular calcium channels that are more easily activated in the presence of SH reagent. These changes in the membrane electrical active and passive properties decrease the quality and reliability of the P/n pulse subtracting procedure normally used for charge movement measurements. These effects can be substantially reduced by cadmium ions (0.1 mM), which has no effect on charge movement. When SH reagents are applied in the presence of cadmium, no effects are observed, indicating that this cation may protect the membrane from the reagent effects. The effects of -SH reagents can be observed by applying them in the absence of cadmium, followed by addition of the cation. Under these conditions the conductance changes are reversed and the effects of the SH reagents on charge movement can be measured with a higher degree of confidence. Maximum charge is reduced by 32% in the presence of 1.5 mM PCMB and by 31% in the presence of 2 mM PHMPS. These effects do not occur in the presence of DTT and in some cases they may be reversed by this agent. Charge 2, recorded in depolarized muscle fibers, is also reduced by these agents.     

K(+)-channel blockers do not decrease acetylcholine depolarizations in canine trachealis.

Using the double sucrose gap, we have examined the role of K+ channels in the cholinergic depolarizations in response to field stimulation and acetylcholine (Ach) in canine trachealis. Acetylcholine-like depolarization per se decreased electrotonic potentials from hyperpolarizing currents. The net effect of acetylcholine (10(-6) M) depolarization on membrane conductance was a small increase after the depolarization was compensated by current clamp. Reversal potentials for acetylcholine depolarization and for the excitatory junction potential (EJP) were determined by extrapolation to be 20-30 mV positive to the resting potential, previously shown to be approximately -55 mV. They were shifted positively by tetraethylammonium ion (TEA) at 20 mM or Ba2+ at 1 mM. TEA or Ba2+ initially depolarized the membrane and increased membrane resistance. Repolarization of the membrane restored any reductions in EJP amplitudes associated with depolarization. After 15 min, the membrane potential partially repolarized, and acetylcholine-induced depolarization and contractions were then increased by TEA. 4-Aminopyridine depolarized the membrane but decreased membrane resistance. Apamin (10(-6) M), charybdotoxin (10(-7) M), and glybenclamide (10(-5) M) each failed to significantly depolarize membranes, increase membrane resistance, or reduce EJP amplitudes or depolarization to 10(-6) M Ach. Glybenclamide reduced depolarizations to added acetylcholine slightly. TEA occasionally reduced the EJP markedly, but this was shown to be most likely a prejunctional effect mediated by norepinephrine release. TEA alone among K(+)-channel blockers slowed the onset and the time courses of the EJP as well as the acetylcholine-induced depolarization. K(+)-channel closure cannot be a complete explanation of acetylcholine-induced membrane effects on this tissue. Acetylcholine must have increased the conductance of an ion with a reversal potential positive to the resting potential in addition to any effect to close K+ channels.     

Electrophysiology of flounder intestinal mucosa. I. Conductance properties of the cellular and paracellular pathways

We evaluated the conductances for ion flow across the cellular and paracellular pathways of flounder intestine using microelectrode techniques and ion-replacement studies. Apical membrane conductance properties are dominated by the presence of Ba-sensitive K channels. An elevated mucosal solution K concentration, [K]m, depolarized the apical membrane potential (psi a) and, at [K]m less than 40 mM, the K dependence of psi a was abolished by 1-2 mM mucosal Ba. The basolateral membrane displayed Cl conductance behavior, as evidenced by depolarization of the basolateral membrane potential (psi b) with reduced serosal Cl concentrations, [Cl]s. psi b was unaffected by changes in [K]s or [Na]s. From the effect of mucosal Ba on transepithelial K selectivity, we estimated that paracellular conductance (Gp) normally accounts for 96% of transepithelial conductance (Gt). The high Gp attenuates the contribution of the cellular pathway to psi t while permitting the apical K and basolateral Cl conductances to influence the electrical potential differences across both membranes. Thus, psi a and psi b (approximately 60 mV, inside negative) lie between the equilibrium potentials for K (76 mV) and Cl (40 mV), thereby establishing driving forces for K secretion across the apical membrane and Cl absorption across the basolateral membrane. Equivalent circuit analysis suggests that apical conductance (Ga approximately equal to 5 mS/cm2) is sufficient to account for the observed rate of K secretion, but that basolateral conductance (Gb approximately equal to 1.5 mS/cm2) would account for only 50% of net Cl absorption. This, together with our failure to detect a basolateral K conductance, suggests that Cl absorption across this barrier involves KCl co-transport.     

