Permeation and gating properties of the L-type calcium channel in mouse pancreatic beta cells

Ba2+ currents through L-type Ca2+ channels were recorded from cell- attached patches on mouse pancreatic beta cells. In 10 mM Ba2+, single- channel currents were recorded at -70 mV, the beta cell resting membrane potential. This suggests that Ca2+ influx at negative membrane potentials may contribute to the resting intracellular Ca2+ concentration and thus to basal insulin release. Increasing external Ba2+ increased the single-channel current amplitude and shifted the current-voltage relation to more positive potentials. This voltage shift could be modeled by assuming that divalent cations both screen and bind to surface charges located at the channel mouth. The single- channel conductance was related to the bulk Ba2+ concentration by a Langmuir isotherm with a dissociation constant (Kd(gamma)) of 5.5 mM and a maximum single-channel conductance (gamma max) of 22 pS. A closer fit to the data was obtained when the barium concentration at the membrane surface was used (Kd(gamma) = 200 mM and gamma max = 47 pS), which suggests that saturation of the concentration-conductance curve may be due to saturation of the surface Ba2+ concentration. Increasing external Ba2+ also shifted the voltage dependence of ensemble currents to positive potentials, consistent with Ba2+ screening and binding to membrane surface charge associated with gating. Ensemble currents recorded with 10 mM Ca2+ activated at more positive potentials than in 10 mM Ba2+, suggesting that external Ca2+ binds more tightly to membrane surface charge associated with gating. The perforated-patch technique was used to record whole-cell currents flowing through L-type Ca2+ channels. Inward currents in 10 mM Ba2+ had a similar voltage dependence to those recorded at a physiological Ca2+ concentration (2.6 mM). BAY-K 8644 (1 microM) increased the amplitude of the ensemble and whole-cell currents but did not alter their voltage dependence. Our results suggest that the high divalent cation solutions usually used to record single L-type Ca2+ channel activity produce a positive shift in the voltage dependence of activation (approximately 32 mV in 100 mM Ba2+).     

Cardiac calcium channels in planar lipid bilayers. L-type channels and calcium-permeable channels open at negative membrane potentials

Planar lipid bilayer recordings were used to study Ca channels from bovine cardiac sarcolemmal membranes. Ca channel activity was recorded in the absence of nucleotides or soluble enzymes, over a range of membrane potentials and ionic conditions that cannot be achieved in intact cells. The dihydropyridine-sensitive L-type Ca channel, studied in the presence of Bay K 8644, was identified by a detailed comparison of its properties in artificial membranes and in intact cells. L-type Ca channels in bilayers showed voltage dependence of channel activation and inactivation, open and closed times, and single-channel conductances in Ba2+ and Ca2+ very similar to those found in cell-attached patch recordings. Open channels were blocked by micromolar concentrations of external Cd2+. In this cell-free system, channel activity tended to decrease during the course of an experiment, reminiscent of Ca2+ channel "rundown" in whole-cell and excised-patch recordings. A purely voltage-dependent component of inactivation was observed in the absence of Ca2+ stores or changes in intracellular Ca2+. Millimolar internal Ca2+ reduced unitary Ba2+ influx but did not greatly increase the rate or extent of inactivation or the rate of channel rundown. In symmetrical Ba2+ solutions, unitary conductance saturated as the Ba2+ concentration was increased up to 500 mM. The bilayer recordings also revealed activity of a novel Ca2+-permeable channel, termed "B-type" because it may contribute a steady background current at negative membrane potentials, which is distinct from L-type or T-type Ca channels previously reported. Unlike L-type channels, B-type channels have a small unitary Ba2+ conductance (7 pS), but do not discriminate between Ba2+ and Ca2+, show no obvious sensitivity to Bay K 8644, and do not run down. Unlike either L- or T-type channels, B-type channels did not require a depolarization for activation and displayed mean open times of greater than 100 ms.     

L-type Ca2+ currents in ascidian eggs.

