Ca channel kinetics during the spontaneous heart beat in embryonic chick ventricle cells.

The ability of Ca ions to inhibit Ca channels presents one of the most intriguing problems in membrane biophysics. Because of this negative feedback, Ca channels can regulate the current that flows through them. The kinetics of the channels depend on voltage, and, because the voltage controls the current, a strong interaction exists between voltage dependence and Ca dependence. In addition to this interaction, the proximity of pores and the local concentration of ions also determine how effectively the Ca ions influence channel kinetics. The present article proposes a model that incorporates voltage-dependent kinetics, current-dependent kinetics, and channel clustering. We have based the model on previous voltage-clamp data and on Ca and Ba action currents measured during the action potential in beating heart cells. In general we observe that great variability exists in channel kinetics from patch to patch: Ba or Ca currents have low or high amplitudes and slow or fast kinetics during essentially the same voltage regime, either applied step-protocols or spontaneous cell action potentials. To explain this variability, we have postulated that Ca channels interact through shared ions. The model we propose expands on our previous model for Ba currents. We use the same voltage-dependent rate constants for the Ca currents that we did for the Ba currents. However, we vary the current-dependent rate constants according to the species of the conducting ion. The model reproduces the main features of our data, and we use it to predict Ca channel kinetics under physiological conditions. Preliminary reports of this work have appeared (DeFelice et al., 1991, Biophys. J. 59:551a; Risso et al., 1992, Biophys. J. 61:248a).     

Fast-deactivating calcium channels in chick sensory neurons

Whole-cell Ca and Ba currents were studied in chick dorsal root ganglion (DRG) cells kept 6-10 in culture. Voltage steps with a 15-microseconds rise time were imposed on the membrane using an improved patch-clamp circuit. Changes in membrane current could be measured 30 microseconds after the initiation of the test pulse. Currents through Ca channels were recorded under conditions that eliminate Na and K currents. Tail currents, associated with Ca channel closing, decayed in two distinct phases that were very well fitted by the sum of two exponentials. The time constants tau f and tau s were near 160 microseconds and 1.5 ms at -80 mV, 20 degrees C. The tail current components, called FD and SD (fast-deactivating and slowly deactivating), are Ca channel currents. They were greatly reduced when Mg2+ replaced all other divalent cations in the bath. The SD component inactivated almost completely as the test pulse duration was increased to 100 ms. It was suppressed when the cell was held at membrane potentials positive to -50 mV and was blocked by 100-200 microM Ni2+. This behavior indicates that the SD component was due to the closing of the low-voltage-activated (LVA) Ca channels previously described in this preparation. The FD component was fully activated with 10-ms test pulses to +20 mV at 20 degrees C, and inactivated to approximately 30% during 500-ms test pulses. It was reduced in amplitude by holding at -40 mV, but was only slightly reduced by micromolar concentrations of Ni2+. Replacement of Ca2+ with Ba2+ increased the FD tail current amplitudes by a factor of approximately 1.5. The deactivation kinetics did not change (a) as channels inactivated during progressively longer pulses or (b) when the degree of activation was varied. Further, tau f was affected neither by changing the holding potential nor by varying the test pulse amplitude. Lowering the temperature from 20 to 10 degrees C decreased tau f by a factor of 2.5. In all cases, the FD component was very well fitted by a single exponential. There was no indication of an additional tail component of significant size. Our findings indicate that the FD component is due to closing of a single class of Ca channels that coexist with the LVA Ca channel type in chick DRG neurons.     

Recovery of Ca currents from inactivation: The roles of Ca influx,membrane potential,and cellular metabolism

