ATP inhibition and rectification of a Ca2+-activated anion channel in sarcoplasmic reticulum of skeletal muscle.

We describe ATP-dependent inhibition of the 75-105-pS (in 250 mM Cl-) anion channel (SCl) from the sarcoplasmic reticulum (SR) of rabbit skeletal muscle. In addition to activation by Ca2+ and voltage, inhibition by ATP provides a further mechanism for regulating SCl channel activity in vivo. Inhibition by the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate (AMP-PNP) ruled out a phosphorylation mechanism. Cytoplasmic ATP (approximately 1 mM) inhibited only when Cl- flowed from cytoplasm to lumen, regardless of membrane voltage. Flux in the opposite direction was not inhibited by 9 mM ATP. Thus ATP causes true, current rectification in SCl channels. Inhibition by cytoplasmic ATP was also voltage dependent, having a K(I) of 0.4-1 mM at -40 mV (Hill coefficient approximately 2), which increased at more negative potentials. Luminal ATP inhibited with a K(I) of approximately 2 mM at +40 mV, and showed no block at negative voltages. Hidden Markov model analysis revealed that ATP inhibition 1) reduced mean open times without altering the maximum channel amplitude, 2) was mediated by a novel, single, voltage-independent closed state (approximately 1 ms), and 3) was much less potent on lower conductance substates than the higher conductance states. Therefore, the SCl channel is unlikely to pass Cl- from cytoplasm to SR lumen in vivo, and balance electrogenic Ca2+ uptake as previously suggested. Possible roles for the SCl channel in the transport of other anions are discussed.     

Properties of single- and double-barreled Cl channels of shark rectal gland in planar bilayers

Chloride channels from the apical plasma membrane fraction of rectal gland of Squalus acanthias were characterized by incorporation into planar bilayers in the presence of cAMP-PK/ATP. In a total of 80 bilayer preparations, 21 Cl-selective channels were observed as single channels and 13 as pairs. This was a significantly greater number of double Cl channels than expected from a binomial distribution. The double Cl channels were divided into two groups based on kinetic and voltage-dependent behavior. One group had properties identical to the single channels (gb1) while the other was consistent with a double-barreled channel (gb2) with coordinated activity between proto-channels. The single-channel slope conductances of gb1 and gb2 from -60 to +20 mV with a 250/70 mM KCl gradient were 41 and 75 pS, respectively. With symmetrical 250 mM KCl, the I-V relation of gb1 showed outward rectification with 47.8 +/- 6.6 pS at cis negative potentials and 68.9 +/- 6.1 pS at cis positive potentials. gb1 was open from 70 to 95% at all electrochemical potentials from -80 to +40 mV. gb2 was steeply voltage dependent between -80 and -20 mV. Both gb1 and gb2 were insensitive to Ca (from 100 nm to 1 microM), blocked by 0.1 mM DIDS and highly selective for chloride. These data suggest that double-barreled Cl channels are related to the family of small, outwardly rectifying Cl channels of epithelial membranes.     

A chloride channel from lobster walking leg nerves. Characterization of single-channel properties in planar bilayers

A novel, small conductance of Cl- channel was characterized by incorporation into planar bilayers from a plasma membrane preparation of lobster walking leg nerves. Under conditions of symmetrical 100 mM NaCl, 10 mM Tris-HCl, pH 7.4, single Cl- channels exhibit rectifying current-voltage (I-V) behavior with a conductance of 19.2 +/- 0.8 pS at positive voltages and 15.1 +/- 1.6 pS in the voltage range of -40 to 0 mV. The channel exhibits a negligible permeability for Na+ compared with Cl- and displays the following sequence of anion permeability relative to Cl- as measured under near bi-ionic conditions: I- (2.7) greater than NO3- (1.8) greater than Br- (1.5) greater than Cl- (1.0) greater than CH3CO2- (0.18) greater than HCO3- (0.10) greater than gluconate (0.06) greater than F- (0.05). The unitary conductance saturates with increasing Cl- concentration in a Michaelis-Menten fashion with a Km of 100 mM and gamma max = 33 pS at positive voltage. The I-V curve is similar in 10 mM Tris or 10 mM HEPES buffer, but substitution of 100 mM NaCl with 100 mM tetraethylammonium chloride on the cis side results in increased rectification with a 40% reduction in current at negative voltages. The gating of the channel is weakly voltage dependent with an open-state probability of 0.23 at -75 mV and 0.64 at +75 mV. Channel gating is sensitive to cis pH with an increased opening probability observed for a pH change of 7.4 to 11 and nearly complete inhibition for a pH change of 7.4 to 6.0. The lobster Cl- channel is reversibly blocked by the anion transport inhibitors, SITS (4-acetamido, 4'-isothiocyanostilbene-2,2'-disulfonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid). Many of these characteristics are similar to those previously described for small conductance Cl- channels in various vertebrate cells, including epithelia. These functional comparisons suggest that this invertebrate Cl- channel is an evolutionary prototype of a widely distributed class of small conductance anion channels.     