We have studied Ca2+ currents in ascidian eggs using the whole-cell clamp technique. T and L components, as observed in somatic cells, are present and the L-type current predominates. Since the IV relationship for these inward currents overlap at -30 mV, separation of the two components using different voltage regimes is not feasible. Increasing external Ca2+ results in larger currents. The L-type current decreases in a dose-dependent fashion in the presence of Mn2+ and Nifedipine, while the T-type current is inhibited in Ni2+. When Ba2+ was used as the carrier ion, channel kinetics and conductance were completely altered. Considering the density and kinetics of L-type channels in unfertilized eggs it is probable they play an important role in regulating cytosolic Ca2+ during early developmental processes.     

Permeation in the dihydropyridine-sensitive calcium channel. Multi-ion occupancy but no anomalous mole-fraction effect between Ba2+ and Ca2+

We investigated the mechanism whereby ions cross dihydropyridine- sensitive (L-type) Ca channels in guinea pig ventricular myocytes. At the single-channel level, we found no evidence of an anomalous mole- fraction effect like that reported previously for whole-cell currents in mixtures of Ba and Ca. With the total concentration of Ba + Ca kept constant at 10 (or 110) mM, neither conductance nor absolute unitary current exhibits a paradoxical decrease when Ba and Ca are mixed, thereby weakening the evidence for a multi-ion permeation scheme. We therefore sought independent evidence to support or reject the multi- ion nature of the L-type Ca channel by measuring conductance at various permeant ion concentrations. Contrary to the predictions of models with only one binding site in the permeation pathway, single-channel conductance does not follow Michaelis-Menten kinetics as Ba activity is increased over three orders of magnitude. Two-fold variation in the Debye length of permeant ion solutions has little effect on conductance, making it unlikely that local surface charge effects could account for these results. Instead, the marked deviation from Michaelis- Menten behavior was best explained by supposing that the permeation pathway contains three or more binding sites that can be occupied simultaneously. The presence of three sites helps explain both a continued rise in conductance as [Ba2+] is increased above 110 mM, and the high single-channel conductance (approximately 7 pS) with 1 mM [Ba2+] as the charge carrier; the latter feature enables the L-type channel to carry surprisingly large currents at physiological divalent cation concentrations. Thus, despite the absence of an anomalous mole- fraction effect between Ba and Ca, we suggest that the L-type Ca channel in heart cells supports ion flux by a single-file, multi-ion permeation mechanism.     

Alamethicin channels incorporated into frog node of ranvier: calcium-induced inactivation and membrane surface charges

Alamethicin, a peptide antibiotic, partitions into artificial lipid bilayer membranes and into frog myelinated nerve membranes, inducing a voltage-dependent conductance. Discrete changes in conductance representing single-channel events with multiple open states can be detected in either frog node or lipid bilayer membranes. In 120 mM salt solution, the average conductance of a single channel is approximately 600 pS. The channel lifetimes are roughly two times longer in the node membrane than in a phosphatidylethanolamine bilayer at the same membrane potential. With 2 or 20 mM external Ca and internal CsCl, the alamethicin-induced conductance of frog nodal membrane inactivates. Inactivation is abolished by internal EGTA, suggesting that internal accumulation of calcium ions is responsible for the inactivation, through binding of Ca to negative internal surface charges. As a probe for both external and internal surface charges, alamethicin indicates a surface potential difference of approximately -20 to -30 mV, with the inner surface more negative. This surface charge asymmetry is opposite to the surface potential distribution near sodium channels.     

S-nitrosation controls gating and conductance of the alpha 1 subunit of class C L-type Ca(2+) channels

