Calcium modulation of mitochondrial inner membrane channel activity.

Protocols were defined that enable patch-clamp studies of the approximately 107 pS voltage dependent channel and a class of activity we refer to as MCC (multiconductance channel) which is characterized by multiple levels and transitions as high as 1 to 1.5 nS. If free calcium was kept at 10(-7) M or lower during mitochondrial isolation, no activity was observed at low voltage (+/- 60 mV). If free calcium levels were higher, MCC activity was observed in about 96% of the patches. The observation of approximately 107 pS channel was enhanced from 2% to 68% of patches by washing isolated mitoplasts (mitochondria stripped of outer membrane) with EGTA. Increasing matrix calcium from 10(-9) to 10(-5) M decreased the probability of opening for the MCC and approximately 107 pS activities.     

Ca2(+)-sensitive K+ channel in aortic smooth muscle of rats

We measured K+ channel activity in inside-out patches of cell membrane from aortic vascular smooth muscle cultured (Passages 1-3) from Wistar, Wistar-Kyoto, and spontaneously hypertensive rats (SHR). With [Ca2+]i between 25 and 100 nm and 150 mm K+ on both sides of the membrane, the conductance of this channel was 55 +/- 7 pS (slope of current-voltage curve through 0 mV) and the current was outwardly rectified. There was no difference in single-channel conductance among the three rat strains. Increasing negative holding voltages or increasing [Ca2+]i, increased the probability of this type channel being open (Npo; P less than 0.01); SHR had a larger NPo (P less than 0.01). Compared with cells from Wistar and Wistar-Kyoto, cells from SHR also had the longest mean open time. The increased NPo and mean open time we observed in this K+ channel of cells from SHR could contribute, at least in part, to the increased membrane K+ permeability, reported previously.     

Single-channel recordings from two types of amiloride-sensitive epithelial Na+ channels

We report here the first evidence in intact epithelial cells of unit conductance events from amiloride-sensitive Na+ channels. The events were observed when patch-clamp recordings were made from the apical surface of cultured epithelial kidney cells (A6). Two types of channels were observed: one with a high selectivity to Na+ and one with relatively low selectivity. The characteristics of the low-selectivity channel are as follows: single-channel conductance ranged between 7 and 10 pS (mean = 8.4 +/- 1.3), the current-voltage (I-V) relationship displayed little if any nonlinearity over a range of +/- 80 mV (with respect to the patch pipette) and the channel Na+/K+ selectivity was approximately 3-4:1. Amiloride, a cationic blocker of the channel, reduced channel mean open time and increased channel mean closed times as the voltage of the cell interior was made more negative. Amiloride induced channel flickering at increased negative potentials (intracellular potential with respect to the patch) but did not alter the single-channel conductance or the I-V relationship from that observed in control patches. The characteristics of the high-selectivity channel are: a single-channel conductance of 1-3 pS (mean = 2.8 +/- 1.2), the current-voltage relationship is markedly nonlinear with a Na+/K+ selectivity greater than 20:1. The mean open and closed times for the two types of channels are quite different, the high-selectivity channel being open only about 10% of the time while the low-selectivity channel is open about 30% of the time.     

Biophysical characterization of a large conductance anion channel in hypodermal membranes of the gastrointestinal nematode,Ascaris suum

Patch-clamp recordings from muscle- and cuticle-facing hypodermal membranes of the gastrointestinal nematode Ascaris suum reveal a high-conductance, voltage- sensitive Ca(2+) -dependent Cl(-) channel. The hypodermal channel has a conductance of 195 pS in symmetrical 160 mM NaCl. The open probability of the channel is highly voltage-sensitive, and channel activity is not observed when Ca(2+) is reduced to <100 microM. The channel is permeable to organic anions that are major end-products of carbohydrate metabolism in A. suum, including acetate, butyrate and 2-methylvalerate. The conductances and relative permeabilities of these organic anions are inversely related to size, with 2-methylvalerate being only approximately 3% as permeable as Cl(-). The diameter of the channel pore was 12.3+/-0.2 A, calculated from the relative permeability coefficients of Cl(-) and the organic anions. Results of this study are consistent with the hypothesis that the large conductance anion channel in A. suum hypodermal membranes provides a low energy pathway for organic anion excretion from the hypodermal compartment, followed by diffusion across the aqueous channels of the cuticle matrix.     

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line.

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.     

