MacKenzie A B, Mahaut-Smith M P
Physiological Laboratory, Cambridge, UK.
Biochim Biophys Acta. 1996 Jan 12;1278(1):131-6. doi: 10.1016/0005-2736(95)00207-3.
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.
采用内面向外膜片钳记录技术对人血小板进行了研究；这些膜片是通过将处于细胞贴附模式的血小板短暂浸入硅酮油脂中形成的。在20℃的对称150 mM NaCl溶液中，在负电位下很少观察到自发通道开放，而在去极化电位（+60至+100 mV）下，38%的膜片中可引发持续的通道活性。在+80 mV（外向电流）时，单通道电导为53±1 pS，在-80 mV（内向电流）时降至20±2 pS。离子置换实验表明，该通道传导Cl-而非Na+。此外，公认的阴离子通道抑制剂5-硝基-2-（3-苯丙基氨基）苯甲酸酯（100 μM）可诱导可逆的“闪烁”通道阻滞。我们估计每个血小板拥有≤30个这样的通道。动力学分析表明至少有两个开放通道状态（τ = 0.8±0.2 ms，τ = 22±14 ms，n = 4）和两个关闭状态（τ = 0.8±0.2 ms，τ = 12±0.6 ms，n = 4）。去极化激活通道后，将[Ca2+]i增加到10 μM对通道动力学或开放概率没有显著影响，然而，升高的[Ca2+]i（300 nM - 10 μM）增加了随后去极化激活的阴离子通道数量。本研究首次记录了人血小板切除的内面向外膜片中的离子电流，并为这些细胞中存在氯离子通道提供了进一步证据。