Ca currents were examined with regard to their recovery from inactivation. The experiments were done on isolated nerve cell bodies of Helix aspersa using a combined suction pipet , microelectrode method for voltage clamp, and internal perfusion. Ca currents were separated by suppressing K and Na currents. The time course of recovery was determined by applying a test pulse at intervals ranging from 1 msec to 20 sec after prepulses varying from 20 to 3000 msec in duration. Each pair of pulses was preceded by a control pulse to ensure that the Ca currents had recovered before the next test pair was applied. Ba and Ca currents were compared and the effects of intracellular perfusion with EGTA, ATP, and vanadate were examined. Ba currents recovered in two stages and this time course was well fit by a sum of two exponentials with amplitudes and time constants given by A1 and tau 1 for the fast component and A2 and tau 2 for the slow component. In Ba the time constants were unchanged when prepulse durations were prolonged from 70 to 700 msec, although the initial amplitudes A1 and A2, particularly A2, were increased. Comparable influxes of Ca during the prepulse caused much more inactivation, but interestingly the recovery occurred at the same rate. The time course of Ca current recovery was also fit by a sum of two exponentials, the time constants of which were both smaller than the time constants of Ba current recovery. However, the time constants of Ca current recovery were increased markedly when prepulse durations were prolonged. Increasing the extracellular Ca concentration had a similar effect. Increasing the Ba influx had no effect on the recovery time constants, and the Ba results are consistent with reversible inactivation gating of potential-dependent membrane Ca channels. The Ca results show that Ca influx enhances inactivation. Intracellular perfusion with EGTA resulted in less inactivation in the cast of Ca but it had no effect on Ba currents. Intracellular ATP increased the rate of recovery of Ca currents, and intracellular vanadate inhibited recovery. It is concluded that recovery of Ca channels depends upon both Ca influx and membrane potential and is modulated by agents which affect Ca metabolism.     

Classification of voltage-dependent Ca2+ channels in trigeminal ganglion neurons from neonatal rats

We examined the subtypes and characteristics of the Ca(2+) channel in small (diameter < 30 microm) trigeminal ganglion (TG) neurons from neonatal rats by means of whole cell patch clamp techniques. There were two current components, low-voltage activated (LVA) and high-voltage activated (HVA) I(Ba), with different activation ranges and waveforms. LVA I(Ba) elicited from a depolarizing step pulse at a holding potential (HP) of -80 mV was inhibited by 0.25 mM amiloride (62%), which did not produce any significant inhibition of the peak amplitude of HVA I(Ba). The application of 0.5 mM amiloride inhibited 10% of the HVA I(Ba). The LVA I(Ba) was also reduced by changing the HP from -80 to -60 mV (61%), and under these conditions the peak amplitude of HVA I(Ba) did not change significantly. In addition, HVA I(Ba) and LVA I(Ba) showed marked differences in their inactivation properties. Experiments with several Ca(2+) channel blockers revealed that on average, 26% of the HVA I(Ba) was nifedipine (10 microM) sensitive, 55% was sensitive to omega-conotoxinGVIA (1 microM), 4% was blocked by omega-agatoxinIVA (1 microM), and the remainder of the current that was resistant to the co-application of all three Ca(2+) channel blockers was 15% of the total current. These results suggest that the application of amiloride and the alteration of the holding potential level can discriminate between HVA and LVA Ba(2+) currents in TG neurons, and that TG neurons expressed T-, L-, N-, P-/Q- and R-type Ca(2+) channels.     

Cardiac Na currents and the inactivating, reopening, and waiting properties of single cardiac Na channels

Tetrodotoxin (TTX)-sensitive Na currents were examined in single dissociated ventricular myocytes from neonatal rats. Single channel and whole cell currents were measured using the patch-clamp method. The channel density was calculated as 2/micron 2, which agreed with our usual finding of four channels per membrane patch. At 20 degrees C, the single channel conductance was 20 pS. The open time distributions were fit by a single-exponential function with a mean open time of approximately 1.0 ms at membrane potentials from -60 to -40 mV. Averaged single channel and whole cell currents were similar when scaled and showed both fast and slow rates of inactivation. The inactivation and activation gating shifted quickly to hyperpolarized potentials for channels in cell-attached as well as excised patches, whereas a much slower shift occurred in whole cells. Slowly inactivating currents were present in both whole cell and single channel current measurements at potentials as positive as -40 mV. In whole cell measurements, the potential range could be extended, and slow inactivation was present at potentials as positive as -10 mV. The curves relating steady state activation and inactivation to membrane potential had very little overlap, and slow inactivation occurred at potentials that were positive to the overlap. Slow inactivation is in this way distinguishable from the overlap or window current, and the slowly inactivating current may contribute to the plateau of the rat cardiac action potential. On rare occasions, a second set of Na channels having a smaller unit conductance and briefer duration was observed. However, a separate set of threshold channels, as described by Gilly and Armstrong (1984. Nature [Lond.]. 309:448), was not found. For the commonly observed Na channels, the number of openings in some samples far exceeded the number of channels per patch and the latencies to first opening or waiting times were not sufficiently dispersed to account for the slowly inactivating currents: the slow inactivation was produced by channel reopening. A general model was developed to predict the number of openings in each sample. Models in which the number of openings per sample was due to a dispersion of waiting times combined with a rapid transition from an open to an absorbing inactivated state were unsatisfactory and a model that was more consistent with the results was identified.     