Ba2+ ions block K+-induced contractures by antagonizing K+-induced membrane depolarization in frog skeletal muscle fibres

The effects of Ba2+ ions on twitches, K+-induced contractures, and on intracellularly recorded membrane potentials (Em) and depolarizations of frog skeletal muscle fibres were investigated. Exposure of toe muscles to choline--Ringer's solution with 10(-3) M Ba2+ with Ca2+ (1.08 mM) eliminated or very greatly reduced contractures produced by 60 mM K+. In contrast, not only did the same concentration of Ba2+ ions fail to depress the twitch tension of isolated semitendinosus fibres when added to Ringer's with Ca2+, but it even restored twitches that had been eliminated in a zero Ca2+ Ringer's solution. The resting Em of sartorius muscle fibres in choline--Ringer's solution was reduced about 20 mV by 10(-3) M Ba2+. This Ba2+ ion concentration also antagonized the K+-induced depolarization. Thus in the presence of 1 mM Ba2+, 20 mM K+ hyperpolarized rather than depolarized the fibres and 60 or 123 mM K+ produced only very slowly developing, small depolarizations. These results suggest that the loss of the K+-induced contracture in choline-Ringer's caused by Ba2+ ions is due to an inhibition of the K+-induced depolarization. The latter result is consistent with previous findings of other workers that Ba2+ ions block membrane K+ channels.     

K+ secretion across frog skin. Induction by removal of basolateral Cl-

We examined the development of K+ secretion after removing Cl- from the basolateral surface of isolated skins of Rana temporaria using noise analysis. K+ secretion was defined by the appearance of a Lorentzian component in the power density spectrum (PDS) when Ba2+ was present in the apical bath (0.5 mM). No Lorentzians were observed when tissues were bathed in control, NaCl Ringer solution. Replacement of basolateral Cl- by gluconate, nitrate, or SO4- (0-Clb) yielded Lorentzians with corner frequencies near 25 Hz, and plateau values (So) that were used to estimate the magnitude of K+ secretion through channels in the apical cell membranes of the principal cells. The response was reversible and reproducible. In contrast, removing apical Cl- did not alter the PDS. Reduction of basolateral Cl- to 11.5 mM induced Lorentzians, but with lower values of So. Inhibition of Na+ transport with amiloride or by omitting apical Na+ depressed K+ secretion but did not prevent its appearance in response to 0-Clb. Using microelectrodes, we observed depolarization of the intracellular voltage concomitant with increased resistance of the basolateral membrane after 0-Clb. Basolateral application of Ba2+ to depolarize cells also induced K+ secretion. Because apical conductance and channel density are unchanged after 0-Clb, we conclude that K+ secretion is "induced" simply by an increase of the electrical driving force for K+ exit across this membrane. Repolarization of the apical membrane after 0-Clb eliminated K+ secretion, while further depolarization increased the magnitude of the secretory current. The cell depolarization after 0-Clb is most likely caused directly by a decrease of the basolateral membrane K+ conductance. Ba2(+)-induced Lorentzians also were elicited by basolateral hypertonic solutions but with lower values of So, indicating that cell shrinkage per se could not entirely account for the response to 0-Clb and that the effects of 0-Clb may be partly related to a fall of intracellular Cl-.     

Effects of barium and bicarbonate on glial cells of Necturus optic nerve. Studies with microelectrodes and voltage-sensitive dyes