Stilbene derivatives inhibit the activity of the inner mitochondrial membrane chloride channels

Ion channels selective for chloride ions are present in all biological membranes, where they regulate the cell volume or membrane potential. Various chloride channels from mitochondrial membranes have been described in recent years. The aim of our study was to characterize the effect of stilbene derivatives on single-chloride channel activity in the inner mitochondrial membrane. The measurements were performed after the reconstitution into a planar lipid bilayer of the inner mitochondrial membranes from rat skeletal muscle (SMM), rat brain (BM) and heart (HM) mitochondria. After incorporation in a symmetric 450/450 mM KCl solution (cis/trans), the chloride channels were recorded with a mean conductance of 155 ± 5 pS (rat skeletal muscle) and 120 ± 16 pS (rat brain). The conductances of the chloride channels from the rat heart mitochondria in 250/50 mM KCl (cis/trans) gradient solutions were within the 70–130 pS range. The chloride channels were inhibited by these two stilbene derivatives: 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS). The skeletal muscle mitochondrial chloride channel was blocked after the addition of 1 mM DIDS or SITS, whereas the brain mitochondrial channel was blocked by 300 μM DIDS or SITS. The chloride channel from the rat heart mitochondria was inhibited by 50–100 μM DIDS. The inhibitory effect of DIDS was irreversible. Our results confirm the presence of chloride channels sensitive to stilbene derivatives in the inner mitochondrial membrane from rat skeletal muscle, brain and heart cells.     

Single channel characteristics of a high conductance anion channel in "sarcoballs"

Previously undescribed high conductance single anion channels from frog skeletal muscle sarcoplasmic reticulum (SR) were studied in native membrane using the "sarcoball" technique (Stein and Palade, 1988). Excised inside-out patches recorded in symmetrical 200 mM TrisCl show the conductance of the channel''s predominant state was 505 +/- 25 pS (n = 35). From reversal potentials, the Pcl/PK ratio was 45. The slope conductance vs. Cl- ion concentration curve saturates at 617 pS, with K0.5 estimated at 77 mM. The steady-state open probability (Po) vs. holding potential relationship produces a bell-shaped curve, with Po values reaching a maximum near 1.0 at 0 mV, and falling off to 0.05 at +/- 25 mV. Kinetic analysis of the voltage dependence reveals that while open time constants are decreased somewhat by increases in potential, the largest effect is an increase in long closed times. Despite the channel''s high conductance, it maintains a moderate selectivity for smaller anions, but will not pass larger anions such as gluconate, as determined by reversal-potential shifts. At least two substates different from the main open level are distinguishable. These properties are unlike those described for mitochondrial voltage- dependent anion channels or skeletal muscle surface membrane Cl channels and since SR Ca channels are present in equally high density in sarcoball patches, we propose these sarcoball anion channels originate from the SR. Preliminary experiments recording currents from frog SR anion channels fused into liposomes indicate that either biochemical isolation and/or alterations in lipid environment greatly decrease the channel''s voltage sensitivity. These results help underline the potential significance of using sarcoballs to study SR channels. The steep voltage sensitivity of the sarcoball anion channel suggests that it could be more actively involved in the regulation of Ca2+ transport by the SR.     

Single chloride-selective channels active at resting membrane potentials in cultured rat skeletal muscle.

