Properties of transient K+ currents and underlying single K+ channels in rat olfactory receptor neurons

The transient potassium current, IK(t), of enzymatically dissociated rat olfactory receptor neurons was studied using patch-clamp techniques. Upon depolarization from negative holding potentials, IK(t) activated rapidly and then inactivated with a time course described by the sum of two exponential components with time constants of 22.4 and 143 ms. Single-channel analysis revealed a further small component with a time constant of several seconds. Steady-state inactivation was complete at -20 mV and completely removed at -80 mV (midpoint -45 mV). Activation was significant at -40 mV and appeared to reach a maximum conductance at +40 mV (midpoint -13 mV). Deactivation was described by the sum of two voltage-dependent exponential components. Recovery from inactivation was extraordinarily slow (50 s at -100 mV) and the underlying processes appeared complex. IK(t) was reduced by 4-aminopyridine and tetraethylammonium applied externally. Increasing the external K+ concentration ([K+]o) from 5 to 25 mM partially removed IK(t) inactivation, usually without affecting activation kinetics. The elevated [K+]o also hyperpolarized the steady-state inactivation curve by 9 mV and significantly depolarized the voltage dependence of activation. Single transient K+ channels, with conductances of 17 and 26 pS, were observed in excised patches and often appeared to be localized into large clusters. These channels were similar to IK(t) in their kinetic, pharmacological, and voltage-dependent properties and their inactivation was also subject to modulation by [K+]o. The properties of IK(t) imply a role in action potential repolarization and suggest it may also be important in modulating spike parameters during neuronal burst firing. A simple method is also presented to correct for errors in the measurement of whole-cell resistance (Ro) that can result when patch-clamping very small cells. The analysis revealed a mean corrected Ro of 26 G omega for these cells.     

Characterization of the inward-rectifying potassium current in cat ventricular myocytes

Whole-cell membrane currents were measured in isolated cat ventricular myocytes using a suction-electrode voltage-clamp technique. An inward-rectifying current was identified that exhibited a time-dependent activation. The peak current appeared to have a linear voltage dependence at membrane potentials negative to the reversal potential. Inward current was sensitive to K channel blockers. In addition, varying the extracellular K+ concentration caused changes in the reversal potential and slope conductance expected for a K+ current. The voltage dependence of the chord conductance exhibited a sigmoidal relationship, increasing at more negative membrane potentials. Increasing the extracellular K+ concentration increased the maximal level of conductance and caused a shift in the relationship that was directly proportional to the change in reversal potential. Activation of the current followed a monoexponential time course, and the time constant of activation exhibited a monoexponential dependence on membrane potential. Increasing the extracellular K+ concentration caused a shift of this relationship that was directly proportional to the change in reversal potential. Inactivation of inward current became evident at more negative potentials, resulting in a negative slope region of the steady state current-voltage relationship between -140 and -180 mV. Steady state inactivation exhibited a sigmoidal voltage dependence, and recovery from inactivation followed a monoexponential time course. Removing extracellular Na+ caused a decrease in the slope of the steady state current-voltage relationship at potentials negative to -140 mV, as well as a decrease of the conductance of inward current. It was concluded that this current was IK1, the inward-rectifying K+ current found in multicellular cardiac preparations. The K+ and voltage sensitivity of IK1 activation resembled that found for the inward-rectifying K+ currents in frog skeletal muscle and various egg cell preparations. Inactivation of IK1 in isolated ventricular myocytes was viewed as being the result of two processes: the first involves a voltage-dependent change in conductance; the second involves depletion of K+ from extracellular spaces. The voltage-dependent component of inactivation was associated with the presence of extracellular Na+.     

Voltage- and time-dependent K+ channel currents in the basolateral membrane of villus enterocytes isolated from guinea pig small intestine