We have studied the effects of Ba++, a known K+ channel blocker, on the electrophysiological properties of the glial cells of Necturus optic nerve. The addition of Ba++ reversibly depolarized glial cells by 25-50 mV; the half maximal deplorization was obtained with a Ba++ concentration of approximately 0.3 mM. In the presence of Ba++, the sensitivity of the membrane to changes in K+ was reduced and there was evidence of competition between K+ and Ba++ for the K+ channel. These effects, which were accompanied by a large increase in the input resistance of the glial cells, indicate that Ba++ blocks the K+ conductance in glial cells of Necturus optic nerve. With the K+ conductance reduced, we were able to investigate the presence of other membrane conductances. We found that in the presence of Ba++, the addition of HCO3- caused a Na+-dependent hyperpolarization that was sensitive to the disulfonic stilbene SITS (4-acetamido-4'-isothiocyanostilbene-2, 2'-disulfonic acid). Removal of Na+ resulted in a HCO3- -dependent, SITS-sensitive depolarization. These results are consistent with the presence in the glial membrane of an electrogenic Na+/HCO3- cotransporter in which Na+, HCO3-, and net negative charge are transported in the same direction. In Cl- -free solutions, the Ba++-induced depolarization increased, suggesting a small permeability to Cl-. Using voltage-sensitive dyes and a photodiode array for multiple site optical recording, the distribution of potential changes in response to square pulses of intracellularly injected current were recorded before and after the addition of increased and the decay of amplitude as a function of distance decreased. Such results indicate that Ba++ increases the membrane resistance more than the resistance of the intercellular junctions.     

A TEA-insensitive flickering potassium channel active around the resting potential in myelinated nerve

A novel potassium-selective channel which is active at membrane potentials between -100 mV and +40 mV has been identified in peripheral myelinated axons of Xenopus laevis using the patch-clamp technique. At negative potentials with 105 mM-K on both sides of the membrane, the channel at 1 kHz resolution showed a series of brief openings and closings interrupted by longer closings, resulting in a flickery bursting activity. Measurements with resolution up to 10 kHz revealed a single-channel conductance of 49 pS with 105 mM-K and 17 pS with 2.5 mM-K on the outer side of the membrane. The channel was selective for K ions over Na ions (PNa/PK = 0.033). The probability of being within a burst in outside-out patches varied from patch to patch (> 0.2, but often > 0.9), and was independent of membrane potential. Open-time histograms were satisfactorily described with a single exponential (tau o = 0.09 msec), closed times with the sum of three exponentials (tau c = 0.13, 5.9, and 36.6 msec). Sensitivity to external tetraethylammonium was comparatively low (IC50 = 19.0 mM). External Cs ions reduced the apparent unitary conductance for inward currents at Em = -90 mV (IC50 = 1.1 mM). Ba and, more potently, Zn ions lowered not only the apparent single-channel conductance but also open probability. The local anesthetic bupivacaine with high potency reduced probability of being within a burst (IC50 = 165 nM). The flickering K channel is clearly different from the other five types of K channels identified so far in the same preparation. We suggest that this channel may form the molecular basis of the resting potential in vertebrate myelinated axons.     

Two types of K+ channels in the apical membrane of rabbit proximal tubule in primary culture

The patch-clamp technique was used to investigate ionic channels in the apical membrane of rabbit proximal tubule cells in primary culture. Cell-attached recordings revealed the presence of a highly selective K+ channel with a conductance of 130 pS. The channel activity was increased with membrane depolarization. Experiments performed on excised patches showed that the channel activity depended on the free Ca2+ concentration on the cytoplasmic face of the membrane and that decreasing the cytoplasmic pH from 7.2 to 6.0 also decreased the channel activity. In symmetrical 140 mM KCl solutions the channel conductance was 200 pS. The channel was blocked by barium, tetraethylammonium and Leiurus quinquestriatus scorpion venom (from which charybdotoxin is extracted) when applied to the extracellular face of the channel. Barium and quinidine also blocked the channel when applied to the cytoplasmic face of the membrane. Another K+ channel with a conductance of 42 pS in symmetrical KCl solutions was also observed in excised patches. The channel was blocked by barium and apamin, but not by tetraethylammonium applied to the extracellular face of the membrane. Using the whole-cell recording configuration we determined a K+ conductance of 4.96 nS per cell that was blocked by 65% when 10 mM tetraethylammonium was applied to the bathing medium.     