The patch-clamp technique was used to characterize channels that could contribute to the resting Cl-conductance in the surface membrane of cultured rat skeletal muscle. Two Cl- -selective channels, in addition to the Cl- -selective channel of large conductance described previously (Blatz and Magleby, 1983), were observed. One of these channels had fast kinetics and a conductance of 45 +/- 1.8 pS (SE) in symmetrical 100 mM KCl. The other had slow kinetics and a conductance of 61 +/- 2.4 pS. The channel with fast kinetics typically closed within 1 ms after opening and flickered between the open and shut states. The channel with slow kinetics typically closed within 10 ms after opening and displayed less flickering. Both channels were active in excised patches of membrane held at potentials similar to resting membrane potentials in intact cells, and both were open a greater percentage of time with depolarization. Under conditions of high ion concentrations, both channels exhibited nonideal selectivity for Cl- over K+ with the permeability ratio PK/PCl of 0.15-0.2. Additional experiments on the fast Cl- channel indicated that its activity decreased with lowered pHi and that SO2-4 and CH3SO-4 were ineffective charge carriers. These findings, plus the observation that the fast Cl- channel was also active in membrane patches on intact cells, suggest that the fast Cl- channel provides a molecular basis for at least some of the resting Cl- conductance. The extent to which the slow Cl- channel contributes is less clear as it was typically active only after excised patches of membrane had been exposed to high concentrations of KCl at the inner membrane surface.     

Reconstitution of ionic channels from inner and outer membranes of mammalian cardiac nuclei.

Recent reports suggest that the nuclear envelope possesses specific ion transport mechanisms that regulate the electrolyte concentrations within the nucleoplasm and perinuclear space. In this work, intact nuclei were isolated from sheep cardiac cells. After chromatin digestion, the nuclear envelopes were sonicated and four nuclear vesicle populations were separated by sucrose step gradients (SF1-SF4). These fractions were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their protein content was analyzed by Western blot, using lamin and SEC 61 antibodies. The lamins, which are associated with the inner nuclear membrane, were present in three fractions, SF2, SF3, and SF4, with a lower amount in SF2. The SEC 61 protein, a marker of the rough endoplasmic reticulum, was detected in small amounts in SF1 and SF2. Upon fusion of vesicles into bilayers, the activities of nuclear ionic channels were recorded in 50 mM trans/250 mM cis KCl or CsCl, pH 7.2. Two types of Cl- selective channels were recorded: a large conducting 150-180-pS channel displaying substates, and a low conducting channel of 30 pS. They were both spontaneously active into bilayers, and their open probability was poorly voltage dependent at negative voltages. Retinoic acid (10(-8) M) increases the po of the large Cl- conducting channel, whereas ATP modifies the kinetics of the low conductance anion selective channel. Our data also suggest that this anionic channel is mainly present in the SF3 and SF4 population. The presence of a 181 +/- 10 pS cation-selective channel was consistently observed in the SF2 population. The behavior of this channel was voltage dependent in the voltage range -80 to +60 mV. Furthermore, we report for the first time the activity of a channel exclusively present in the SF3 and SF4 fractions, shown to contain mainly inner membrane vesicles. This cation selective channel displays a 75-pS conductance in 50 mM trans/250 mM cis K-gluconate. It is concluded that the bilayer reconstitution technique is an attractive approach to studying the electrophysiological properties of the inner and outer membranes of the nuclear envelope.     

Single-channel calcium and barium currents of large and small conductance from sarcoplasmic reticulum.

Two types of divalent cation conducting channels from rabbit skeletal muscle sarcoplasmic reticulum (SR) were incorporated into planar lipid bilayers. A high conductance (100 pS in 53 mM trans Ca2+) Ca2+ channel was incorporated from heavy density SR fractions. The 100-pS channel was activated by adenine nucleotides and Ca2+ and inhibited by Mg2+ and ruthenium red. A 10-pS calcium and barium conducting channel could be incorporated into planar lipid bilayers from light, intermediate, and heavy density SR vesicles. 10-pS channel activity in bilayers was not dependent on cis Ca2+ and was only weakly dependent on adenine nucleotides. Ruthenium red at concentrations up to 1 mM had no effect and Mg2+ was only marginally effective in inhibiting macroscopic Ba2+ currents from this channel. Calcium releasing activity in intermediate and heavy density SR fractions was assayed according to a rapid quench protocol and compared with the results obtained in the bilayer. Results from this comparison indicate that the 10-pS channel is probably not involved in rapid Ca2+- and adenine nucleotide-induced Ca2+ release from isolated SR vesicles.     