Patch-clamp studies were carried out in villus enterocytes isolated from the guinea pig proximal small intestine. In the whole-cell mode, outward K+ currents were found to be activated by depolarizing command pulses to -45 mV. The activation followed fourth order kinetics. The time constant of K+ current activation was voltage-dependent, decreasing from approximately 3 ms at -10 mV to 1 ms at +50 mV. The K+ current inactivated during maintained depolarizations by a voltage- independent, monoexponential process with a time constant of approximately 470 ms. If the interpulse interval was shorter than 30 s, cumulative inactivation was observed upon repeated stimulations. The steady state inactivation was voltage-dependent over the voltage range from -70 to -30 mV with a half inactivation voltage of -46 mV. The steady state activation was also voltage-dependent with a half- activation voltage of -22 mV. The K+ current profiles were not affected by chelation of cytosolic Ca2+. The K+ current induced by a depolarizing pulse was suppressed by extracellular application of TEA+, Ba2+, 4-aminopyridine or quinine with half-maximal inhibitory concentrations of 8.9 mM, 4.6 mM, 86 microM and 26 microM, respectively. The inactivation time course was accelerated by quinine but decelerated by TEA+, when applied to the extracellular (but not the intracellular) solution. Extracellular (but not intracellular) applications of verapamil and nifedipine also quickened the inactivation time course with 50% effective concentrations of 3 and 17 microM, respectively. Quinine, verapamil and nifedipine shifted the steady state inactivation curve towards more negative potentials. Outward single K+ channel events with a unitary conductance of approximately 8.4 pS were observed in excised inside-out patches of the basolateral membrane, when the patch was depolarized to -40 mV. The ensemble current rapidly activated and thereafter slowly inactivated with similar time constants to those of whole-cell K+ currents. It is concluded that the basolateral membrane of guinea pig villus enterocytes has a voltage-gated, time-dependent, Ca(2+)-insensitive, small-conductance K+ channel. Quinine, verapamil, and nifedipine accelerate the inactivation time course by affecting the inactivation gate from the external side of the cell membrane.     

Characterization of the calcium-sensitive voltage-gated delayed rectifier potassium channel in isolated guinea pig hepatocytes

The voltage-dependent K+ channel was examined in enzymatically isolated guinea pig hepatocytes using whole-cell, excised outside-out and inside- out configurations of the patch-clamp technique. The resting membrane potential in isolated hepatocytes was -25.3 +/- 4.9 mV (n = 40). Under the whole-cell voltage-clamp, the time-dependent delayed rectifier outward current was observed at membrane potentials positive to -20 mV at physiological temperature (37 degrees C). The reversal potential of the current, as determined from tail current measurements, shifted by approximately 57 mV per 10-fold change in the external K+ concentration. In addition, the current did not appear when K+ was replaced with Cs+ in the internal and external solutions, indicating that the current was carried by K+ ions. The envelope test of the tails demonstrated that the growth of the tail current followed that of the current activation. The ratio between the activated current and the tail amplitude was constant during the depolarizing step. The time course of growth and deactivation of the tail current were best described by a double exponential function. The current was suppressed in Ca(2+)-free, 5 mM EGTA internal or external solution (pCa > 9). The activation curve (P infinity curve) was not shifted by changing the internal Ca2+ concentration ([Ca2+]i). The current was inhibited by bath application of 4-aminopyridine or apamin. alpha 1-Adrenergic stimulation with noradrenaline enhanced the current but beta-adrenergic stimulation with isoproterenol had no effect on the current. In single- channel recordings from outside-out patches, unitary current activity was observed by depolarizing voltage-clamp steps whose slope conductance was 9.5 +/- 2.2 pS (n = 10). The open time distribution was best described by a single exponential function with the mean open lifetime of 18.5 +/- 2.6 ms (n = 14), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 2.0 +/- 0.3 ms (n = 14) and that for the slow component of 47.7 +/- 5.9 ms (n = 14). Ensemble averaged current exhibited delayed rectifier nature which was consistent with whole-cell measurements. In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The concentration of Ca2+ at the half-maximal activation was 0.031 microM. These results suggest that guinea pig hepatocytes possess voltage-gated delayed rectifier K+ channels which are modified by intracellular Ca2+.     

A delayed rectifier potassium current in Xenopus oocytes.

A delayed voltage-dependent K+ current endogenous to Xenopus oocytes has been investigated by the voltage-clamp technique. Both activation and inactivation of the K+ current are voltage-dependent processes. The K+ currents were activated when membrane potential was depolarized from a holding potential of -90 to -50 mV. The peak current was reached within 150 ms at membrane potential of +30 mV. Voltage-dependent inactivation of the current was observed by depolarizing the membrane potential from -50 to 0 mV at 10-mV increments. Voltage-dependent inactivation was a slow process with a time constant of 16.5 s at -10 mV. Removal of Ca2+ from the bath has no effect on current amplitudes, which indicates that the current is Ca2+)-insensitive. Tail current analysis showed that reversal potentials were shifted by changing external K+ concentration, as would be expected for a K(+)-selective channel. The current was sensitive to quinine, a K+ channel blocker, with a Ki of 35 microM. The blockade of quinine is voltage-independent in the range of -20 to +60 mV. Whereas oocytes from the same animal have a relatively homogeneous current distribution, average amplitude of the K+ current varied among oocytes from different animals from 30 to 400 nA at membrane potential of +30 mV. Our results indicate the presence of the endogenous K+ current in Xenopus oocytes with characteristics of the delayed rectifier found in some nerve and muscle cells.     