K+-selective channel from sarcoplasmic reticulum of split lobster muscle fibers

The patch clamp technique has been used to study channels in a membrane inside a cell. A single muscle fiber is skinned in relaxing saline (high K+, low Ca2+ with EGTA and ATP), leaving the native sarcoplasmic reticulum (SR) membrane exposed for patching. Fibers are dissected from the second antenna remotor muscles of the American lobster, Homarus americanus. Transmission and scanning electron microscopy confirm the large volume fraction of SR (approximately 70%) and absence of sarcolemma in this unusual skinned preparation. The resting potential of the SR was measured after the resistance of the patch of membrane was broken down. It is near 0 mV (-0.4 +/- 0.6 mV). The average input resistance of the SR is 842 +/- 295 M omega. Some 25% of patches contain a K+-selective channel with a mean open time of seconds and the channel displays at least two conducting states. The open probability is weakly voltage dependent, large at zero and positive potentials (cytoplasm minus SR lumen), and decreasing at negative potentials. The maximal conductance of this channel is 200 +/- 1 pS and the substate conductance is 170 +/- 3 pS in symmetrical 480 mM K+ solution. The current-voltage relation of the open channel is linear over a range of +/- 100 mV. The selectivity is similar to the SR K+ channel of vertebrates: PK/PNa is 3.77 +/- 0.03, determined from reversal potential measurements, whereas gamma K/gamma Na is 3.28 +/- 0.06, determined from open-channel conductance measurements in symmetrical 480 mM solutions. Voltage-dependent block in the lobster SR K+ channel is similar to, but distinct from, that reported for the vertebrate channels. It occurs asymmetrically when hexamethonium is added to both sides of the membrane. The block is more effective from the cytoplasmic side of the channel.     

Batrachotoxin-modified sodium channels from squid optic nerve in planar bilayers. Ion conduction and gating properties

Squid optic nerve sodium channels were characterized in planar bilayers in the presence of batrachotoxin (BTX). The channel exhibits a conductance of 20 pS in symmetrical 200 mM NaCl and behaves as a sodium electrode. The single-channel conductance saturates with increasing the concentration of sodium and the channel conductance vs. sodium concentration relation is well described by a simple rectangular hyperbola. The apparent dissociation constant of the channel for sodium is 11 mM and the maximal conductance is 23 pS. The selectivity determined from reversal potentials obtained in mixed ionic conditions is Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+. Calcium blocks the channel in a voltage-dependent manner. Analysis of single-channel membranes showed that the probability of being open (Po) vs. voltage relation is sigmoidal with a value of 0.5 between -90 and -100 mV. The fitting of Po requires at least two closed and one open state. The apparent gating charge required to move through the whole transmembrane voltage during the closed-open transition is four to five electronic charges per channel. Distribution of open and closed times are well described by single exponentials in most of the voltage range tested and mean open and mean closed times are voltage dependent. The number of charges associated with channel closing is 1.6 electronic charges per channel. Tetrodotoxin blocked the BTX-modified channel being the blockade favored by negative voltages. The apparent dissociation constant at zero potential is 16 nM. We concluded that sodium channels from the squid optic nerve are similar to other BTX-modified channels reconstituted in bilayers and to the BTX-modified sodium channel detected in the squid giant axon.     

Basolateral outward rectifier chloride channel in isolated crypts of mouse colon

Single channel patch-clamp techniques were used to demonstrate the presence of outwardly rectifying chloride channels in the basolateral membrane of crypt cells from mouse distal colon. These channels were rarely observed in the cell-attached mode and, in the inside-out configuration, only became active after a delay and depolarizing voltage steps. Single channel conductance was 23.4 pS between -100 and -40 mV and increased to 90.2 pS between 40 and 100 mV. The channel permeability sequence for anions was: I(-) > SCN(-) > Br(-) > Cl(-) > NO(3)(-) > F(-)> SO(4)(2-) approximately gluconate. In inside-out patches, the channel open probability was voltage dependent but insensitive to intracellular Ca(2+) concentration. In cell-attached mode, forskolin, histamine, carbachol, A-23187, and activators of protein kinase C all failed to activate the channel, and activity could not be evoked in inside-out patches by exposure to the purified catalytic subunit of cAMP-dependent protein kinase A. The channel was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, 9-anthracenecarboxylic acid, and DIDS. Stimulation of G proteins with guanosine 5'-O-(3-thiotriphosphate) decreased the channel open probability and conductance, whereas subsequent addition of guanosine 5'-O-(2-thiodiphosphate) reactivated the channel.     