Effect of phosphatidylserine on unitary conductance and Ba2+ block of the BK Ca2+-activated K+ channel: re-examination of the surface charge hypothesis

Incorporation of BK Ca2+-activated K+ channels into planar bilayers composed of negatively charged phospholipids such as phosphatidylserine (PS) or phosphatidylinositol (PI) results in a large enhancement of unitary conductance (gch) in comparison to BK channels in bilayers formed from the neutral zwitterionic lipid, phospatidylethanolamine (PE). Enhancement of gch by PS or PI is inversely dependent on KCl concentration, decreasing from 70% at 10 mM KCl to 8% at 1,000 mM KCl. This effect was explained previously by a surface charge hypothesis (Moczydlowski, E., O. Alvarez, C. Vergara, and R. Latorre. 1985. J. Membr. Biol. 83:273-282), which attributed the conductance enhancement to an increase in local K+ concentration near the entryways of the channel. To test this hypothesis, we measured the kinetics of block by external and internal Ba2+, a divalent cation that is expected to respond strongly to changes in surface electrostatics. We observed little or no effect of PS on discrete blocking kinetics by external and internal Ba2+ at 100 mM KCl and only a small enhancement of discrete and fast block by external Ba2+ in PS-containing membranes at 20 mM KCl. Model calculations of effective surface potential sensed by the K+ conduction and Ba2+-blocking reactions using the Gouy-Chapman-Stern theory of lipid surface charge do not lend support to a simple electrostatic mechanism that predicts valence-dependent increase of local cation concentration. The results imply that the conduction pore of the BK channel is electrostatically insulated from the lipid surface, presumably by a lateral distance of separation (>20 A) from the lipid head groups. The lack of effect of PS on apparent association and dissociation rates of Ba2+ suggest that lipid modulation of K+ conductance is preferentially coupled through conformational changes of the selectivity filter region that determine the high K+ flux rate of this channel relative to other cations. We discuss possible mechanisms for the effect of anionic lipids in the context of specific molecular interactions of phospholipids documented for the KcsA bacterial potassium channel and general membrane physical properties proposed to regulate membrane protein conformation via energetics of bilayer stress.     

A calcium-activated cation-selective channel in rat cultured Schwann cells

Calcium-activated channels, in the plasma membrane of rat cultured Schwann cells were studied in isolated 'inside-out' membrane patches. With identical (150 mM NaCl) solutions on either side of the membrane, a single channel conductance of 32 pS was calculated for inward current; the conductance was somewhat less for outward current. The channel is about equally permeable to sodium and potassium ions, but is not detectably permeable to either chloride or calcium. Under our experimental conditions the channel is activated by high (more than 10(-4) M) concentrations of calcium and is sensitive to voltage, channel activity increasing with membrane depolarization.     

Dihydropyridine-sensitive skeletal muscle Ca channels in polarized planar bilayers. 1. Kinetics and voltage dependence of gating.

Rabbit skeletal muscle transverse tubule (T) membranes were fused with planar bilayers. Ca channel activity was studied with a "cellular" approach, using solutions that were closer to physiological than in previous studies, including asymmetric extracellular divalent ions as current carriers. The bilayer was kept polarized at -80 mV and depolarizing pulses were applied under voltage clamp. Upon depolarization the channels opened in a steeply voltage-dependent manner, and closed rapidly at the end of the pulses. The activity was characterized at the single-channel level and on macroscopic ensemble averages of test-minus-control records, using as controls the null sweeps. The open channel events had one predominant current corresponding to a conductance of 9 pS (100 mM Ba2+). The open time histogram was fitted with two exponentials, with time constants of 5.8 and 30 ms (23 degrees C). Both types of events were virtually absent at -80 mV. The average open probability (fractional open time) increased sigmoidally from 0 to a saturation level of 0.08, following a Boltzmann function centered at -25 mV and with a steepness factor of 7 mV. Ensemble averages of test-minus-control currents showed a sigmoidal activation followed by inactivation during the pulse and deactivation (closing) after the pulse. The ON time course was well fitted with "m3h" kinetics, with tau m = 120 ms and tau h = 1.2 s. Deactivation was exponential with tau = 8 ms. This study demonstrates a technique for obtaining Ca channel events in lipid bilayers that are strictly voltage dependent and exhibit most of the features of the macroscopic ICa. The technique provides a useful approach for further characterization of channel properties, as exemplified in the accompanying paper, that describes the consequences on channel properties of phosphorylation by cAMP dependent protein kinase.     