Modulation of smooth muscle, L-type Ca(2+) channels (class C, Ca(V)1.2b) by thionitrite S-nitrosoglutathione (GSNO) was investigated in the human embryonic kidney 293 expression system at the level of whole-cell and single-channel currents. Extracellular administration of GSNO (2 mM) rapidly reduced whole-cell Ba(2+) currents through channels derived either by expression of alpha1C-b or by coexpression of alpha1C-b plus beta2a and alpha2-delta. The non-thiol nitric oxide (NO) donors 2,2-diethyl-1-nitroso-oxhydrazin (2 mM) and 3-morpholinosydnonimine-hydrochloride (2 mM), which elevated cellular cGMP levels to a similar extent as GSNO, failed to affect Ba(2+) currents significantly. Intracellular administration of copper ions, which promote decomposition of the thionitrite, antagonized its inhibitory effect, and loading of cells with high concentrations of dithiothreitol (2 mM) prevented the effect of GSNO on alpha1C-b channels. Intracellular loading of cells with oxidized glutathione (2 mM) affected neither alpha1C-b channel function nor their modulation by GSNO. Analysis of single-channel behavior revealed that GSNO inhibited Ca(2+) channels mainly by reducing open probability. The development of GSNO-induced inhibition was associated with the transient occurrence of a reduced conductance state of the channel. Our results demonstrate that GSNO modulates the alpha1 subunit of smooth muscle L-type Ca(2+) channels by an intracellular mechanism that is independent of NO release and stimulation of guanylyl cyclase. We suggest S-nitrosation of intracellularly located sulfhydryl groups as an important determinant of Ca(2+) channel gating and conductance.     

Calcium permeability and modulation of nicotinic acetylcholine receptor-channels in rat parasympathetic neurons.

Neuronal nicotinic acetylcholine (ACh)-activated currents in rat parasympathetic ganglion cells were examined using whole-cell and single-channel patch clamp recording techniques. The whole-cell current-voltage (I-V) relationship exhibited strong inward rectification and a reversal (zero current) potential of -3.9 mV in nearly symmetrical Na+ solutions (external 140 mM Na+/internal 160 mM Na+). Isosmotic replacement of extracellular Na+ with either Ca2+ or Mg2+ yielded the permeability (Px/PNa) sequence Mg2+ (1.1) > Na+ (1.0) > Ca2+ (0.65). Whole-cell ACh-induced current amplitude decreased as [Ca2+]0 was raised from 2.5 mM to 20 mM, and remained constant at higher [Ca2+]0. Unitary ACh-activated currents recorded in excised outside-out patches had conductances ranging from 15-35 pS with at least three distinct conductance levels (33 pS, 26 pS, 19 pS) observed in most patches. The neuronal nicotinic ACh receptor-channel had a slope conductance of 30 pS in Na+ external solution, which decreased to 20 pS in isotonic Ca2+ and was unchanged by isosmotic replacement of Na+ with Mg2+. ACh-activated single channel currents had an apparent mean open time (tau 0) of 1.15 +/- 0.16 ms and a mean burst length (tau b) of 6.83 +/- 1.76 ms at -60 mV in Na+ external solution. Ca(2+)-free external solutions, or raising [Ca2+]0 to 50-100 mM decreased both the tau 0 and tau b of the nAChR channel. Varying [Ca2+]0 produced a marked decrease in NP0, while substitution of Mg2+ for Na+ increased NP0. These data suggest that activation of the neuronal nAChR channel permits a substantial Ca2+ influx which may modulate Ca(2+)-dependent ion channels and second messenger pathways to affect neuronal excitability in parasympathetic ganglia.     

Block of T-type Ca channels in guinea pig atrial cells by antiarrhythmic agents and Ca channel antagonists

