Sivaramakrishnan S, Brodwick M S, Bittner G D
Department of Zoology, College of Pharmacy, University of Texas, Austin 78712.
J Gen Physiol. 1991 Dec;98(6):1181-96. doi: 10.1085/jgp.98.6.1181.
Membrane potential was recorded intracellularly near presynaptic terminals of the excitor axon of the crayfish opener neuromuscular junction (NMJ), while transmitter release was recorded postsynaptically. This study focused on the effects of a presynaptic calcium-activated potassium conductance, gK(Ca), on the transmitter release evoked by single and paired depolarizing current pulses. Blocking gK(Ca) by adding tetraethylammonium ion (TEA; 5-20 mM) to a solution containing tetrodotoxin and aminopyridines caused the relation between presynaptic potential and transmitter release to steepen and shift to less depolarized potentials. When two depolarizing current pulses were applied at 20-ms intervals with gK(Ca) not blocked, the presynaptic voltage change to the second (test) pulse was inversely related to the amplitude of the first (conditioning) pulse. This effect of the conditioning prepulse on the response to the test pulse was eliminated by 20 mM TEA and by solutions containing 0 mM Ca2+/1 mM EGTA, suggesting that the reduction in the amplitude of the test pulse was due to activation of gK(Ca) by calcium remaining from the conditioning pulse. In the absence of TEA, facilitation of transmitter release evoked by a test pulse increased as the conditioning pulse grew from -40 to -20 mV, but then decreased with further increase in the conditioning depolarization. A similar nonmonotonic relationship between facilitation and the amplitude of the conditioning depolarization was reported in previous studies using extracellular recording, and interpreted as supporting an additional voltage-dependent step in the activation of transmitter release. We suggest that this result was due instead to activation of a gK(Ca) by the conditioning depolarization, since facilitation of transmitter release increased monotonically with the amplitude of the conditioning depolarization, and the early time course of the decay of facilitation was prolonged when gK(Ca) was blocked. The different time courses for decay of the presynaptic potential (20 ms) and facilitation (greater than 50 ms) suggest either that residual free calcium does not account for facilitation at the crayfish NMJ or that the transmitter release mechanism has a markedly higher affinity or stoichiometry for internal free calcium than does gK(Ca). Finally, our data suggest that the calcium channels responsible for transmitter release at the crayfish NMJ are not of the L, N, or T type.
在小龙虾开肌神经肌肉接头（NMJ）的兴奋性轴突的突触前终末附近，通过细胞内记录膜电位，同时在突触后记录递质释放。本研究聚焦于突触前钙激活钾电导gK(Ca)对单个和成对去极化电流脉冲诱发的递质释放的影响。通过向含有河豚毒素和氨基吡啶的溶液中添加四乙铵离子（TEA；5 - 20 mM）来阻断gK(Ca)，导致突触前电位与递质释放之间的关系变陡，并向去极化程度较小的电位偏移。当在不阻断gK(Ca)的情况下以20毫秒的间隔施加两个去极化电流脉冲时，对第二个（测试）脉冲的突触前电压变化与第一个（条件）脉冲的幅度呈负相关。20 mM TEA和含有0 mM Ca2+/1 mM EGTA的溶液消除了条件预脉冲对测试脉冲反应的这种影响，这表明测试脉冲幅度的降低是由于条件脉冲残留钙激活了gK(Ca)。在没有TEA的情况下，随着条件脉冲从 - 40 mV增加到 - 20 mV，测试脉冲诱发的递质释放促进作用增强，但随着条件去极化的进一步增加而降低。在先前使用细胞外记录的研究中也报道了促进作用与条件去极化幅度之间类似的非单调关系，并解释为支持递质释放激活过程中额外的电压依赖性步骤。我们认为，该结果相反是由于条件去极化激活了gK(Ca)，因为递质释放促进作用随条件去极化幅度单调增加，并且当gK(Ca)被阻断时，促进作用衰减的早期时间进程延长。突触前电位衰减（20毫秒）和促进作用（大于50毫秒）的不同时间进程表明，要么残留游离钙不能解释小龙虾NMJ处的促进作用，要么递质释放机制对内部游离钙的亲和力或化学计量比明显高于gK(Ca)。最后，我们的数据表明，小龙虾NMJ处负责递质释放的钙通道不是L型、N型或T型。
