We used a two-microelectrode current clamp to investigate various characteristics of the Ca(2+)-activated K+ conductance [gK(Ca)] and Ca2+ conductance (gCa), and transmitter release in presynaptic terminals of excitatory neuromuscular junctions in the crayfish walking leg. 2. Voltage-activated Na+ conductances (gNa) and K+ conductances [gK(v)] were blocked with tetrodotoxin and 3,4-diaminopyridine, respectively. Under these conditions, presynaptic depolarization produced by a first (conditioning) pulse admitted Ca2+ into the presynaptic terminals and activated gK(Ca), which modulated the amplitude of the depolarization produced by a second (test) pulse. The relative amount of gK(Ca) measured at the test pulse increased with increased magnitude or duration of the conditioning pulse. 3. A brief hyperpolarization immediately after a conditioning pulse substantially reduced gK(Ca). 4. gK(Ca) activation was blocked by funnel web spider toxin (a Ca2+ channel blocker) or by injection of the presynaptic terminal region with a calcium chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). Under current-clamp conditions, gK(Ca) was not blocked by charybdotoxin or iberiotoxin [specific gK(Ca) blockers]. 5. When gK(Ca) was blocked or reduced, the amplitude of the depolarizing afterpotential of action potentials was increased. When gK(v) was blocked or reduced, the duration of action potentials was increased. 6. Intracellular injection of BAPTA into the presynaptic terminal region eliminated evoked neurotransmitter release before test pulse modulation was affected, suggesting that the K(Ca) channel had a greater sensitivity (greater affinity or lower stoichiometry) for Ca2+ than did the transmitter release machinery. BAPTA reduced neurotransmitter release by 66-78%, but did not affect facilitation of neurotransmitter release. 7. When gNa, gK(v), and gK(Ca) were blocked, we detected a membrane depolarization produced by an increase in presynaptic gCa that was eliminated by 2 mM Cd2+ or 0 mM Ca2+.
摘要
我们使用双微电极电流钳来研究小龙虾步足兴奋性神经肌肉接头突触前终末中钙激活钾电导[gK(Ca)]和钙电导(gCa)的各种特性，以及递质释放情况。2. 电压激活的钠电导(gNa)和钾电导[gK(v)]分别用河豚毒素和3,4 - 二氨基吡啶阻断。在这些条件下，由第一个（条件性）脉冲产生的突触前去极化使钙离子进入突触前终末并激活gK(Ca)，后者调节由第二个（测试）脉冲产生的去极化幅度。在测试脉冲时测得的gK(Ca)相对量随条件性脉冲幅度或持续时间的增加而增加。3. 条件性脉冲后紧接着的短暂超极化显著降低了gK(Ca)。4. gK(Ca)的激活被漏斗网蜘蛛毒素（一种钙通道阻滞剂）或通过向突触前终末区域注射钙螯合剂双 -（邻氨基苯氧基）- N,N,N',N'-四乙酸（BAPTA）所阻断。在电流钳条件下，gK(Ca)不被蝎毒素或iberiotoxin[特异性gK(Ca)阻滞剂]阻断。5. 当gK(Ca)被阻断或降低时，动作电位去极化后电位的幅度增加。当gK(v)被阻断或降低时，动作电位的持续时间增加。6. 向突触前终末区域细胞内注射BAPTA在测试脉冲调制受影响之前消除了诱发的神经递质释放，这表明钾钙通道对钙离子的敏感性（更高的亲和力或更低的化学计量比）比递质释放机制更高。BAPTA使神经递质释放减少了66 - 78%，但不影响神经递质释放的易化作用。7. 当gNa、gK(v)和gK(Ca)被阻断时，我们检测到由突触前gCa增加产生的膜去极化，该去极化被2 mM Cd2+或0 mM Ca2+消除。