Single channel studies of the phosphorylation of K+ channels in the squid giant axon. I. Steady-state conditions

Phosphorylation of the delayed rectifier channel of squid potentiates the macroscopic K+ current and slows its activation kinetics. We have studied this phenomenon at the single channel level using the cut-open axon technique under steady-state conditions. In 10 mM external K+/310 mM internal K+ there are predominantly two types of channels present, a 20-pS and a 40-pS channel. In steady state at depolarized potentials, the 40-pS channel was most active, whereas the 20-pS channel tended to disappear due to a slow inactivation process. Two methods were developed to shift the population of channels toward a dephosphorylated state. One method consisted of predialyzing a whole axon with solutions containing no ATP, while recording the currents under axial-wire voltage clamp. A piece of axon was then removed and cut open, and single channel currents were recorded from the cut-open axon. A second method was based on the difference in diffusion coefficients for ATP and proteins such as the endogenous phosphatase. The axon was cut open in a solution that did not contain Ca2+ or Cl- in order to maintain the axoplasm structurally intact and permit endogenous phosphatase to act on the membrane while ATP diffused away, before removing the axoplasm and forming a membrane patch. When dephosphorylating conditions were used, the steady-state open probability of the 40-pS channel at 42 mV was very low (less than 0.0002), and the channel openings appeared as a series of infrequent, short-duration events. The channel activity was increased up to 150-fold by photoreleasing caged ATP inside the patch pipette in the presence of the catalytic subunit of protein kinase A. The sharp increase in open probability could be accounted for by a decrease of the slow component of the closed time distribution from 23 s to 170 ms with little change in the distribution of open times (1-2 ms) and no change in the single channel current amplitude. In voltage-jump experiments the contribution of the 40-pS channel to the delayed rectifier current was often small due to the large values of the latency to the first opening.     

Expression of calcium channels in Xenopus oocyte injected with crab skeletal muscle fibre mRNA

Summary— Using the whole cell voltage-clamp technique and a Cl free and Na free Ba methane sulfonate solution, stage V and VI Xenopus oocytes demonstrated a Ba current (endogenous component) with a peak amplitude average of 6 nA (6 ± 2 nA). When oocytes were injected with crustacean skeletal muscle mRNA, an additional component of I_Ba could be detected (exogenous I_Ba). The latter current could be distinguished from the native one by several electrophysiological means: a peak amplitude average of 90 nA (90 ± 4 nA), activation potential threshold, steady state inactivation properties and sensitivity to Ca blockers. As shown by Jdaïâa and Guilbault in crustacean skeletal muscle fibres, exogenous I_Ba could be divided into two components: a “fast component” and a “slow component” probably passing through two types of Ca channels (fast and slow) since the peak Ba current voltage relationship was biphasic and the fast component of exogenous I_Ba was less sensitive than the slow to nifedipine. The features of the newly synthesized channels incorporated in the Xenopus oocyte membrane suggest that they may be associated with fast and slow channels, previously described in many preparations, particularly in crustacean skeletal muscle fibres.     