Reconstitution of the ATP-sensitive potassium channel of skeletal muscle. Activation by a G protein-dependent process

Potassium channels inhibited by adenosine-5'-trisphosphate, K(ATP), found in the transverse tubular membrane of rabbit skeletal muscle were studied using the planar bilayer recording technique. In addition to the single-channel properties of K(ATP) we report its regulation of Mg2+ and by the guanosine-5'-trisphosphate analogue, GTP-y(gamma)-S. The K(ATP) channel (a) has a conductance of 67 pS in 250 mM internal, 50 mM external KCl, and rectifies weakly at holding potentials more positive than 50 mV, (b) is not activated by internal Ca2+ or membrane depolarization, (c) has a permeability ratio PK/PNa greater than 50, and (d) is inhibited by millimolar internal ATP. Activity of K(ATP), measured as open channel probability as a function of time, was unstable at all holding potentials and decreases continuously within a few minutes after a recording is initiated. After a decrease in activity, GTP-y-S (100 microM) added to the internal side reactivated K(ATP) channels but only transiently. In the presence of internal 1 mM Mg2+, GTP-y-S produced a sustained reactivation lasting 20-45 min. Incubation of purified t-tubule vesicles with AlF4 increased the activity of K(ATP) channels, mimicking the effect of GTP-y-S. The effect of AlF4 and the requirement of GTP-y-S plus Mg2+ for sustained channel activation suggests that a nucleotide-binding G protein regulates ATP-sensitive K channels in the t-tuble membrane of rabbit skeletal muscle.     

Voltage-activated ionic channels and conductances in embryonic chick osteoblast cultures

Patch-clamp measurements were made on osteoblast-like cells isolated from embryonic chick calvaria. Cell-attached-patch measurements revealed two types of high conductance (100-250 pS) channels, which rapidly activated upon 50-100 mV depolarization. One type showed sustained and the other transient activation over a 10-sec period of depolarization. The single-channel conductances of these channel types were about 100 or 250 pS, depending on whether the pipettes were filled with a low K+ (3 mM) or high K+ (143 mM) saline, respectively. The different reversal potentials under these conditions were consistent with at least K+ conduction. Whole-cell measurements revealed the existence of two types of outward rectifying conductances. The first type conducts K+ ions and activates within 20-200 msec (depending on the stimulus) upon depolarizing voltage steps from less than -60 mV to greater than -30 mV. It inactivates almost completely with a time constant of 2-3 sec. Recovery from inactivation is biphasic with an initial rapid phase (1-2 sec) followed by a slow phase (greater than 20 sec). The second whole-cell conductance activates at positive membrane potentials of greater than +50 mV. It also rapidly turns on upon depolarizing voltage steps. Activation may partly disappear at the higher voltages. Its single channels of 140 pS conductance were identified in the whole cell and did conduct K+ ions but were not highly Cl- or Na+ selective. The results show that osteoblasts may express various types of voltage controlled ionic channels. We predict a role for such channels in mineral metabolism of bone tissue and its control by osteoblasts.     

Chloride-dependent sarcoplasmic reticulum Ca2+ release correlates with increased Ca2+ activation of ryanodine receptors.

The mechanism by which chloride increases sarcoplasmic reticulum (SR) Ca2+ permeability was investigated. In the presence of 3 microM Ca2+, Ca2+ release from 45Ca(2+)-loaded SR vesicles prepared from procine skeletal muscle was increased approximately 4-fold when the media contained 150 mM chloride versus 150 mM propionate, whereas in the presence of 30 nM Ca2+, Ca2+ release was similar in the chloride- and the propionate-containing media. Ca(2+)-activated [3H]ryanodine binding to skeletal muscle SR was also increased (2- to 10-fold) in media in which propionate or other organic anions were replaced with chloride; however, chloride had little or no effect on cardiac muscle SR 45Ca2+ release or [3H]ryanodine binding. Ca(2+)-activated [3H]ryanodine binding was increased approximately 4.5-fold after reconstitution of skeletal muscle RYR protein into liposomes, and [3H]ryanodine binding to reconstituted RYR protein was similar in chloride- and propionate-containing media, suggesting that the sensitivity of the RYR protein to changes in the anionic composition of the media may be diminished upon reconstitution. Together, our results demonstrate a close correlation between chloride-dependent increases in SR Ca2+ permeability and increased Ca2+ activation of skeletal muscle RYR channels. We postulate that media containing supraphysiological concentrations of chloride or other inorganic anions may enhance skeletal muscle RYR activity by favoring a conformational state of the channel that exhibits increased activation by Ca2+ in comparison to the Ca2+ activation exhibited by this channel in native membranes in the presence of physiological chloride (< or = 10 mM). Transitions to this putative Ca(2+)-activatable state may thus provide a mechanism for controlling the activation of RYR channels in skeletal muscle.     

