Abstract
Salivary acini were enzymatically isolated from submandibular glands of adult male mice. The patch-clamp technique was employed to investigate the conductive properties and activities of a large-conductance K+ channel in both cell-attached and in excised patches of basolateral acinar cell membranes. In excised, inside out, patches with identical high-K+ solutions (145 mM-KCl) on either side of the membrane the current-voltage (I-V) plot was linear. The mean single-channel conductance was 245 +/- 4.8 ps with a single-channel permeability of 4.6 X 10(-13) cm3 s-1. At Ca2+ concentrations of 10(-9)-10(-8) M bathing the intracellular membrane face the channel was exquisitely sensitive to changes in transmembrane potential, i.e. voltage sensitive. At 10(-7) M-Ca the channel was almost always open and displayed little sensitivity to voltage. Single-channel currents were recorded in cell-attached patches. When the recording pipettes contained the high-K+ solution the I-V plots were linear and the mean single-channel conductance and permeability almost identical to that in the excised patches. The mean spontaneous resting potential of the acinar cells bathed in physiological saline (140 mM-NaCl, 4.5 mM-KCl) was -43 +/- 1.8 mV. The voltage sensitivity of the in situ K+ channel was very similar to that recorded in excised patches at 10(-9)-10(-8) M-Ca. In experiments designed to reproduce the physiological ionic gradients across the patch membrane pipettes were filled with the high-Na+ solution. The I-V plot was not linear but showed pronounced rectification at negative membrane potentials. The channel is K+ selective and the extrapolated reversal potential was close to -90 mV. The single-channel conductance at the spontaneous resting membrane potential was about 35 pS. The single-channel permeability was however only slightly reduced at 4.29 X 10(-13) cm3 s-1. It was demonstrated that current flow through the open K+ channel could be accurately modelled using constant field electrodiffusion theory. Continuous in situ recordings before and after application of the agonist acetylcholine to the solution bathing the acini revealed that acetylcholine stimulation is associated with a marked increase in the frequency and duration of K+ currents in the patch membrane. The increased current activity in the patch membrane during acetylcholine application must be mediated via an intracellular second messenger and was very similar to that observed in the excised patches on increasing ionized Ca2+ concentrations from 10(-8) to 10(-7) M.(ABSTRACT TRUNCATED AT 400 WORDS)