Multiple conductance levels in rat heart inner mitochondrial membranes studied by patch clamping.

The behavior of the mitochondrial inner membrane multiple conductance channel (MCC) which has a peak conductance of 1-1.5 nS has been examined in rat heart mitochondria. MCC can display several unique characteristics: (a) prolonged open and closed times on the order of seconds to minutes, (b) a voltage dependence in which MCC opens (negative potential) or closes (positive potential) generally in steps, (c) a response to inhibitors such as amiodarone in steps corresponding at least approximately to those in (b), (d) a 'free-running mode' in which the current level rapidly fluctuates between a minimum of nine conductance levels but with a preferred occupation of the 0.5-0.7 nS levels, and (e) very large transitions (1-1.5 nS) resolved at 4 kHz bandwidth as single events with variable mean open time.     

Ca2+ currents in cerebral artery smooth muscle cells of rat at physiological Ca2+ concentrations

Single Ca2+ channel and whole cell currents were measured in smooth muscle cells dissociated from resistance-sized (100-microns diameter) rat cerebral arteries. We sought to quantify the magnitude of Ca2+ channel currents and activity under the putative physiological conditions of these cells: 2 mM [Ca2+]o, steady depolarizations to potentials between -50 and -20 mV, and (where possible) without extrinsic channel agonists. Single Ca2+ channel conductance was measured over a broad range of Ca2+ concentrations (0.5-80 mM). The saturating conductance ranged from 1.5 pS at 0.5 mM to 7.8 pS at 80 mM, with a value of 3.5 pS at 2 mM Ca (unitary currents of 0.18 pA at -40 mV). Both single channel and whole cell Ca2+ currents were measured during pulses and at steady holding potentials. Ca2+ channel open probability and the lower limit for the total number of channels per cell were estimated by dividing the whole-cell Ca2+ currents by the single channel current. We estimate that an average cell has at least 5,000 functional channels with open probabilities of 3.4 x 10(-4) and 2 x 10(-3) at -40 and -20 mV, respectively. An average of 1-10 (-40 mV and -20 mV, respectively) Ca2+ channels are thus open at physiological potentials, carrying approximately 0.5 pA steady Ca2+ current at -30 mV. We also observed a very slow reduction in open probability during steady test potentials when compared with peak pulse responses. This 4- 10-fold reduction in activity could not be accounted for by the channel's normal inactivation at our recording potentials between -50 and -20 mV, implying that an additional slow inactivation process may be important in regulating Ca2+ channel activity during steady depolarization.     

Pharmacological and biophysical properties of swelling-activated chloride channel in mouse cardiac myocytes

The present study was designed to observe the properties of swelling-activated chloride channel (ICl.swell) in mouse cardiac myocytes using patch clamp techniques. In whole-cell recordings, hypotonic solution activated a chloride current that exhibited outward rectification, weak voltage-dependent inactivation, and anion selectivity with permeability sequence of I- > Br- > Cl-. The current was sensitive to Cl- channel blockers tamoxifen, NPPB and DIDS. In single-channel recordings, cell swelling activated a single channel current which showed outward rectification with open probability of 0.76 +/- 0.08 and conductance of 38.1 +/- 2.5 pS at +100 mV under [Cl-] symmetrical condition. I-V relation revealed the reversal potential as expected for a Cl(-)-selective channel. These results suggested that in mouse cardiac myocytes, swelling-activated, outward rectifying chloride channel with a single channel conductance of 38.1 +/- 2.5 pS (at +100 mV under [Cl-] symmetrical condition) underlies the volume regulatory Cl- channel.     