Calcium- and voltage-activated plateau currents of cardiac Purkinje fibers

We have used the two-microelectrode voltage-clamp technique to investigate the components of membrane current that contribute to the formation of the early part of the plateau phase of the action potential of calf cardiac Purkinje fibers. 3,4-Diaminopyridine (50 microM) reduced the net transient outward current elicited by depolarizations to potentials positive to -30 mV but had no consistent effect on contraction. We attribute this effect to the blockade of a voltage-activated transient potassium current component. Ryanodine (1 microM), an inhibitor of sarcoplasmic reticulum calcium release and intracellular calcium oscillations in Purkinje fibers (Sutko, J.L., and J.L. Kenyon. 1983. Journal of General Physiology. 82:385-404), had complex effects on membrane currents as it abolished phasic contractions. At early times during a depolarization (5-30 ms), ryanodine reduced the net outward current. We attribute this effect to the loss of a component of calcium-activated potassium current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. At later times during a depolarization (50-200 ms), ryanodine increased the net outward current. This effect was not seen in low-sodium solutions and we could not observe a reversal potential over a voltage range of -100 to +75 mV. These data suggest that the effect of ryanodine on the late membrane current is attributable to the loss of sodium-calcium exchange current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. Neither effect of ryanodine was dependent on chloride ions, which suggests that chloride ions do not carry the ryanodine-sensitive current components. Strontium (2.7 mM replacing calcium) and caffeine (10 mM), two other treatments that interfere with sarcoplasmic reticulum function, had effects in common with ryanodine. This supports the hypothesis that the effects of ryanodine may be attributed to the inhibition of sarcoplasmic reticulum calcium release.     

Swelling and potassium uptake in cultured astrocytes

The intracellular water content of astrocytes in primary cultures shows a biphasic swelling pattern on exposure to various increased external K+ concentrations over the range of 1.5-100 mM. The two phases (physiological, 1.5-12 mM K+; pathological, 25-100 mM K+) are based on two different mechanisms. Both can be blocked by low Cl- solutions and involve intensive net uptake of K+. However, the physiological phase consists of the activation of a KCl + NaCl carrier, while the Na+ in turn is pumped out by Na+-K+ ATPase, with a resultant net accumulation of KCl. At pathological K+ concentrations the KCl + NaCl carrier is less active because the Na+ driving force, its energy source, is reduced (owing to depolarization by K+). However, the Donnan equilibrium across the cell membrane is heavily disturbed, which leads to passive KCl accumulation. The results suggest that volume changes in cultured glial cells during exposure to high K+ should be taken into consideration since they disguise K+ accumulation when only ion activity is measured.     

The low conductance K(+) channel in human colonic crypt cells has a voltage-dependent permeability not affected by Mg(++)

The low conductance K(+) channel found in human colonocytes was investigated using the patch-clamp technique. The channel is Ca(++)-dependent and is blocked by Ba(++) (5 mM) with a decrease in open probability from 0.42 to 0.19. At -40 mV the slope conductance was 29 pS (using intracellular solution in the pipette). In inside-out patches, inward rectification was seen both with KCl (pipette)/NaCl (bath) solutions as well as KCl/KCl solutions. The rectification could not be affected by omitting Mg(++) from the pipette or the bath solution, neither by exposing the patches to the polyamine spermine (1 mM). Using the Goldman-Hodgkin-Katz equation we show that the permeability decreased in a linear fashion from approximately 5.2 x 10(-14) cm(3)/s to 1.8 x 10(-14) cm(3)/s (-100 to +100 mV), both with and without Mg(++) in the solutions. There was no significant difference in the nominal values of permeability. This property of the K(+) channel may facilitate the hyperpolarization needed to sustain a chloride secretion.     

Newly identified steady-state potassium channels in rat hippocampal neurons.

We report two new types of potassium channels in cultured hippocampal neurons of rat. Both channels occurred in the soma membrane of these cells at very low density. They were active in steady-state conditions, within a wide voltage range that included the resting membrane potential. Their open probability was enhanced by membrane depolarization, but not influenced by Ca ions. In symmetrical 150 mM KCl the channels showed a slope conductance of ca. 40 and 80 pS, respectively. Current-voltage relations of both K channels show a negative slope at high positive voltages.     

Internal citrate ions reduce the membrane potential for contraction threshold in mammalian skeletal muscle fibers.