Inositol (1,4,5)-trisphosphate activates a calcium channel in isolated sarcoplasmic reticulum membranes.

Sarcoplasmic reticulum membrane vesicles isolated from frog skeletal muscle display high conductance calcium channels when fused into phospholipid bilayers. The channels are selective for calcium and barium over Tris. The fractional open time was voltage-independent (-40 to +25 mV), but was steeply dependent on the free cis [Ca2+] (P0 = 0.02 at 10 microM cis Ca2+ and 0.77 at 150 microM Ca2+; estimated Hill coefficient: 1.6). Addition of ATP (1 mM; cis) further increased P0 from 0.77 to 0.94. Calcium activation was reversed by addition of EGTA to the cis compartment. Magnesium (2 mM) increased the frequency of rapid closures and 8 mM magnesium decreased the current amplitude from 3.4 to 1.2 pA at 0 mV, suggesting a reversible fast blockade. Addition of increasing concentrations of inositol (1, 4, 5)-triphosphate (cis), increased P0 from 0.10 +/- 0.01 (mean +/- SEM) in the control to 0.85 +/- 0.02 at 50 microM in an approximately sigmoidal fashion, with an apparent half-maximal activation at 15 microM inositol (1, 4, 5)-trisphosphate in the presence of 40 microM cis Ca2+. Lower concentrations of this agonist were required to produce a significant increase in P0 when 10 microM or less cis Ca2+ were used. The channel was blocked by the addition to the cis compartment of either 0.5 mM lanthanum, 0.5 microM ruthenium red, or 200 nM ryanodine, all known inhibitors of Ca2+ release from sarcoplasmic reticulum vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Ion channels on synaptic vesicle membranes studied by planar lipid bilayer method.

An anion selective channel and three types of cation selective channels were found in planar lipid bilayers incorporating synaptic vesicles from rat brains. In asymmetric KCl solutions (cis: 300 mM/trans: 150 mM), the anion selective channel showed a single-channel conductance of 94 pS and was inactivated by negative voltages and by 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid disodium salt (SITS). In the same solution, single-channel conductances of three types of cation selective channels were 250 pS (Type 1), 248 pS (Type 2), and 213 pS (Type 3), respectively. These channels resembled one another in single-channel conductances but were different in gating behaviors. Type 1 channel, which was most frequently observed, had a remarkable subconducting state (175 pS). Type 2 channel had a flickering state that increased as the potential became more positive, and a long inactive state that increased as the potentials were more negative. Type 3 channel, which was also sensitive to the potentials, had the open-channel probability increased as the potential became more positive.     

Quinine inhibits chloride and nonselective cation channels in isolated rat distal colon cells.

Isolated cells from rat distal colon were investigated with the patch-clamp technique. In cell-attached and cell-excised patches (inside-out) single chloride channels with outward-rectifying properties were observed. In excised patches the single-channel conductance g was 47 +/- 5 pS at positive and 22 +/- 2 pS at negative clamp potentials (n = 6). The Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10 microM) induced fast closing events, whereas 10 microM of 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) had no effect when applied to the cytosolic side. Quinine in the bath inhibited the Cl- channel by reducing its single-channel amplitude and increased open channel noise. With 0.1 mM the current amplitude decreased by 54% and with 1 mM quinine by 67%. Ca2(+)-dependent nonselective cation channels where observed after excision of the membrane patch. This channel was completely and reversibly inhibited by 100 microM DCDPC. Application of 1 mM quinine to the bath induced flickering and reduced the open-state probability from 0.94 to 0.44. In summary, besides its well established effects on K+ channels, quinine also inhibits nonselective cation channels and chloride channels by inducing fast closing events.     

CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is regulated by phosphorylation of the R domain and ATP hydrolysis at two nucleotide-binding domains (NBDs). It is controversial whether CFTR conducts ATP or whether CFTR might be closely associated with a separate ATP conductance. To characterize ATP channels associated with CFTR, we analyzed Cl- and ATP single channel-currents in excised inside-out membrane patches from MDCK epithelial cells transiently expressing CFTR. With 100 mM ATP in the pipette and 140 mM Cl- in the bath, ATP channels were associated with CFTR Cl- channels in two-thirds of patches that included CFTR. CFTR Cl- channels and CFTR-associated ATP channels had slope conductances of 7.4 pS and 5.2 pS, respectively, and had distinct reversal potentials and sensitivities to channel blockers. CFTR-associated ATP channels exhibited slow gating kinetics that depended on the presence of protein kinase A and cytoplasmic ATP, similar to CFTR Cl- channels. Gating kinetics of the ATP channels as well as the CFTR Cl- channels were similarly affected by non-hydrolyzable ATP analogues and mutations in the CFTR R domain and NBDs. Our results indicate that phosphorylation- and nucleotide-hydrolysis-dependent gating of CFTR is directly involved in gating of an associated ATP channel. However, the permeation pathways for Cl- and ATP are distinct and the ATP conduction pathway is not obligatorily associated with the expression of CFTR.     

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.     

Properties and modulation of alpha human atrial natriuretic peptide (alpha-hANP)-formed ion channels

Using the lipid bilayer technique we have optimized recording conditions and confirmed that alpha human atrial natriuretic peptide [alpha-hANP(1-28)] forms single ion channels. The single channel currents recorded in 250/50 mM KCl cis/trans chambers show that the ANP-formed channels were heterogeneous, and differed in their conductance, kinetic, and pharmacological properties. The ANP-formed single channels were grouped as: (i) H202- and Ba2+-sensitive channel with fast kinetics; the nonlinear current-voltage (I-V) relationship of this channel had a reversal potential (Erev) of -28.2 mV, which is close to the equilibrium potential for K+ (EK = -35 mV) and a maximal slope conductance (gmax) of 68 pS at positive potentials. Sequential ionic substitution (KCl, K gluconate and choline Cl) of the cis solution suggests that the current was carried by cations. The fast channel had three modes (spike mode, burst mode, and open mode) that differed in their kinetics but not in their conductance properties. (ii) A large conductance channel possessing several subconductance levels that showed time-dependent inactivation at positive and negative membrane potentials (Vm). The inactivation ratio of the current at the end of the voltage step (Iss) to the initial current (Ii) activated immediately after the voltage step, (Iss/Ii), was voltage dependent and described by a bell-shaped curve. The maximal current-voltage (I-V) relationship of this channel, which had an Erev of +17.2 mV, was nonlinear and the value of gmax was 273 pS at negative voltages. (iii) A transiently-activated channel: the nonlinear I-V relationship of this channel had an Erev of -29.8 mV and the value of gmax was 160 pS at positive voltages. We propose that the voltage-dependence of the ionic currents and the kinetic parameters of these channel types indicate that if they were formed in vivo and activated by cytosolic factors they could change the membrane potential and the electrolyte homeostasis of the cell.     

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.     

Desensitization of the skeletal muscle ryanodine receptor: evidence for heterogeneity of calcium release channels.

Ca release channels from the junctional sarcoplasmic reticulum (SR) membranes of rabbit skeletal muscle were incorporated into the lipid bilayer membrane, and the inactivation kinetics of the channel were studied at large membrane potentials. The channels conducting Cs currents exhibited a characteristic desensitization that is both ligand and voltage dependent: 1) with a test pulse to -100 mV (myoplasmic minus luminal SR), the channel inactivated with a time constant of 3.9 s; 2) the inactivation had an asymmetric voltage dependence; it was only observed at voltages more negative than -80 mV; and 3) repetitive tests to -100 mV usually led to immobilization of the channel, which could be recovered by a conditioning pulse to positive voltages. The apparent desensitization was seen in approximately 50% of the experiments, with both the native Ca release channel (in the absence of ryanodine) and the ryanodine-activated channel (1 microM ryanodine). The native Ca release channels revealed heterogeneous gating with regard to activation by ATP and binding to ryanodine. Most channels had high affinity to ATP activation (average open probability (po) = 0.55, 2 mM ATP, 100 microM Ca), whereas a small portion of channels had low affinity to ATP activation (po = 0.11, 2 mM ATP, 100 microM Ca), and some channels bound ryanodine faster (< 2 min), whereas others bound much slower (> 20 min). The faster ryanodine-binding channels always desensitized at large negative voltages, whereas those that bound slowly did not show apparent desensitization. The heterogeneity of the reconstituted Ca release channels is likely due to the regulatory roles of other junctional SR membrane proteins on the Ca release channel.