Myocardial cells have two types of Ca channels commonly called T-type and L-type. Whole cell Ca channel currents in guinea pig atrial myocytes can be separated and quantitated by analyzing channel closing kinetics after a brief depolarization (tail current analysis). L-type Ca channels deactivate rapidly when the membrane is repolarized and T-type Ca channels deactivate relatively slowly. Ca channel block by the therapeutically useful Ca channel antagonists is voltage dependent, so it is desirable to study block of both channel types over an extended voltage range. Tail current analysis allows this and was used to study block of both types of Ca channels under identical conditions. Amiodarone, bepridil, and cinnarizine block T-type Ca channels more potently than L-type Ca channels when binding equilibrates at normal diastolic potentials (approximately -90 mV). None of these drugs is a selective blocker of T-type Ca channels because block of L-type Ca channels is enhanced when cells are almost completely depolarized. Although weak block of T-type Ca channels by 1,4-dihydropyridines has usually been reported, we found that felodipine blocks these channels with high affinity. When most T-type Ca channels are inactivated, the apparent dissociation constant (KI) is 13 nM. Felodipine also blocks T-type Ca channels in GH3 cells (a cell line derived from rat anterior pituitary), but KI = 700 nM. Thus, T-type Ca channels in different cell types are pharmacologically distinct. Felodipine can block L-type Ca channels in atrial cells more potently than T-type Ca channels, but block of L-type Ca channels is potent only at depolarized potentials; block of both channel types is comparable at normal diastolic membrane potentials. Felodipine and the 1,4-dihydropyridines isradipine and (-)-202-791 are approximately equipotent at blocking T-type Ca channels, but differ substantially in potency for block of L-type Ca channels. Block of T-type Ca channels may account for some of the pharmacological effects of 1,4-dihydropyridines and for the antiarrhythmic activity of amiodarone and bepridil.     

On the block of outward potassium current in rabbit Schwann cells by internal sodium ions.

Currents through delayed rectifier-type K+ channels in Schwann cells cultured from rabbit sciatic nerve were studied with patch-clamp techniques. When the internal and external solutions contained physiological concentrations of sodium, the amplitude of these outward currents declined as the cell was depolarized to potentials above about +40 mV, despite the increased driving force. This reduction in the amplitude of outward K+ currents was observed in many cells before the subtraction of leakage currents; it was also observed for ensemble currents recorded in outside-out patches. It was therefore not the result of a leak-subtraction artefact nor of inadequate voltage-clamp control. Several lines of evidence also suggested that it was not the result of the extracellular accumulation of K+. By contrast, when the Na+ ion concentration of the internal solution was nominally zero, the reduction in the amplitude of outward K+ currents at positive membrane potentials was not observed. The apparent amplitude of single-channel currents through two types of K+ channel was reduced by 30 mM internal Na+, apparently as the result of a rapid 'flickery' block. The results suggest that channel block by internal Na+ is largely responsible for the negative slope conductance seen in current-voltage plots of whole-cell K+ currents at positive membrane potentials. In addition, our analysis of single-channel currents suggests that the current-voltage curve for a delayed rectifier channel in rabbit Schwann cells (in the absence of internal Na+) is roughly linear with internal and external K+ concentrations of 140 mM and 5.6 mM, respectively.     

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

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

L-type Ca2+ channels access multiple open states to produce two components of Bay K 8644-dependent current in GH3 cells

To determine the number of L-channel populations responsible for producing the two components of whole-cell L-type Ca2+ channel current revealed by Bay K 8644 (Fass, D.M., and E.S. Levitan. 1996. J. Gen. Physiol. 108:1-11), L-type Ca2+ channel activity was recorded in cell- attached patches. Ensemble tail currents from most (six out of nine) single-channel patches had double-exponential time courses, with time constants that were similar to whole-cell tail current decay values. Also, in single-channel patches subjected to two different levels of depolarization, ensemble tail currents exactly reproduced the voltage dependence of activation of the two whole-cell components: The slow component is activated at more negative potentials than the fast component. In addition, deactivation of Bay K 8644-modified whole-cell L-current was slower after long (100-ms) depolarizations than after short (20-ms) depolarizations, and this phenomenon was also evident in ensemble tail currents from single L-channels. Thus, a single population of L-channels can produce the two components of macroscopic L-current deactivation. To determine how individual L-channels produce multiple macroscopic tail current components, we constructed ensemble tail currents from traces that contained a single opening upon repolarization and no reopenings. These ensemble tails were biexponential. This type of analysis also revealed that reopenings do not contribute to the slowing of tail current deactivation after long depolarizations. Thus, individual L-channels must have access to several open states to produce multiple macroscopic current components. We also obtained evidence that access to these open states can vary over time. Use of several open states may give L-channels the flexibility to participate in many cell functions.     