Expression of calcium channels in Xenopus oocyte injected with crab skeletal muscle fibre mRNA

Using the whole cell voltage-clamp technique and a Cl free and Na free Ba methane sulfonate solution, stage V and VI Xenopus oocytes demonstrated a Ba current (endogenous component) with a peak amplitude average of 6 nA (6 ± 2 nA). When oocytes were injected with crustacean skeletal muscle mRNA, an additional component of I_Ba could be detected (exogenous I_Ba). The latter current could be distinguished from the native one by several electrophysiological means: a peak amplitude average of 90 nA (90 ± 4 nA), activation potential threshold, steady state inactivation properties and sensitivity to Ca blockers. As shown by Jdaïâa and Guilbault in crustacean skeletal muscle fibres, exogenous I_Ba could be divided into two components: a “fast component” and a “slow component” probably passing through two types of Ca channels (fast and slow) since the peak Ba current voltage relationship was biphasic and the fast component of exogenous I_Ba was less sensitive than the slow to nifedipine. The features of the newly synthesized channels incorporated in the Xenopus oocyte membrane suggest that they may be associated with fast and slow channels, previously described in many preparations, particularly in crustacean skeletal muscle fibres.     

Gating and permeation properties of two types of calcium channels in neuroblastoma cells.

The gating and permeation properties of two types of calcium channels were studied in the neuroblastoma cell line N1E-115. Calcium channel currents as carried by Ba2+ (50 mM) were recorded using the whole-cell variation of the patch electrode voltage-clamp technique. The two types of calcium channels showed similar membrane potential dependence with respect to the steady-state activation and inactivation gating properties. However, the properties of the long-lasting type II channels were shifted approximately 30 mV in the depolarizing direction compared with those of the transient type I channels. Activation of type I channels developed with a sigmoidal time course which was described by m2 kinetics, whereas the activation of type II channels was described by a single exponential function. Tail current upon repolarization followed an exponential decay in either type of calcium channels. In comparison to type I channels, the activation process of type II channels was shifted approximately 30 mV in the positive direction, while the deactivation process showed a 60 mV shift in the positive direction. The rate constants of activation obtained from the activation and deactivation processes indicated that under comparable membrane potential conditions, type II channels close 2.4 times faster than type I channels upon repolarization. When external 50 mM Ba2+ was replaced with Ca2+ or Sr2+ on the equimolar basis, the amplitudes of transient and long-lasting currents were altered without a significant change in their time courses. The ion permeability ratios determined from the maximum amplitude of the inward current were as follows: Ba2+ (1.0) = Sr2+ (1.0) greater than Ca2+ (0.7) for type I channels, and Ba2+ (1.0) greater than Sr2+ (0.7) greater than Ca2+ (0.3) for type II channels. Replacement of Ba2+ with Ca2+ caused a 10-12 mV positive shift in the current-voltage relation for type II channels. However, the shift for type I channels was much less. This suggests that negative surface charges are present around type II channels. After correction for the surface charge effect on the ion permeation, there was no significant difference between the permeability ratios of these cations for the two channel types. It was concluded that the two types of calcium channels have many common properties in their gating and permeation mechanisms despite their differential voltage sensitivity and ion selectivity.     

Cyclic adenosine monophosphate regulates calcium channels in the plasma membrane of Arabidopsis leaf guard and mesophyll cells

The effect of cAMP on Ca(2+)-permeable channels from Arabidopsis thaliana leaf guard cell and mesophyll cell protoplasts was studied using the patch clamp technique. In the whole cell configuration, dibutyryl cAMP was found to increase a hyperpolarization-activated Ba(2+) conductance (I(Ba)). The increase of I(Ba) was blocked by the addition of GdCl(3). In excised outside-out patches, the addition of dibutyryl cAMP consistently activated a channel with particularly fast gating kinetics. Current/voltage analyses indicated a single channel conductance of approximately 13 picosiemens. In patches where we measured some channel activity prior to cAMP application, the data suggest that cAMP enhances channel activity without affecting the single channel conductance. The cAMP activation of these channels was reversible upon washout. The results obtained with excised patches indicate that the cAMP-activated I(Ba) seen in the whole cell configuration could be explained by a direct effect of cAMP on the Ca(2+) channel itself or a close entity to the channel. This work represents the first demonstration using patch clamp analysis of the presence in plant cell membranes of an ion channel directly activated by cAMP.     

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.     

