The stoichiometry and voltage dependence of the Na/K pump were studied in internally dialyzed, voltage-clamped squid giant axons by simultaneously measuring, at various membrane potentials, the changes in Na efflux (delta phi Na) and holding current (delta I) induced by dihydrodigitoxigenin (H2DTG). H2DTG stops the Na/K pump without directly affecting other current pathways: (a) it causes no delta I when the pump lacks Na, K, Mg, or ATP, and (b) ouabain causes no delta I or delta phi Na in the presence of saturating H2DTG. External K (Ko) activates Na efflux with Michaelis-Menten kinetics (Km = 0.45 +/- 0.06 mM [SEM]) in Na-free seawater (SW), but with sigmoid kinetics in approximately 400 mM Na SW (Hill coefficient = 1.53 +/- 0.08, K1/2 = 3.92 +/- 0.29 mM). H2DTG inhibits less strongly (Ki = 6.1 +/- 0.3 microM) in 1 or 10 mM K Na-free SW than in 10 mM K, 390 mM Na SW (1.8 +/- 0.2 microM). Dialysis with 5 mM each ATP, phosphoenolpyruvate, and phosphoarginine reduced Na/Na exchange to at most 2% of the H2DTG- sensitive Na efflux. H2DTG sensitive but nonpump current caused by periaxonal K accumulation upon stopping the pump, was minimized by the K channel blockers 3,4-diaminopyridine (1 mM), tetraethylammonium (approximately 200 mM), and phenylpropyltriethylammonium (20-25 mM) whose adequacy was tested by varying [K]o (0-10 mM) with H2DTG present. Two ancillary clamp circuits suppressed stray current from the axon ends. Current and flux measured from the center pool derive from the same membrane area since, over the voltage range -60 to +20 mV, tetrodotoxin-sensitive current and Na efflux into Na-free SW, under K- free conditions, were equal. The stoichiometry and voltage dependence of pump Na/K exchange were examined at near-saturating [ATP], [K]o and [Na]i in both Na-free and 390 mM Na SW. The H2DTG-sensitive F delta phi Na/delta I ratio (F is Faraday's constant) of paired measurements corrected for membrane area match, was 2.86 +/- 0.09 (n = 8) at 0 mV and 3.05 +/- 0.13 (n = 6) at -60 to -90 mV in Na-free SW, and 2.72 +/- 0.09 (n = 7) at 0 mV and 2.91 +/- 0.21 (n = 4) at -60 mV in 390 mM Na SW. Its overall mean value was 2.87 +/- 0.07 (n = 25), which was not significantly different from the 3.0 expected of a 3 Na/2 K pump.(ABSTRACT TRUNCATED AT 400 WORDS)
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机译:通过同时测量各种膜电位下的Na流出量(delphi phi Na)和保持电流(delta I)的变化,研究了内部透析的电压钳制鱿鱼巨型轴突中Na / K泵的化学计量和电压依赖性。由双氢氧还蛋白(H2DTG)。 H2DTG在不直接影响其他电流路径的情况下停止了Na / K泵浦:(a)当泵浦中缺少Na,K,Mg或ATP时,它不引起δI;(b)哇巴因在水中不引起δI或δphi Na。 H2DTG饱和的存在。外部钾(Ko)在无钠海水(SW)中以Michaelis-Menten动力学(Km = 0.45 +/- 0.06 mM [SEM])激活Na外排,但在大约400 mM Na SW中具有S形动力学(山系数= 1.53) +/- 0.08,K1 / 2 = 3.92 +/- 0.29 mM)。与不含10 mM K,390 mM Na SW(1.8 +/- 0.2 microM)的10 mM K,不含Na的SW相比,H2DTG的抑制作用较小(Ki = 6.1 +/- 0.3 microM)。分别使用5 mM的ATP,磷酸烯醇丙酮酸和磷酸精氨酸进行透析,将Na / Na交换减少到H2DTG敏感的Na外排的最多2%。由K通道阻滞剂3,4-二氨基吡啶(1 mM),四乙铵(约200 mM)和苯丙基三乙铵(20-25 mM)使H2DTG敏感但非泵浦电流由停止泵时轴周K积累引起,但最小通过在存在H2DTG的情况下改变[K] o(0-10 mM)进行测试。两个辅助钳位电路抑制了来自轴突端的杂散电流。从中心池测得的电流和通量来自相同的膜面积,因为在-60至+20 mV的电压范围内,河豚毒素敏感电流和在无K条件下流入Na无SW的Na相等。在无钠和390 mM Na SW中,在接近饱和的[ATP],[K] o和[Na] i下检查泵Na / K交换的化学计量和电压依赖性。对H2DTG敏感的F delta phi Na / delta I比值(F为法拉第常数),针对膜面积匹配进行校正,在0 mV时为2.86 +/- 0.09(n = 8),在3.05 +/- 0.13(n = 6)在无钠SW中在-60至-90 mV时,在390 mM Na SW中在-60 mV时在0 mV时为2.72 +/- 0.09(n = 7),在-60 mV时为2.91 +/- 0.21(n = 4)。它的整体平均值为2.87 +/- 0.07(n = 25),与3 Na / 2 K泵的3.0预期值没有显着差异(抽象截断为400字)
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