Modulation of single cardiac sodium channels by DPI 201-106

Single sodium channel currents were analysed in cell attached patches from single ventricular cells of guinea pig hearts in the presence of a novel cardiotonic compound DPI 201-106. The mean single channel conductance of DPI-treated Na channels was not changed by DPI (20.8 +/- 4 pS, control, 3 patches; 21.3 +/- 1 pS with DPI, 5 mumol/1,3 patches). DPI voltage-dependently prolongs the cardiac sodium channel openings by removal of inactivation at potentials positive to -40 mV. At potentials negative to -40 mV a clustering of short openings at the very beginning of the depolarizing voltage steps can be observed causing a transient time course of the averaged currents. Long openings induced an extremely slow inactivation. Short openings, long openings and nulls appeared in groups referring to a modal gating behaviour of DPI-treated sodium channels. DPI-modified Na channels showed a monotonously prolonged mean open time with increased depolarizing voltage steps, e.g. the open state probability within a sweep was increased. However, the number of non-empty sweeps was decreased with the magnitude of the depolarizing steps, e.g. the probability of the channel being open as calculated from the averaged currents was voltage-dependently decreased by DPI (50% decrease at -50.7 +/- 9 9 mV, 3 patches). Short and long openings of DPI-modified channels could be separated by variation of the holding potential. The occurrence of long Na channel openings was much more suppressed by reducing the holding potential (half maximum inactivation at -112 +/- 8 mV, 4 patches) than that of short openings (half maximum inactivation at -88 +/- 8 mV, 4 patches). Otherwise, short living openings completely disappeared at potentials positive to -40 mV where the occurrence of long openings was favoured. The differential voltage dependence of blocking and activating effects of DPI on cardiac Na channels as well as the differential voltage dependence of the appearance of short and long openings refers to a modal gating behaviour of cardiac Na channels.     

A mechanosensitive K+ channel in heart cells. Activation by arachidonic acid

Mechanosensitive ion channels have been described in many types of cells. These channels are believed to transduce pressure signals into intracellular biochemical and physiological events. In this study, the patch-clamp technique was used to identify and characterize a mechanosensitive ion channel in rat atrial cells. In cell-attached patches, negative pressure in the pipette activated an ion channel in a pressure-dependent manner. The pressure to induce half-maximal activation was 12 +/- 3 mmHg at +40 mV, and nearly full activation was observed at approximately 20 mmHg. The probability of opening was voltage dependent, with greater channel activity at depolarized potentials. The mechanosensitive channel was identical to the K+ channel previously shown to be activated by arachidonic acid and other lipophilic compounds, as judged by the outwardly rectifying current-voltage relation, single channel amplitude, mean open time (1.4 +/- 0.3 ms), bursty openings, K+ selectivity, insensitivity to any known organic inhibitors of ion channels, and pH sensitivity. In symmetrical 140 mM KCl, the slope conductance was 94 +/- 11 pS at +60 mV and 64 +/- 8 pS at -60 mV. Anions and cations such as Cl-, glutamate, Na+, Cs+, Li+, Ca2+, and Ba2+ were not permeant. Extracellular Ba2+ (1 mM) blocked the inward K+ current completely. GdCl3 (100 microM) or CaCl2 (100 microM) did not alter the K+ channel activity or amplitude. Lowering of intracellular pH increased the pressure sensitivity of the channel. The K+ channel could be activated in the presence of 5 mM intracellular [ATP] or 10 microM glybenclamide in inside-out patches. In the absence of ATP, when the ATP-sensitive K+ channel was active, the mechanosensitive channel could further be activated by pressure, suggesting that they were two separate channels. The ATP-sensitive K+ channel was not mechanosensitive. Pressure activated the K+ channel in the presence of albumin, a fatty acid binding protein, suggesting that pressure and arachidonic acid activate the K+ channel via separate pathways.     

Two novel cardiac atrial K+ channels, IK.AA and IK.PC

Two K(+)-selective channels in neonatal rat atrial cells activated by lipophilic compounds have been characterized in detail. The arachidonic acid-stimulated channel (IK.AA) had a slope conductance of 124 +/- 17 pS at +30 mV in symmetrical 140 mM potassium and a mean open time of approximately 1 ms, and was relatively voltage independent. IK.AA activity was reversibly increased by lowering pH to 6.0. Arachidonic acid was most effective in activating this channel, although a number of lipophilic compounds resulted in activation. Surprisingly, choline, a polar molecule, also activated the channel. A second K+ channel was activated by 10 microM phosphatidylcholine applied to the intracellular surface of inside-out atrial patches. This channel (IK.PC) had a slope conductance of 60 +/- 6 pS at +40 mV and a mean open time of approximately 0.6 ms, and was also relatively voltage independent. Fatty acids are probably monomeric in the membrane under the conditions of our recording; thus detergent effects are unlikely. Since a number of compounds including fatty acids and prostaglandins activated these two channels, an indirect, channel-specific mechanism may account for activation of these two cardiac K+ channels.     