Cav1.4 encodes a calcium channel with low open probability and unitary conductance

When transiently expressed in tsA-201 cells, Ca(v)1.4 calcium channels support only modest whole-cell currents with unusually slow voltage-dependent inactivation kinetics. To examine the basis for this unique behavior we used cell-attached patch single-channel recordings using 100 mM external barium as the charge carrier to determine the single-channel properties of Ca(v)1.4 and to compare them to those of the Ca(v)1.2. Ca(v)1.4 channel openings occurred infrequently and were of brief duration. Moreover, openings occurred throughout the duration of the test depolarization, indicating that the slow inactivation kinetics observed at the whole-cell level are caused by sustained channel activity. Ca(v)1.4 and Ca(v)1.2 channels displayed similar latencies to first opening. Because of the rare occurrence of events, the probability of opening could not be precisely determined but was estimated to be <0.015 over a voltage range of -20 to +20 mV. The single-channel conductance of Ca(v)1.4 channels was approximately 4 pS compared with approximately 20 pS for Ca(v)1.2 under the same experimental conditions. Additionally, in the absence of divalent cations, Ca(v)1.4 channels pass cesium ions with a single-channel conductance of approximately 21 pS. Although Ca(v)1.2 opening events were best described kinetically with two open time constants, Ca(v)1.4 open times were best described by a single time constant. BayK8644 slightly enhanced the single-channel conductance in addition to increasing the open time constant for Ca(v)1.4 channels by approximately 45% without, however, causing the appearance of an additional slower gating mode. Overall, our data indicate that single Ca(v)1.4 channels support only minute amounts of calcium entry, suggesting that large numbers of these channels are needed to allow for significant whole-cell current activity, and providing a mechanism to reduce noise in the visual system.     

Patch-clamp studies in human macrophages: Single-channel and whole-cell characterization of two K+ conductances

Summary Human peripheral blood monocytes cultured for varying periods of time were studied using whole-cell and single-channel patch-clamp recording techniques. Whole-cell recordings revealed both an outward K current activating at potentials >20 mV and an inwardly rectifying K current present at potentials negative to –60 mV. Tail currents elicited by voltage steps that activated outward current reversed nearE _K, indicating that the outward current was due to a K conductance. TheI–V curve for the macroscopic outward current was similar to the mean single-channelI–V curve for the large conductance (240 pS in symmetrical K) calcium-activated K channel present in these cells. TEA and charybdotoxin blocked the whole-cell outward current and the single-channel current. Excised and cell-attached single-channel data showed that calcium-activated K channels were absent in freshly isolated monocytes but were present in >85% of patches from macrophages cultured for >7 days. Only 35% of the human macrophages cultured for >7 days exhibited whole-cell inward currents. The inward current was blocked by external barium and increased when [K] _o increased. Inward-rectifying single-channel currents with a conductance of 28 pS were present in cells exhibiting inward whole-cell currents. These single-channel currents are similar to those described in detail in J774.1 cells (L.C. McKinney & E.K. Gallin,J. Membrane Biol. 103:41–53, 1988).     

Inhibition of Ca2+-activated K+ channels in pig pancreatic acinar cells by Ba2+, Ca2+, quinine and quinidine

Patch-clamp whole-cell and single-channel current recordings were made from pig pancreatic acinar cells to test the effects of quinine, quinidine, Ba2+ and Ca2+. Voltage-clamp current recordings from single isolated cells showed that high external concentrations of Ba2+ or Ca2+ (88 mM) abolished the outward K+ currents normally associated with depolarizing voltage steps. Lower concentrations of Ca2+ only had small inhibitory effects whereas 11 mM Ba2+ almost blocked the K+ current. 5.5 mM Ba2+ reduced the outward K+ current to less than 30% of the control value. Both external quinine and quinidine (200-500 microM) markedly reduced whole-cell outward K+ currents. In single-channel current studies it was shown that external Ba2+ (1-5 mM) markedly reduced the probability of opening of high-conductance Ca2+ and voltage-activated K+ channels whereas internal Ba2+ (6 X 10(-6) to 3 X 10(-5) M) caused activation at negative membrane potentials and inhibition at positive potentials. Quinidine (200-400 microM) evoked rapid chopping of single K+ channel openings acting both from the outside and inside of the membrane and in this way markedly reduced the total current passing through the channels.     

