Voltage-dependent inactivation of inward-rectifying single-channel currents in the guinea-pig heart cell membrane.

Inward currents through single K+ channels in isolated ventricular heart cells of the guinea-pig were recorded using the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The voltage-dependent gating properties of the channels were examined in the potential range between 0 and -120 mV with 145 mM-KCl on the extracellular side of the membrane patch, i.e. with approximately symmetrical transmembrane K+ concentrations. When voltage pulses from 0 mV to negative test potentials were applied to patches containing several channels, more channels were open at the beginning of the pulses than in the steady state. Averages of many current responses showed inactivation of the mean current in response to the hyperpolarizing voltage pulses. The inactivation was stronger and faster at larger hyperpolarization. The lifetimes of the open and closed states of the channel and the probability of the open state p were estimated from records of the elementary currents at various constant potentials. As indicated by the inactivation of the averaged currents, the value of p was smaller at more negative potentials, approximately 0.15 at -50 mV and 0.02 at -110 mV. This caused a negative slope in the current-voltage relation of the time-averaged current at potentials more negative than -50 mV. The channel openings were grouped in complex bursts. At least three exponentials were needed to fit the frequency histogram of the lifetimes of all closed states (time constants at -50 mV: 1.1 ms, 16 ms and 3.2 s). The lifetimes of the individual openings were exponentially distributed (time constant: 70 ms). The kinetics of the channel were interpreted by two different models involving three states of a channel (closed-closed-open or closed-open-closed). The rate constants and their voltage dependence were estimated for both models. Both models describe the data equally well; the reason for this ambiguity is discussed. The channels are blocked by Cs+ or Ba2+. Cs+ (0.1 mM) caused frequent and short interruptions of the individual channel openings. Ba2+ (0.5 mM) also shortened the openings and in addition decreased the number of openings per burst. The results suggest that the inward-rectifying current IK1 in heart ventricular cells is partially inactivated by hyperpolarization. The inactivation could account for part of the time-dependent decrease in the whole-cell current previously ascribed to depletion of K+

Conductance properties of single inwardly rectifying potassium channels in ventricular cells from guinea-pig heart

Single ventricular cells were enzymatically isolated from adult guinea-pig hearts (Isenberg & Klöckner, 1982). The patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) was used to examine the conductance properties of an inward-rectifying K+ channel present in their sarcolemmal membrane. When the K+ concentration on the extracellular side of the patch was between 10.8 and 300 mM, inward current steps were observed at potentials more negative than the K+ equilibrium potential (EK). At more positive potentials no current steps were detectable, demonstrating the strong rectification of the channel. The zero-current potential extrapolated from the voltage dependence of the inward currents depends on the external K4 concentration [K+]o in a fashion expected for a predominantly K+-selective ion channel. It is shifted by 49 mV for a tenfold change in [K+]o. The conductance of the channel depends on the square root of [K+]o. In approximately symmetrical transmembrane K+ concentrations (145 mM-external K+), the single-channel conductance is 27 pS (at 19-23 degrees C). In normal Tyrode solution (5.4 mM-external K+) we calculate a single-channel conductance of 3.6 pS. The size of inward current steps at a fixed negative membrane potential V increases with [K+]o. The relation between step size and [K+]o shows saturation. Assuming a Michaelis-Menten scheme for binding of permeating K+ to the channel, an apparent binding constant of 210 mM is calculated for a membrane potential of -100 mV. For this potential the current at saturating [K+]o is estimated as 6.5 pA. The rectification of the single-channel conductance at membrane potentials positive to EK occurs within 1.5 ms of stepping the membrane potential from a potential of high conductance to one of low conductance. In addition to the main conductance state, the channel can adopt several substates of conductance. The main state could be the result of the simultaneous opening of four conducting subunits, each of which has a conductance of about 7 pS in 145 mM-external K+. The density of the inward-rectifying K+ channels in the ventricular sarcolemma is 0-10 channel/10 micron2 of surface membrane; the average of twenty-eight patches was 1 channel/1.8 micron2. It is concluded that the inward-rectifying K+ channels mediate the resting K+ conductance of ventricular heart muscle and the current termed IK1 in conventional voltage-clamp experiments

Calcium and delayed potassium currents in mouse pancreatic beta-cells under voltage-clamp conditions.

Pancreatic islets of NMRI mice were dissociated into single cells which were kept in tissue culture for 1-3 days. The whole-cell configuration of the patch-clamp technique was used to study inward and delayed outward currents of beta-cells under voltage-clamp conditions at 20-22 degrees C. Outward currents were suppressed by substituting the impermeant cation N-methyl-D-glucamine for intracellular K+. The remaining inward current had a V-shaped current-voltage relation reaching a peak value of 39 +/- 4 pA (mean +/- S.E. of mean) around -15 mV. It was identified as a Ca2+ current, because the peak amplitude was increased 1.6 times by increasing external [Ca2+] ([Ca2+]o) from 2.6 mM to 10 mM and it was blocked by Co2+ (5 mM) or nifedipine (5 microM) but not by TTX (20 microM). The activation time constant of the inward current at -10 mV was 1.28 +/- 0.08 ms. The relation between the degree of activation (estimated from the size of the tail currents) and membrane potential V followed the sigmoidal function f = 1/(1 + exp [(Vh-V)/k]) with half-maximal activation potential, Vh = 4 +/- 1 mV and slope factor, k = 14 +/- 1 mV (for [Ca2+]o 10 mM). The inward current inactivated only weakly during depolarizing pulses of 0.1-1 s duration. The delayed outward current (in experiments with 155 mM-internal [K+] ([K+]i)) had a linear voltage dependence at potentials above -20 mV; its amplitude at -10 mV was 210 +/- 30 pA. Tail currents related to the activation of the outward current had K+-dependent reversal potentials. The current was blocked by extracellularly applied tetraethylammonium (20 mM) and 4-aminopyridine (2 mM). It was not affected by glibenclamide (3 microM), tolbutamide (0.2 mM) and alterations of intracellular [Ca2+] (1 nM-1 microM). The activation time constant of the outward current at -10 mV was 21 +/- 3 ms. The voltage dependence of activation could be described by the sigmoidal function (see above) with Vh = 19 +/- 1 mV and k = 5.6 +/- 0.4 mV. The outward current inactivated during long (15 s) depolarizing pre-pulses (time constant at -10 mV: 2.6 +/- 0.6 s). 50% inactivation occurred at Vh = -36 +/- 2 mV, k was -4.1 +/- 0.2 mV. Inward and outward currents during depolarizing voltage pulses in beta-cells are similar to Ca2+ and delayed K+ currents in other cell types. These currents seem sufficient to generate the action potentials of the beta-cell