Single channel properties of P2X2 purinoceptors

The single channel properties of cloned P2X2 purinoceptors expressed in human embryonic kidney (HEK) 293 cells and Xenopus oocytes were studied in outside-out patches. The mean single channel current-voltage relationship exhibited inward rectification in symmetric solutions with a chord conductance of approximately 30 pS at -100 mV in 145 mM NaCl. The channel open state exhibited fast flickering with significant power beyond 10 kHz. Conformational changes, not ionic blockade, appeared responsible for the flickering. The equilibrium constant of Na+ binding in the pore was approximately 150 mM at 0 mV and voltage dependent. The binding site appeared to be approximately 0.2 of the electrical distance from the extracellular surface. The mean channel current and the excess noise had the selectivity: K+ > Rb+ > Cs+ > Na+ > Li+. ATP increased the probability of being open (Po) to a maximum of 0.6 with an EC50 of 11.2 microM and a Hill coefficient of 2.3. Lowering extracellular pH enhanced the apparent affinity of the channel for ATP with a pKa of approximately 7.9, but did not cause a proton block of the open channel. High pH slowed the rise time to steps of ATP without affecting the fall time. The mean single channel amplitude was independent of pH, but the excess noise increased with decreasing pH. Kinetic analysis showed that ATP shortened the mean closed time but did not affect the mean open time. Maximum likelihood kinetic fitting of idealized single channel currents at different ATP concentrations produced a model with four sequential closed states (three binding steps) branching to two open states that converged on a final closed state. The ATP association rates increased with the sequential binding of ATP showing that the binding sites are not independent, but positively cooperative. Partially liganded channels do not appear to open. The predicted Po vs. ATP concentration closely matches the single channel current dose-response curve.     

Evidence for KCNQ1 K+ channel expression in rat zymogen granule membranes and involvement in cholecystokinin-induced pancreatic acinar secretion

Secretion of enzymes and fluid induced by Ca(2+) in pancreatic acini is not completely understood and may involve activation of ion conductive pathways in zymogen granule (ZG) membranes. We hypothesized that a chromanol 293B-sensitive K(+) conductance carried by a KCNQ1 protein is expressed in ZG membranes (ZGM). In suspensions of rat pancreatic ZG, ion flux was determined by ionophore-induced osmotic lysis of ZG suspended in isotonic salts. The KCNQ1 blocker 293B selectively blocked K(+) permeability (IC(50) of approximately 10 microM). After incorporation of ZGM into planar bilayer membranes, cation channels were detected in 645/150 mM potassium gluconate cis/trans solutions. Channels had linear current-voltage relationships, a reversal potential (E(rev)) of -20.9 +/- 0.9 mV, and a single-channel K(+) conductance (g(K)) of 265.8 +/- 44.0 pS (n = 39). Replacement of cis 500 mM K(+) by 500 mM Na(+) shifted E(rev) to -2.4 +/- 3.6 mV (n = 3), indicating K(+) selectivity. Single-channel analysis identified several K(+) channel groups with distinct channel behaviors. K(+) channels with a g(K) of 651.8 +/- 88.0 pS, E(rev) of -22.9 +/- 2.2 mV, and open probability (P(open)) of 0.43 +/- 0.06 at 0 mV (n = 6) and channels with a g(K) of 155.0 +/- 11.4 pS, E(rev) of -18.3 +/- 1.8 mV, and P(open) of 0.80 +/- 0.03 at 0 mV (n = 3) were inhibited by 100 microM 293B or by the more selective inhibitor HMR-1556 but not by the maxi-Ca(2+)-activated K(+) channel (BK channel) inhibitor charybdotoxin (5 nM). KCNQ1 protein was demonstrated by immunoperoxidase labeling of pancreatic tissue, immunogold labeling of ZG, and immunoblotting of ZGM. 293B also inhibited cholecystokinin-induced amylase secretion of permeabilized acini (IC(50) of approximately 10 microM). Thus KCNQ1 may account for ZG K(+) conductance and contribute to pancreatic hormone-stimulated enzyme and fluid secretion.     