Effect of mibefradil on sodium and calcium currents

Interstitial cells of Cajal (ICC) generate the electrical slow wave. The ionic conductances that contribute to the slow wave appear to vary among species. In humans, a tetrodotoxin-resistant Na+ current (Na(V)1.5) encoded by SCN5A contributes to the rising phase of the slow wave, whereas T-type Ca2+ currents have been reported from cultured mouse intestine ICC and also from canine colonic ICC. Mibefradil has a higher affinity for T-type over L-type Ca2+ channels, and the drug has been used in the gastrointestinal tract to identify T-type currents. However, the selectivity of mibefradil for T-type Ca2+ channels over ICC and smooth muscle Na+ channels has not been clearly demonstrated. The aim of this study was to determine the effect of mibefradil on T-type and L-type Ca2+ and Na+ currents. Whole cell currents were recorded from HEK-293 cells coexpressing green fluorescent protein with either the rat brain T-type Ca2+ channel alpha(1)3.3b + beta(2), the human intestinal L-type Ca2+ channel subunits alpha(1C) + beta(2), or Na(V)1.5. Mibefradil significantly reduced expressed T-type Ca2+ current at concentrations > or = 0.1 microM (IC(50) = 0.29 microM), L-type Ca2+ current at > 1 microM (IC(50) = 2.7 microM), and Na+ current at > or = 0.3 microM (IC(50) = 0.98 microM). In conclusion, mibefradil inhibits the human intestinal tetrodotoxin-resistant Na+ channel at submicromolar concentrations. Caution must be used in the interpretation of the effects of mibefradil when several ion channel classes are coexpressed.     

Characterization of a chloroplast inner envelope K+ channel.

A K(+)-conducting protein of the chloroplast inner envelope was characterized as a K+ channel. Studies of this transport protein in the native membrane documented its sensitivity to K+ channel blockers. Further studies of native membranes demonstrated a sensitivity of K+ conductance to divalent cations such as Mg2+, which modulate ion conduction through interaction with negative surface charges on the inner-envelope membrane. Purified chloroplast inner-envelope vesicles were fused into an artificial planar lipid bilayer to facilitate recording of single-channel K+ currents. These single-channel K+ currents had a slope conductance of 160 picosiemens. Antibodies generated against the conserved amino acid sequence that serves as a selectivity filter in the pore of K+ channels immunoreacted with a 62-kD polypeptide derived from the chloroplast inner envelope. This polypeptide was fractionated using density gradient centrifugation. Comigration of this immunoreactive polypeptide and K+ channel activity in sucrose density gradients further suggested that this polypeptide is the protein facilitating K+ conductance across the chloroplast inner envelope.     

Properties of the Ca-activated K conductance of human red cells as revealed by the patch-clamp technique

Application of Ca2+ to the inner surface of red-cell membranes activates unitary currents that can be measured in cell-attached and cell-free membrane patches. Ca2+ can be replaced by Pb2+ to activate the single channels. In addition to internal Ca2+ external K+ has to be present. The channels are preferentially permeable to K+ with a selectivity ratio PK:PNa of about 15:1 as estimated from measurement of reversal potentials. The dependence of channel activity on Ca2+ is compatible with the conception that the binding of two Ca2+ is necessary to open a single channel. Both the channel activity and the single-channel conductance exhibit inward rectification. External and internal Na+ inhibit the K+ currents. The reported results suggest that the unitary current events are responsible for the Ca2+-dependent K+ permeability known from measurement on cell suspensions. Therefore, comparison of the two techniques allows calculation of the number of K+ channels per red cell, which on average is about 10.     