Slow cholinergic and peptidergic transmission in sympathetic ganglia

Experiments of voltage-clamped bullfrog sympathetic neurons suggest that the "slow depolarization" produced by orthodromic stimulation, by muscarinic agonists, or by the peptide luteinizing hormone-releasing hormone (LHRH), results from the suppression of a time- and voltage-dependent outward K+ current, the "M current" (IM). This current is activated between -60 and -10mV, with a half-maximal activation voltage of -35 mV, a minimum time constant (TM) of 150 ms at -35 mV, and a voltage sensitivity corresponding to a single gating particle with a minimum valency of 4.IM does not show time-dependent inactivation within its activation range and provides the sole potential-sensitive component of the steady outward membrane conductances between -60 and -25 mV. Muscarinic agonists and LHRH selectively depress IM via different receptors, without altering their voltage sensitivity. Although not dependent on external Ca2+ ion, IM is also selectively depressed by Ba2+ ions, so accounting for the cholinomimetic action of Ba2+. It is suggested that IM acts as a braking control on spike discharges and that removal of this control during slow cholinergic and peptidergic transmission provides a unique synaptic tuning mechanism.     

Voltage-dependent block of cardiac inward-rectifying potassium current by monovalent cations

The inward-rectifying K+ current (IK1) in cat ventricular myocytes, like inward-rectifying K+ currents in many other preparations, exhibited a negative slope conductance region at hyperpolarized membrane potentials that was time-dependent. This was evident as an inactivation of inward current elicited by hyperpolarizing voltage-clamp pulses resulting in a negative slope region of the steady-state current-voltage relationship at potentials negative to -140 mV. Removing extracellular Na+ prevented the development of the negative slope in this voltage region, suggesting that Na+ can block IK1 channels in a time- and voltage-dependent manner. The time and voltage dependence of Cs+-induced block of IK1 was also examined. Cs+ blocked inward current in a manner similar to that of Na+, but the former was much more potent. The fraction of current blocked by Cs+ in the presence of Na+ was reduced in a time- and voltage-dependent manner, which suggested that these blocking ions compete for a common or at least similar site of action. In the absence of Na+, inactivation of IK1 could also be induced by both Cs+ and Li+. However, Li+ was less potent than Na+ in this respect. Calculation of the voltage sensitivity of current block by each of these ions suggests that the mechanism of block by each is similar.     

Two components of cardiac delayed rectifier K+ current. Differential sensitivity to block by class III antiarrhythmic agents

An envelope of tails test was used to show that the delayed rectifier K+ current (IK) of guinea pig ventricular myocytes results from the activation of two outward K+ currents. One current was specifically blocked by the benzenesulfonamide antiarrhythmic agent, E-4031 (IC50 = 397 nM). The drug-sensitive current, "IKr" exhibits prominent rectification and activates very rapidly relative to the slowly activating drug-insensitive current, "IKs." IKs was characterized by a delayed onset of activation that occurs over a voltage range typical of the classically described cardiac IK. Fully activated IKs, measured as tail current after 7.5-s test pulses, was 11.4 times larger than the fully activated IKr. IKr was also blocked by d-sotalol (100 microM), a less potent benzenesulfonamide Class III antiarrhythmic agent. The activation curve of IKr had a steep slope (+7.5 mV) and a negative half-point (-21.5 mV) relative to the activation curve of IKs (slope = +12.7 mV, half-point = +15.7 mV). The reversal potential (Erev) of IKr (-93 mV) was similar to EK (-94 mV for [K+]o = 4 mM), whereas Erev of IKs was -77 mV. The time constants for activation and deactivation of IKr made up a bell-shaped function of membrane potential, peaking between -30 and -40 mV (170 ms). The slope conductance of the linear portion of the fully activated IKr-V relation was 22.5 S/F. Inward rectification of this relation occurred at potentials greater than -50 mV, resulting in a voltage-dependent decrease in peak IKr at test potentials greater than 0 mV. Peak IKr at 0 mV averaged 0.8 pA/pF (n = 21). Although the magnitude of IKr was small relative to fully activated IKs, the two currents were of similar magnitude when measured during a relatively short pulse protocol (225 ms) at membrane potentials (-20 to +20 mV) typical of the plateau phase of cardiac action potentials.     