Single- and multi-channel currents recorded with patch electrodes in mouse eggs

Zona-free mouse eggs are amenable to patch recording as well as to whole-cell recording (Bland et al., 1984; Peres, 1986a). Although many patches are silent, others show spontaneous channel opening; the single channel current is 1.5 pA and inwardly directed at resting potential (RP). Current amplitudes distribute normally as a single population at RP, however at RP-50 mV the amplitude histogram indicates two channel populations. Open time distribution is exponential with a mean open time between 4 and 7 msec at RP. In three cases out of thirty-eight, in which depolarizations were given from a holding potential of RP-50 mV, microscopic currents very similar in kinetics and voltage-dependence to the whole-cell Ca2+ current were observed. No single-channel currents could be resolved in these traces. The results reported here indicate that the voltage-dependent Ca2+ current of the mouse egg goes through low-conductance channels located in high density spots on the egg surface.     

Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9- anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.     

Membrane currents in taste cells of the rat fungiform papilla. Evidence for two types of Ca currents and inhibition of K currents by saccharin

Taste buds were isolated from the fungiform papilla of the rat tongue and the receptor cells (TRCs) were patch clamped. Seals were obtained on the basolateral membrane of 281 TRCs, protruding from the intact taste buds or isolated by micro-dissection. In whole-cell configuration 72% of the cells had a TTX blockable transient Na inward current (mean peak amplitude 0.74 nA). All cells had outward K currents. Their activation was slower than for the Na current and a slow inactivation was also noticeable. The K currents were blocked by tetraethylammonium, Ba, and 4-aminopyridine, and were absent when the pipette contained Cs instead of K. With 100 mM Ba or 100 mM Ca in the bath, two types of inward current were observed. An L-type Ca current (ICaL) activated at -20 mV had a mean peak amplitude of 440 pA and inactivated very slowly. At 3 mM Ca the activation threshold of ICaL was near -40 mV. A transient T-type current (ICaT) activated at -50 mV had an average peak amplitude of 53 pA and inactivated with a time constant of 36 ms at -30 mV. ICaL was blocked more efficiently by Cd and D600 than ICaT. ICaT was blocked by 0.2 mM Ni and half blocked by 200 microM amiloride. In whole-cell voltage clamp, Na-saccharin caused (in 34% of 55 cells tested) a decrease in outward K currents by 21%, which may be expected to depolarize the TRCs. Also, Na-saccharin caused some taste cells to fire action potentials (on-cell, 7 out of 24 cells; whole-cell, 2 out of 38 cells responding to saccharin) of amplitudes sufficient to activate ICaL. Thus the action potentials will cause Ca inflow, which may trigger release of transmitter.     

Calcium channel currents in pars intermedia cells of the rat pituitary gland. Kinetic properties and washout during intracellular dialysis

Ca channel currents in primary cultured pars intermedia cells were studied using whole-cell recording with patch pipettes. Experiments were carried out at 18-21 degrees C in cells internally dialyzed with K-free, EGTA-containing solutions and in the presence of 10 mM Ca or 10 mM Ba in the external solution. Ca and Ba currents depended on the activity of two main populations of channels, SD and FD. With Ca as the charge carrier, these two populations differed in their closing time constants at -80 mV (SD, 1.8 ms; FD, 110 microseconds), apparent activation levels (SD, -40 mV; FD, -5 mV), half-maximal activation levels (SD, +5 to +10 mV; FD, +20 to +25 mV), half-times of activation at +20 mV (SD, 2.5-3.5 ms; FD, 1.0-1.3 ms), and time courses of inactivation (SD, fast; FD, slow). Functional FD channels were almost completely lost within 20-25 min of breaking into a cell, whereas SD channels retained most of their functional activity. In addition, the conductance-voltage curve for FD channels shifted approximately 15 mV toward more negative membrane potentials within 11-14 min under whole-cell recording. At that time, 60-70% of the FD channel maximum conductance was lost. However, the conductance-voltage curve for SD channels shifted less than 5 mV within 25 min. The addition of 3 mM MgATP and 40 microM GTP to the internal solution slowed down the loss of FD channels and prevented the shift in their activation curve. It was also found that the amplitude of the current carried by FD channels tends to increase as a function of the age of the culture, with no obvious changes in the kinetic properties of the channels or in SD channel activity.     

Two modes of gating during late Na+ channel currents in frog sartorius muscle

Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.     