Changes in the conductance of the neuronal nicotinic acetylcholine receptor channel induced by magnesium

We have investigated the effect of magnesium on the single-channel conductance of neuronal nicotinic acetylcholine receptors (nAChRs) in nerve growth-factor treated rat pheochromocytoma (PC12) cells. The patch-clamp technique was used to record single-channel currents from cell attached and excised, outside-out patches in the presence of various internal and external Mg2+ concentrations. Mg2+ reduced the single-channel conductance in a concentration-dependent manner with an IC50 of 9.2 mM for external Mg2+ (inward conductance) and 0.69 mM for external Mg2+ (outward conductance). Both estimated and measured conductances for divalent cation-free CsCl solutions were around 60 pS. We also find that divalent cations are not involved in the inward rectification of whole-cell ACh-induced currents in these cells. Our results imply that the amino acids screened by divalent cations sense electric fields only weakly and are presumably outside the lipid bilayer. They also suggest that the density and the number of charges (or both) differ on either side of the ion pore.     

Both T- and L-type Ca2+ channels can contribute to excitation-contraction coupling in cardiac Purkinje cells.

Although L-type Ca2+ channels have been shown to play a central role in cardiac excitation-contraction (E-C) coupling, little is known about the role of T-type Ca2+ channels in this process. We used the amphotericin B perforated patch method to study the possible role of T-type Ca2+ current in E-C coupling in isolated canine Purkinje myocytes where both Ca2+ currents are large. T-type Ca2+ current was separated from L-type Ca2+ current using protocols employing the different voltage dependencies of the channel types and their different sensitivities to pharmacological blockade. We showed that Ca2+ admitted through either T- or L-type Ca2+ channels is capable of initiating contraction and that the contractions depended on Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR). The contractions, however, had different properties. Those initiated by Ca2+ entry through T-type Ca2+ channels had a longer delay to the onset of shortening, slower rates of shortening and relaxation, lower peak shortening, and longer time to peak shortening. These differences were present even when L-type Ca2+ current amplitude, or charge entry, was less than that of T-type Ca2+ current, suggesting that Ca2+ entry through the T-type Ca2+ channel is a less effective signal transduction mechanism to the SR than is Ca2+ entry through the L-type Ca2+ channel. We conclude that under our experimental conditions in cardiac Purkinje cells Ca2+ entry through the T-type Ca2+ channel can activate cell contraction. However, Ca2+ entry through the L-type Ca2+ channel is a more effective signal transduction mechanism. Our findings support the concept that different structural relationships exist between these channel types and the SR Ca2+ release mechanism.     

Effects of external protons on single cardiac sodium channels from guinea pig ventricular myocytes.

The effects of external protons on single sodium channel currents recorded from cell-attached patches on guinea pig ventricular myocytes were investigated. Extracellular protons reduce single channel current amplitude in a dose-dependent manner, consistent with a simple rapid channel block model where protons bind to a site within the channel with an apparent pKH of 5.10. The reduction in single channel current amplitude by protons is voltage independent between -70 and -20 mV. Increasing external proton concentration also shifts channel gating parameters to more positive voltages, consistent with previous macroscopic results. Similar voltage shifts are seen in the steady-state inactivation (h infinity) curve, the time constant for macroscopic current inactivation (tau h), and the first latency function describing channel activation. As pHo decreases from 7.4 to 5.5 the midpoint of the h infinity curve shifts from -107.6 +/- 2.6 mV (mean +/- SD, n = 16) to -94.3 +/- 1.9 mV (n = 3, P less than 0.001). These effects on channel gating are consistent with a reduction in negative surface potential due to titration of negative external surface charge. The Gouy-Chapman-Stern surface charge model incorporating specific proton binding provides an excellent fit to the dose-response curve for the shift in the midpoint of the h infinity curve with protons, yielding an estimate for total negative surface charge density of -1e/490 A2 and a pKH for proton binding of 5.16. By reducing external surface Na+ concentration, titration of negative surface charge can also quantitatively account for the reduction in single Na+ channel current amplitude, although we cannot rule out a potential role for channel block. Thus, titration by protons of a single class of negatively charged sites may account for effects on both single channel current amplitude and gating.