An effect of internal citrate ions on excitation-contraction coupling in skeletal muscle is described. The threshold for contraction was measured in rat extensor digitorum longus, (EDL), and soleus muscle fibers using a two microelectrode voltage clamp technique with either KCl-filled or K3 citrate-filled current electrodes. Contraction thresholds were stable for many minutes with KCl current electrodes. In contrast, thresholds fell progressively towards the resting membrane potential, by as much as -15 mV over a period of 10 to 20 min of voltage-clamp with citrate current electrodes. In addition, prepulse inhibition was suppressed, subthreshold activation enhanced and steady-state inactivation shifted to more negative potentials. Fibers recovered slowly from these effects when the citrate electrode was withdrawn and replaced with a KCl electrode. The changes in contraction threshold suggest that citrate ions act on the muscle activation system at an intracellular site, since the citrate permeability of the surface membrane is probably very low. An internal citrate concentration of 5 mM was calculated to result from citrate diffusion out of the microelectrode into the recording area for 20 min. 5 mM citrate added to an artificial cell lowered the free calcium concentration from 240 to 31 microM. It is suggested that citrate modifies excitation-contraction coupling either by acting upon an anion-dependent step in activation or by reducing the free calcium and/or free magnesium concentration in the myoplasm.     

Effects of Cu2+-o-phenanthroline on gastric (H+ + K+)-ATPase. Evidence for opening of a closed anion conductance by S-S cross-linkings

Effects of the S-S cross-linking reagent, Cu2+-o-phenanthroline (CuP), on salt conductances of gastric vesicle membranes in which the (H+ + K+)-ATPase is present were studied. CuP caused a dose-dependent increase in the KCl conductance of the vesicle membrane. The increase of the KCl conductance caused by 10 microM CuP was completely prevented by 0.3 mM ATP or 0.3 mM adenyl 5'-yl imidodiphosphate and partially prevented by ADP. The NaCl conductance was also increased by the CuP reaction. However, CuP has no effect on the K2SO4 conductance. Pretreatments of vesicles with 0.1 mM 4-acetoamide-4'-isothiocyanostilbene-2,2'-disulfonate, an anion channel inhibitor, completely blocked the effect of CuP. Thus, these effects of CuP are ascribable to the increase in the anion conductance of the vesicle membrane produced by S-S cross-linking. Furthermore, tyrosine-tyrosine cross-linking with tetranitromethane also increases the anion conductance. Probable roles of the opening of the closed anion channel of the ATPase were discussed in regard to the acid secretory mechanism of gastric mucosa.     

Whole-cell and single channel K+ and Cl- currents in epithelial cells of frog skin.

Whole-cell and single channel currents were studied in cells from frog (R. pipiens and R. catesbiana) skin epithelium, isolated by collagenase and trypsin treatment, and kept in primary cultures up to three days. Whole-cell currents did not exhibit any significant time-dependent kinetics under any ionic conditions used. With an external K gluconate Ringer solution the currents showed slight inward rectification with a reversal potential near zero and an average conductance of 5 nS at reversal. Ionic substitution of the external medium showed that most of the cell conductance was due to K and that very little, if any, Na conductance was present. This confirmed that most cells originate from inner epithelial layers and contain membranes with basolateral properties. At voltages more positive than 20 mV outward currents were larger with K in the medium than with Na or N-methyl-D-glucamine. Such behavior is indicative of a multi-ion transport mechanism. Whole-cell K current was inhibited by external Ba and quinidine. Blockade by Ba was strongly voltage dependent, while that by quinidine was not. In the presence of high external Cl, a component of outward current that was inhibited by the anion channel blocker diphenylamine-2-carboxylate (DPC) appeared in 70% of the cells. This component was strongly outwardly rectifying and reversed at a potential expected for a Cl current. At the single channel level the event most frequently observed in the cell-attached configuration was a K channel with the following characteristics: inward-rectifying I-V relation with a conductance (with 112.5 mM K in the pipette) of 44 pS at the reversal potential, one open and at least two closed states, and open probability that increased with depolarization. Quinidine blocked by binding in the open state and decreasing mean open time. Several observations suggest that this channel is responsible for most of the whole-cell current observed in high external K, and for the K conductance of the basolateral membrane of the intact epithelium. On a few occasions a Cl channel was observed that activated upon excision and brief strong depolarization. The I-V relation exhibited strong outward rectification with a single channel conductance of 48 pS at 0 mV in symmetrical 112 mM Cl solutions. Kinetic analysis showed the presence of two open and at least two closed states. Open time constants and open probability increased markedly with depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)