Alteration of the transmembrane K+ gradient during development of delayed rectifier in isolated rat pulmonary arterial cells

The properties of the tail current associated with the delayed rectifier K+ current (IK) in isolated rat pulmonary artery smooth muscle cells were examined using the whole cell patch clamp technique. The tail currents observed upon repolarization to -60 mV after brief (e.g., 20 ms) or small (i.e. to potentials negative of 0 mV) depolarizations were outwardly directed, as expected given the calculated K+ reversal potential of -83 mV. The tail currents seen upon repolarization after longer (e.g., 500 ms) and larger (e.g., to +60 mV) depolarizations tended to be inwardly directed. Depolarizations of intermediate strength and/or duration were followed by biphasic tail currents, which were inwardly directed immediately upon repolarization, but changed direction and became outwardly directed before deactivation was complete. When cells were depolarized to +60 mV for 500 ms both IK and the subsequent inward tail current at -60 mV were similarly blocked by phencyclidine. Both IK and the inward tail current were also blocked by 4-aminopyridine. Application of progressively more depolarized 30 s preconditioning potentials inactivated IK, and reduced the inward tail current amplitude with a similar potential dependency. These results indicated that the inward tail current was mediated by IK. The reversal potential of the tail current became progressively more positive with longer depolarizations to +60 mV, shifting from -76.1 +/- 2.2 mV (n = 10) after a 20-ms step to -57.7 +/- 3.5 mV (n = 9) after a 500-ms step. Similar effects occurred when extracellular K+ and Na+ were replaced by choline. When extracellular K+ was raised to 50 mM, the tail current was always inwardly directed at -60 mV, but showed little change in amplitude as the duration of depolarization was increased. These observations are best explained if the dependencies of tail current direction and kinetics upon the duration of the preceding depolarization result from an accumulation of K+ at the external face of the membrane, possibly in membrane invaginations. A mathematical model which simulates the reversal potential shift and the biphasic kinetics of the tail current on this basis is presented.     

Time-dependent outward current in guinea pig ventricular myocytes. Gating kinetics of the delayed rectifier

Several conflicting models have been used to characterize the gating behavior of the cardiac delayed rectifier. In this study, whole-cell delayed rectifier currents were measured in voltage-clamped guinea pig ventricular myocytes, and a minimal model which reproduced the observed kinetic behavior was identified. First, whole-cell potassium currents between -10 and +70 mV were recorded using external solutions designed to eliminate Na and Ca currents and two components of time-dependent outward current were found. One component was a La3(+)-sensitive current which inactivated and resembled the transient outward current described in other cell types; single-channel observations confirmed the presence of a transient outward current in these guinea pig ventricular cells (gamma = 9.9 pS, [K]o = 4.5 mM). Analysis of envelopes of tail amplitudes demonstrated that this component was absent in solutions containing 30-100 microM La3+. The remaining time-dependent current, IK, activated with a sigmoidal time course that was well-characterized by three time constants. Nonlinear least-squares fits of a four-state Markovian chain model (closed - closed - closed - open) to IK activation were therefore compared to other models previously used to characterize IK gating: n2 and n4 Hodgkin-Huxley models and a Markovian chain model with only two closed states. In each case the four-state model was significantly better (P less than 0.05). The failure of the Hodgkin-Huxley models to adequately describe the macroscopic current indicates that identical and independent gating particles should not be assumed for this K channel. The voltage-dependent terms describing the rate constants for the four-state model were then derived using a global fitting approach for IK data obtained over a wide range of potentials (-80 to +70 mV). The fit was significantly improved by including a term representing the membrane dipole forces (P less than 0.01). The resulting rate constants predicted long single-channel openings (greater than 1 s) at voltages greater than 0 mV. In cell-attached patches, single delayed rectifier channels which had a mean chord conductance of 5.4 pS at +60 mV ([K]o = 4.5 mM) were recorded for brief periods. These channels exhibited behavior predicted by the four-state model: long openings and latency distributions with delayed peaks. These results suggest that the cardiac delayed rectifier undergoes at least two major transitions between closed states before opening upon depolarization.     