Stretch-sensitive switching among different channel sublevels of an endothelial cation channel

A mechanosensitive Ca(2+)-permeable cation channel was recorded by patch clamp in isolated rat aortic endothelial cells. A low level of channel activity could be observed after seal formation. The channel displayed some inward rectification and had a conductance for inward current of approx. 32 pS in Ca(2+)-free pipette and bath solutions. Negative suction of -10 to -20 mmHg increased the probability of the channel being open. When the negative pressure in the pipette was raised to -35 to -45 mmHg, the channel underwent an abrupt transition to a large conductance substate that was interrupted occasionally by two other low conductance levels. Under this condition, the overwhelming majority of openings and closings were between a main level of 83 pS and the closed level. Compared to the 32 pS substate, the 83 pS large conductance substate had shorter mean open and closed times. The two channel substates had similar ionic selectivity and both were sensitive to the inhibition of cGMP and protein kinase G. This is the first demonstration showing that mechanostress can change the single channel conductance level of an ion channel in eukaryotic cells.     

GABA increases both the conductance and mean open time of recombinant GABAA channels co-expressed with GABARAP

The single channel properties of recombinant gamma-aminobutyric acid type A (GABA(A))alphabetagamma receptors co-expressed with the trafficking protein GABARAP were investigated using membrane patches in the outside-out patch clamp configuration from transiently transfected L929 cells. In control cells expressing alphabetagamma receptors alone, GABA activated single channels whose main conductance was 30 picosiemens (pS) with a subconductance state of 20 pS, and increasing the GABA concentration did not alter their conductance. In contrast, when GABA(A) receptors were co-expressed with GABARAP, the GABA-activated single channels displayed multiple, high conductances (> or =40 pS), and GABA (> or =10 microM) was able to increase their conductance, up to a maximum of 60 pS. The mean open time of GABA-activated channels in control cells expressing alphabetagamma receptors alone was 2.3 +/- 0.1 ms for the main 30-pS channel and shorter for the subconductance state (20 pS, 0.8 +/- 0.1 ms). Similar values were measured for the 30- and 20-pS channels active in patches from cells co-expressing GABARAP. However higher conductance channels (> or =40 pS) remained open longer, irrespective of whether GABA or GABA plus diazepam activated them. Plotting mean open times against mean conductances revealed a linear relationship between these two parameters. Since high GABA concentrations increase both the maximum single channel conductance and mean open time of GABA(A) channels co-expressed with GABARAP, trafficking processes must influence ion channel properties. This suggests that the organization of extrasynaptic GABA(A) receptors may provide a range of distinct inhibitory currents in the brain and, further, provide differential drug responses.     

Properties of a potassium channel in the basolateral membrane of renal proximal convoluted tubule and the effect of cyclosporine on it

We studied the potassium channel in the basolateral membrane of the rat proximal convoluted tubule as affected by cyclosporine A. Proximal convoluted tubules were dissected from the rat kidney under a stereoscopic microscope, without a preliminary enzyme treatment. The standard configuration for single-channel tight seal patch-clamp technique was used to record channel currents. A small conductance, stretch-sensitive potassium channel could be observed spontaneously in most of the cell-attached patches as the gigaohm seal was formed. In the inside-out configuration, channel activity was diminished. The K(+) channel appeared to be an inward rectifier. The limiting inward slope conductance was 28.3+/-1.7 pS (Vp was between 40 mV and 80 mV, n=6) and the outward chord conductance was 5.6+/-0.3 pS (Vp was between -40 and -60 mV, n=5). The open dwell time constants of the potassium channel were 0.524 ms and 5.087 ms, while the closed dwell time constants were 1.029 ms and 16.500 ms. The opening probability of the channel decreased when the extracellular fluid was acidified. Cyclosporine A had no significant effect on the potassium channel of the proximal tubular cell in the basolateral membrane at concentrations of 10 and 50 microg/ml, while at 100 microg/ml, it decreased the opening probability.     

Heterogeneity of calcium channels from a purified dihydropyridine receptor preparation.

Dihydropyridine receptors were purified from rabbit skeletal muscle transverse tubule membranes and incorporated into planar lipid bilayers. Calcium channels from both the purified dihydropyridine receptor preparation and the intact transverse tubule membranes exhibited two sizes of unitary currents, corresponding to conductances of 7 +/- 1 pS and 16 +/- 3 pS in 80 mM BaCl2. Both conductance levels were selective for divalent cations over monovalent cations and anions. Cadmium, an inorganic calcium channel blocker, reduced the single channel conductance of calcium channels from the purified preparation. The organic calcium channel antagonist nifedipine reduced the probability of a single channel being open with little effect on the single channel conductance. The presence of two conductance levels in both the intact transverse tubule membranes and the purified dihydropyridine receptor preparation suggests that the calcium channel may have multiple conductance levels or that multiple types of calcium channels with closely related structures are present in transverse tubule membranes.