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

Abstract

Human platelets were studied by patch clamp recordings from inside-out membranes; these were formed by briefly dipping the platelet, in cell-attached mode, into silicone grease. At 20°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 ± 1 pS at + 80 mV (outward current), decreasing to 20 ± 2 pS at −80 mV (inward current). Io substitution experiments indicated that this channel conducts Cl − and not Na +. Furthermore, 5-nitro-2-(3-phenylpropylamino)benzoate (100 μM), a recognized inhibitor of anion channels, induced a reversible ‘flickery‘ channel block. We estimate that each platelet possesses ≥30 such channels. Kinetic analysis suggested at least two open channel states ( τ = 0.8 ± 0.2 ms, τ= 22 ± 14 ms, n = 4) and two closed states ( τ = 0.8 ± 0.2 ms, τ = 12 ± 0.6 ms, n = 4). Increasing [Ca 2+] i to 10 μM, following channel activation by depolarisation, had no significant effect on channel kinetics or open probability, however, elevated [Ca 2+] i (300 nM–10 μM) 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.