The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. I. Basic characterization and kinetic analysis

Enzymatically isolated myocytes from ferret right ventricles (12-16 wk, male) were studied using the whole cell patch clamp technique. The macroscopic properties of a transient outward K+ current I(to) were quantified. I(to) is selective for K+, with a PNa/PK of 0.082. Activation of I(to) is a voltage-dependent process, with both activation and inactivation being independent of Na+ or Ca2+ influx. Steady-state inactivation is well described by a single Boltzmann relationship (V1/2 = -13.5 mV; k = 5.6 mV). Substantial inactivation can occur during a subthreshold depolarization without any measurable macroscopic current. Both development of and recovery from inactivation are well described by single exponential processes. Ensemble averages of single I(to) channel currents recorded in cell-attached patches reproduce macroscopic I(to) and indicate that inactivation is complete at depolarized potentials. The overall inactivation/recovery time constant curve has a bell-shaped potential dependence that peaks between -10 and -20 mV, with time constants (22 degrees C) ranging from 23 ms (-90 mV) to 304 ms (-10 mV). Steady-state activation displays a sigmoidal dependence on membrane potential, with a net aggregate half- activation potential of +22.5 mV. Activation kinetics (0 to +70 mV, 22 degrees C) are rapid, with I(to) peaking in approximately 5-15 ms at +50 mV. Experiments conducted at reduced temperatures (12 degrees C) demonstrate that activation occurs with a time delay. A nonlinear least- squares analysis indicates that three closed kinetic states are necessary and sufficient to model activation. Derived time constants of activation (22 degrees C) ranged from 10 ms (+10 mV) to 2 ms (+70 mV). Within the framework of Hodgkin-Huxley formalism, Ito gating can be described using an a3i formulation.     

Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes

Depolarization-activated outward K+ currents in isolated adult rat ventricular myocytes were characterized using the whole-cell variation of the patch-clamp recording technique. During brief depolarizations to potentials positive to -40 mV, Ca(2+)-independent outward K+ currents in these cells rise to a transient peak, followed by a slower decay to an apparent plateau. The analyses completed here reveal that the observed outward current waveforms result from the activation of two kinetically distinct voltage-dependent K+ currents: one that activates and inactivates rapidly, and one that activates and inactivates slowly, on membrane depolarization. These currents are referred to here as Ito (transient outward) and IK (delayed rectifier), respectively, because their properties are similar (although not identical) to these K+ current types in other cells. Although the voltage dependences of Ito and IK activation are similar, Ito activates approximately 10-fold and inactivates approximately 30-fold more rapidly than IK at all test potentials. In the composite current waveforms measured during brief depolarizations, therefore, the peak current predominantly reflects Ito, whereas IK is the primary determinant of the plateau. There are also marked differences in the voltage dependences of steady-state inactivation of these two K+ currents: IK undergoes steady-state inactivation at all potentials positive to -120 mV, and is 50% inactivated at -69 mV; Ito, in contrast, is insensitive to steady-state inactivation at membrane potentials negative to -50 mV. In addition, Ito recovers from steady-state inactivation faster than IK: at -90 mV, for example, approximately 70% recovery from the inactivation produced at -20 mV is observed within 20 ms for Ito; IK recovers approximately 25-fold more slowly. The pharmacological properties of Ito and IK are also distinct: 4-aminopyridine preferentially attenuates Ito, and tetraethylammonium suppresses predominantly IK. The voltage- and time-dependent properties of these currents are interpreted here in terms of a model in which Ito underlies the initial, rapid repolarization phase of the action potential (AP), and IK is responsible for the slower phase of AP repolarization back to the resting membrane potential, in adult rat ventricular myocytes.     

Kinetic analysis of a transient potassium current in rat cerebellar granule cells in vitro]

Thin slices were prepared from cerebella of 10-24 day old rats and examined with whole-cell patch-clamp methods. Depolarizing steps from holding potentials negative to -60 mV elicited an early transient outward current, identified as IA, and a late outward K+ current. Depolarizations from -50 mV failed to evoke any A current and gave only a slowly rising component similar to the delayed K+ current, which inactivated thereafter with a time constant of 2.5 s at -30 mV. The IA peaked in 1-2 ms, decayed following a double exponential with time constants of 8.1 and 53.2 ms at +20 mV and was half-inactivated at -82.5 mV. 4-AP 4 mM depressed both K+ currents showing little specificity between them, while TEA 20 mM selectively abolished only the delayed K+ current.     

