Currents In Cell-attached Patches Research Articles

Anoxia opens ATP regulated K channels in isolated heart cells of the guinea pig

We studied single channel ionic currents in cell-attached patches of guinea pig heart cells under conditions of anoxia (pO2 less than 0.1 torr) to identify the type of channels which contribute to the anoxia-induced time-independent K current in whole cells. In most experiments, K currents were recorded at negative potentials as inward currents with 150 mmol/l KCl in the pipette. After periods of 5-60 minutes of anoxia, opening events of one to four voltage-independent 83 pS channels developed whose open probability reached a steady state value between 0.6 and 0.95 (T = 35 degrees C). The reversal potential of the unitary currents, determined at 150 mmol/l and 10.8 mmol/l K+ in the pipette, showed that the channels were highly selective for K+ ions. Open time histograms were fitted by two or three exponentials of which the fast time constant (tau O1 = 0.46 +/- 0.20 ms, mean +/- SD) was bandwidth-limited by our filter and the slow components substantially varied (tau O2 = 1.5-19 ms; tau O3 = 23-200 ms). Voltage ramp experiments showed that the channels were slightly rectifying in an inward direction. The unitary conductance of anoxia-induced outward currents at reduced K+ in the pipette was smaller (11 pS at 5.4 mmol K+, 25 pS at 10.8 mmol/l K+) than in excised patches. It is concluded that in isolated cardiocytes substrate-free anoxia causes opening of ATP regulated K channels whose conductance is reduced at physiological levels of [K+]O by a fast block, most likely by intracellular Mg++ and Na+.

The receptor‐regulated calcium influx in mouse submandibular acinar cells is sodium dependent: a patch‐clamp study.

1. Salivary acini were isolated enzymatically from submandibular glands of adult male mice. The patch-clamp technique was employed to record K+ currents in cell-attached patches of basolateral membrane. Application of acetylcholine (10(-5) M) to the medium bathing the cells results in a pronounced and sustained activation of the K+ channels in the cell-attached patches, an effect mediated by an intracellular second messenger. In the present study we investigate the mechanism by which acetylcholine achieves activation of K+ channels. 2. The effects of acetylcholine on single-channel activity were shown to be dependent on extracellular Ca2+, i.e. due to Ca2+ influx. In cells bathed in Ca2+-free medium acetylcholine activation resulted in no increase in single-channel open probability. This blockade could be reversed by reintroduction of Ca2+ to the extracellular fluid in the continued presence of the agonist. The effects of acetylcholine in control medium (1.2 mM-Ca2+) were mimicked by the Ca2+ ionophore, A23187 (10(-8) M). 3. In K+-depolarized cells (bathed in a Na+-free, 145 mM-KCl solution) there was no evidence of any voltage-activated Ca2+ influx pathway. In the K+-depolarized cells acetylcholine application was no longer associated with any increase in the open probability of the K+ channels. K+ channels could be activated by adding A23187 (10(-8) M) to the high-K+ solution. 4. Cells bathed in another Na+-free (N-methyl-D-glucamine substituted for Na+) but non-depolarizing solution were also refractory to acetylcholine. K+ currents could, however, be activated in patches attached to these cells by application of A23187 (10(-8) M) or by the introduction of 20 mM-Na+ to the extracellular fluid in the presence of acetylcholine. The increased activity associated with the reintroduction of Na+ was totally reversed by atropine, i.e. it was receptor regulated. 5. The data presented above indicate that the cholinergic regulation of K+ channels is secondary to the receptor-regulated activation of a Ca2+ influx pathway. There is no evidence of voltage-activated Ca2+ influx in these cells. The cholinergic activation of Ca2+ influx is abolished in Na+-depleted cells. We conclude that the Na+ dependency indicates either that Na+ is involved in the gating of some voltage-independent Ca2+ channel or that Ca2+ entry is via a coupled Na+-Ca2+ co- or countertransport pathway.

Calcium channels of amphibian stomach and mammalian aorta smooth muscle cells

Whole-cell and single-channel calcium currents were studied using single smooth muscle cells enzymatically-isolated from stomach of Amphiuma tridactylum and from guinea-pig aorta. These cells have a high specific resistance and can sustain calcium action potentials after suppression of potassium currents. Dialyzed Amphiuma smooth muscle cells had calcium currents which were stable for several hours whereas the calcium currents of aortic cells ran down quickly. Single channel calcium currents in cell-attached patches behaved similarly for the two cell types. Calcium channel conductance in 110 mM barium was 12 pS and the mean open time was 1.4 ms at a nominal membrane potential of +10 mV. Exposure of both cell types to BAY K8644 resulted in a dramatic prolongation of the calcium channel open times and a shift in the probability of opening to more negative potentials. Low-threshold calcium channels were not identified in the extensively studied amphibian cells. High-threshold calcium channels therefore appear to be the primary pathway for the calcium influx that produces contraction in these smooth muscle cells.