Interaction of Ca2+ agonist and depolarization on Ca2+ channel current in guinea pig detrusor cells [published erratum appears in J Gen Physiiol 1996 Jun;107(6):755]

The interaction of large depolarization and dihydropyridine Ca2+ agonists, both of which are known to enhance L-type Ca2+ channel current, was examined using a conventional whole-cell clamp technique. In guinea pig detrusor cells, only L-type Ca2+ channels occur. A second open state (long open state: O2) of the Ca2+ channels develops during large depolarization (at +80 mV, without Ca2+ agonists). This was judged from lack of inactivation of the Ca2+ channel current during the large depolarizing steps (5 s) and slowly deactivating inward tail currents (= 10-15 ms) upon repolarization of the cell membrane to the holding potential (-60 mV). Application of Bay K 8644 (in 2.4 mM Ca(2+)- containing solutions) increased the amplitude of the Ca2+ currents evoked by simple depolarizations, and made it possible to observe inward tail currents (= 2.5-5 ms at -60 mV). The open state induced by large depolarization (O2*) in the Bay K 8644 also seemed hardly to inactivate. After preconditioning with large depolarizing steps, the decay time course of the inward tail currents upon repolarization to the holding potential (-60 mV) was significantly slowed, and could be fitted reasonably with two exponentials. The fast and slow time constants were 10 and 45 ms, respectively, after 2 s preconditioning depolarizations. Qualitatively the same results were obtained using Ba2+ as a charge carrier. Although the amplitudes of the inward currents observed in the test step and the subsequent repolarization to the holding potential were decreased in the same manner by additional application of nifedipine (in the presence of Bay K 8644), the very slow deactivation time course of the tail current was little changed. The additive enhancement by large depolarization and Ca2+ agonists of the inward tail current implies that two mechanisms separately induce long opening of the Ca2+ channels: i.e., that there are four open states.     

大鼠胰腺β细胞离子通道的一些特性

实验以单个Ｗｉｓｔａｒ大鼠胰腺β细胞为对象,用穿孔膜片箝和细胞贴附式记录技术研究ＡＴＰ敏感Ｋ＾＋通道（ＫＡＴＰ）、延迟整流型Ｋ＾＋通道（ＫＤＲ）、Ｃａ＾２＋通道和Ｎａ＾＋通道的有关特性。结果表明：⑴ＫＡＴＰ通道的内流电导约６５ｐＳ,外流电导约３１ｐＳ,反转电位在－６０ｍＶ左右;⑵ＫＤＲ通道在延迟２０ｍｓ后达到最大激活,ＫＤＲ电流约为ＫＡＴＰ的１／３;⑶钙电流在０ｍＶ左右达到４０－６０ｐＡ的峰值,Ｌ     

Properties of two types of calcium channels in clonal pituitary cells

The calcium currents of GH3 cells have been studied using the whole cell variant of the patch-clamp technique. Under conditions that eliminate sodium and potassium currents, we observed inward currents that activated within a few milliseconds, and deactivated with two time constants, approximately 150 microseconds and 3 ms at -80 mV, 18-20 degrees C. The components are called FD and SD (fast deactivating and slow deactivating). Both components are calcium currents, and are greatly reduced when magnesium is substituted for most of the calcium in the bath. In addition to (a) their different rates of deactivation, the two components differ in a number of other properties. (b) The SD component inactivates almost completely, with a time constant of 23 ms at 20 mV, 19 degrees C. The FD component, on the other hand, shows little or no sign of inactivation, and is almost the same in amplitude from 10 to 100 ms. The components thus seem quite independent of each other, and must arise from two independent sets of channels. (c) The FD channels activate more rapidly than SD at 20 mV, by a factor of approximately 2 as is shown in several ways. (d) In 10 Ca or 10 Ba, the activation curve for SD channels is approximately 20 mV more negative than for FD or Na channels. (e) FD channels conduct barium ions more effectively than calcium by a ratio of approximately 2. (f) FD channels "wash out" within minutes after the patch electrode breaks into a cell, whereas SD channel current remains relatively stable. It is argued that SD channels, because of their negative activation threshold, are involved in electrical events near threshold, and that FD channels are best suited for calcium injection once a spike has been initiated.     

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+).