Inward current in single smooth muscle cells of the guinea pig taenia coli

Using the tight-seal voltage-clamp method, the ionic currents in the enzymatically dispersed single smooth muscle cells of the guinea pig taenia coli have been studied. In a physiological medium containing 3 mM Ca2+, the cells are gently tapering spindles, averaging 201 (length) x 8 microns (largest diameter in center of cell), with a volume of 5 pl. The average cell capacitance is 50 pF, and the specific membrane capacitance 1.15 microF/cm2. The input impedance of the resting cell is 1-2 G omega. Spatially uniform voltage-control prevails after the first 400 microseconds. There is much overlap of the inward and outward currents, but the inward current can be isolated by applying Cs+ internally to block all potassium currents. The inward current is carried by Ca2+. Activation begins at approximately -30 mV, maximum ICa occurs at +10-+20 mV, and the reversal potential is approximately +75 mV. The Ca2+ channel is permeable to Sr2+ and Ba2+, and to Cs+ moving outwards, but not to Na+ moving inwards. Activation and deactivation are very rapid at approximately 33 degrees C, with time-constants of less than 1 ms. Inactivation has a complex time course, resolvable into three exponential components, with average time constants (at 0 mV) of 7, 45, and 400 ms, which are affected differently by voltage. Steady-state inactivation is half-maximal at -30 mV for all components combined, but -36 mV for the fast component and -26 and -23 mV for the other two components. The presence of multiple forms of Ca2+ channel is inferred from the inactivation characteristics, not from activation properties. Recovery of the fast channel occurs with a time-constant of 72 ms (at +10 mV). Ca2+ influx during an action potential can transfer approximately 9 pC of charge, which could elevate intracellular Ca2+ concentration adequately for various physiological functions.     

A slowly inactivating potassium current in native oocytes of Xenopus laevis

Membrane currents were recorded in voltage-clamped oocytes of Xenopus laevis in response to voltage steps. We describe results obtained in oocytes obtained from one donor frog, which showed an unusually large outward current upon depolarization. Measurements of reversal potentials of tail currents in solutions of different K+ concentration indicated that this current is carried largely by K+ ions. It was strongly reduced by extracellular application of tetraethylammonium, though not by Ba2+ or 4-aminopyridine. Removal of surrounding follicular cells did not reduce the K+ current, indicating that it arises across the oocyte membrane proper. Activation of the K+ conductance was first detected with depolarization to about -12 mV, increased with a limiting voltage sensitivity of 3 mV for an e-fold change in current, and was half-maximally activated at about +10 mV. The current rose following a single exponential timecourse after depolarization, with a time constant that shortened from about 400 ms at -10 mV to about 15 ms at +80 mV. During prolonged depolarization the current inactivated with a time constant of about 4 s, which did not alter greatly with potential. The K+ current was independent of Ca2+, as it was not altered by addition of 10 mM Mn2+ to the bathing medium, or by intracellular injection of EGTA. Noise analysis of K+ current fluctuations indicated that the current is carried by channels with a unitary conductance of about 20 ps and a mean open lifetime of about 300 ms (at room temperature and potential of +10 to +20 mV).     

Calcium current activated upon hyperpolarization of Paramecium tetraurelia

Hyperpolarization of Paramecium tetraurelia under conditions where K+ currents are suppressed elicits an inward current that activates rapidly toward a peak at 25-80 ms and decays thereafter. This peak current (Ihyp) is not affected by removing Cl ions from the microelectrodes used to clamp membrane potential, or by changing extracellular Cl- concentration, but is lost upon removing extracellular Ca2+. Ihyp is also lost upon replacing extracellular Ca2+ with equimolar concentrations of Ba2+, Co2+, Mg2+, Mn2+, or Sr2+, suggesting that the permeability mechanism that mediates Ihyp is highly selective for Ca2+. Divalent cations also inhibit Ihyp when introduced extracellularly, in a concentration- and voltage-dependent manner. Ba2+ inhibits Ihyp with an apparent dissociation constant of 81 microM at -110 mV, and with an effective valence of 0.42. Ihyp is also inhibited reversibly by amiloride, with a dissociation constant of 0.4 mM. Ihyp is not affected significantly by changes in extracellular Na+, K+, or H+ concentration, or by EGTA injection. Also, it is unaffected by manipulations or mutations that suppress the depolarization-activated Ca2+ current or the various Ca(2+)-dependent currents of Paramecium. We suggest that Ihyp is mediated by a novel, hyperpolarization-activated calcium conductance that is distinct from the one activated by depolarization.     

