Ca(2+)-dependent K+ Channels of High Conductance in Smooth Muscle Cells Isolated from Rat Cerebral Arteries

1. Cerebrovascular smooth muscle cells (CVSMCs) were dispersed from cerebral arteries of adult rats using collagenase and trypsin. The extracellular patch clamp technique was used to study single calcium-activated potassium channels, KCa+ channels, in these cells at 21-23 degrees C. 2. Whole-cell, current clamp recordings showed that isolated CVSMCs possessed a mean resting potential of -41 +/- 7.4 mV (n = 69), an input resistance of 3.2 +/- 0.49 G omega (n = 20) and a capacitance of 24 +/- 2.3 pF (n = 7). 3. Inside-out patches displayed a calcium-dependent potassium channel, KCa+ channel, of mean conductance 207 +/- 10 pS (n = 16) and potassium permeability 3.9 x 10(-13) cm s-1 (n = 16) in symmetrical 140 mM K+ solutions. No substate conductance level was evident. 4. This channel was highly selective for K+ over Na+ or Cs+ (permeability ratio PNa/PK < 0.05; PCs/PK < 0.05, n = 5 patches in each case). Cs+ caused a voltage-dependent block of the open channel. 5. Channel openings were detected at a threshold level of free internal calcium, [Ca2+]i = 10(-8) M, and channels were open half of the time at [Ca2+]i = 2.3 x 10(-5) M (membrane potential, Vm = +40 mV, n = 5). Over the probable physiological range of [Ca2+]i, the open probability of the KCa+ channel increased with the second power of calcium concentration. 6. Open time distributions were well fitted by the sum of two exponential terms, showing the occurrence of at least two kinetically distinguishable open states. Raising [Ca2+]i increased the time constant of the slow exponential component, but had no effect on that of the fast component. 7. At [Ca2+]i < 5 x 10(-5) M, a 14 mV depolarization in membrane potential resulted in an e-fold increase in the probability of KCa+ channels adopting an open state (n = 5). The slow time constant of the open time distributions also increased on membrane depolarization. 8. Tetraethylammonium ions applied to the cytoplasmic membrane face caused a reversible, dose-dependent reduction in current flow through the KCa+ channel. This block was characterized by a dissociation constant of 0.83 +/- 0.09 mM at Vm = +40 mV and [Ca2+]i = 10(-4) M (n = 5). 9. The lower calcium sensitivity and higher sensitivity to tetraethylammonium block distinguish this from other large conductance KCa+ channels in smooth muscle cells.