Chloride channels in excised membrane patches from human platelets: effect of intracellular calcium

Human platelets were studied by patch clamp recordings from inside-out membranes; there were formed by briefly dipping the platelet, in cell-attached mode, into silicone grease. At 20 degrees C in symmetrical 150 mM NaCl, spontaneous channel openings were rarely observed at negative potentials, whereas depolarised potentials (+ 60 to + 100 mV) elicited sustained channel activity in 38% of patches. The single channel conductance was 53 +A- 1 pS at + 80 mV (outward current), decreasing to 20 +/- 2 pS at -80 mV (inward current). Ion substitution experiments indicated that this channel conducts Cl- and not Na+. Furthermore, 5-nitro-2-(3-phenylpropylamino)benzoate (100 microM), a recognized inhibitor of anion channels, induced a reversible 'flickery' channel block. We estimate that each platelet possesses < or = 30 such channels. Kinetic analysis suggested at least two open channel states (tau = 0.8 +/- 0.2 ms, tau = 22 +/- 14 ms, n = 4) and two closed states (tau = 0.8 +/- 0.2 ms, tau = 12 +/- 0.6 ms, n = 4). Increasing [Ca2+]i to 10 microM, following channel activation by depolarisation, had no significant effect on channel kinetics or open probability, however, elevated [Ca2+]i (300 nM-10 microM) increased the number of anion channels activated by subsequent depolarisation. This study represents the first recordings of ionic currents in excised, inside-out membrane patches from human platelets, and provides further evidence for the existence of chloride channels in these cells.