Voltage-gated transient currents in bovine adrenal fasciculata cells. I. T-type Ca2+ current

The whole cell version of the patch clamp technique was used to identify and characterize voltage-gated Ca2+ channels in enzymatically dissociated bovine adrenal zona fasciculata (AZF) cells. The great majority of cells (84 of 86) expressed only low voltage-activated, rapidly inactivating Ca2+ current with properties of T-type Ca2+ current described in other cells. Voltage-dependent activation of this current was fit by a Boltzmann function raised to an integer power of 4 with a midpoint at -17 mV. Independent estimates of the single channel gating charge obtained from the activation curve and using the "limiting logarithmic potential sensitivity" were 8.1 and 6.8 elementary charges, respectively. Inactivation was a steep function of voltage with a v1/2 of -49.9 mV and a slope factor K of 3.73 mV. The expression of a single Ca2+ channel subtype by AZF cells allowed the voltage-dependent gating and kinetic properties of T current to be studied over a wide range of potentials. Analysis of the gating kinetics of this Ca2+ current indicate that T channel activation, inactivation, deactivation (closing), and reactivation (recovery from inactivation) each include voltage-independent transitions that become rate limiting at extreme voltages. Ca2+ current activated with voltage- dependent sigmoidal kinetics that were described by an m4 model. The activation time constant varied exponentially at test potentials between -30 and +10 mV, approaching a voltage-independent minimum of 1.6 ms. The inactivation time constant (tau i) also decreased exponentially to a minimum of 18.3 ms at potentials positive to 0 mV. T channel closing (deactivation) was faster at more negative voltages; the deactivation time constant (tau d) decreased from 8.14 +/- 0.7 to 0.48 +/- 0.1 ms at potentials between -40 and -150 mV. T channels inactivated by depolarization returned to the closed state along pathways that included two voltage-dependent time constants. tau rec-s ranged from 8.11 to 4.80 s when the recovery potential was varied from - 50 to -90 mV, while tau rec-f decreased from 1.01 to 0.372 s. At potentials negative to -70 mV, both time constants approached minimum values. The low voltage-activated Ca2+ current in AZF cells was blocked by the T channel selective antagonist Ni2+ with an IC50 of 20 microM. At similar concentrations, Ni2+ also blocked cortisol secretion stimulated by adrenocorticotropic hormone. Our results indicate that bovine AZF cells are distinctive among secretory cells in expressing primarily or exclusively T-type Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.     

A delayed rectifier current is modulated by the circadian pacemaker in Bulla

Basal retinal neurons of the marine mollusc Bulla gouldiana continue to express a circadian modulation of their membrane conductance for at least two cycles in cell culture. Voltage-dependent currents of these pacemaker cells were recorded using the whole-cell perforated patch-clamp technique to characterize outward currents and investigate their putative circadian modulation. Three components of the outward potassium current were identified. A transient outward current (IA) was activated after depolarization from holding potentials greater than -30 mV, inactivated with a time constant of 50 ms, and partially blocked by 4-aminopyridine (1-5 mM). A Ca(2+)-dependent potassium current (IK(Ca)) was activated by depolarization to potentials more positive than -10 mV and was blocked by removing Ca2+ from the bath or by applying the Ca2+ channel blockers Cd2+ (0.1-0.2 mM) and Ni2+ (1-5 mM). A sustained Ca(2+)-independent current component including the delayed rectifier current (IK) was recorded at potentials positive to -20 mV in the absence of extracellular Na+ and Ca2+ and was partially blocked by tetraethylammonium chloride (TEA, 30mM). Whole-cell currents recorded before and after the projected dawn and normalized to the cell capacitance revealed a circadian modulation of the delayed rectifier current (IK). However, the IA and IK(Ca) currents were not affected by the circadian pacemaker.