Khodakhah K, Melishchuk A, Armstrong C M
Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and Marine Biological Laboratory, Woods Hole, Massachusetts 02543 USA.
Biophys J. 1998 Dec;75(6):2821-9. doi: 10.1016/S0006-3495(98)77725-2.
We studied the effects of modification of native cysteines present in squid giant axon Na channels with methanethiosulfonates. We find that intracellular, but not extracellular, perfusion of axons with positively charged [(2-trimethylammonium)-ethyl]methanethiosulfonate (MTSET), or 3(triethylammonium)propyl]methanethiosulfonate (MTS-PTrEA) irreversibly reduces sodium ionic (INa) and gating (Ig) currents. The rate of modification of Na channels was dependent on the concentration of the modifying agent and the transmembrane voltage. Hyperpolarized membrane potentials (e.g., -110 mV) protected the channels from modification by MTS-PTrEA. In addition to reducing the amplitudes of INa and Ig, MTS-PTrEA also altered their kinetics such that the remaining INa did not appear to inactivate, whereas Ig was made sharper and declined to baseline more quickly. The shape and amplitude of Ig after modification of channels with MTS-PTrEA appeared to be "charge-immobilized," as if the modified channels were inactivated. MTS-PTrEA did not affect INa or Ig when inactivation was removed by internal perfusion of the axon with pronase. In addition, we find that the steady-state inactivation curve of modified Na channels is made much shallower and is markedly shifted to hyperpolarized potentials. The rates of activation, deactivation, or open-state inactivation were not altered in MTS-PTrEA-modified channels. The uncharged sulfhydryl reagent methymethanethiosulfonate (MMTS) did not affect either INa or Ig, but prevented the irreversible effects of MTS-PTrEA or MTSET on Na channels. It is proposed that the positively charged methanethiosulfonates MTS-PTrEA and MTSET modify a native internal cysteine(s) in squid Na channels, and by doing so promote inactivation from closed states, resulting in charge immobilization and reduction of INa.
我们研究了用甲硫基磺酸盐修饰枪乌贼巨大轴突钠通道中天然存在的半胱氨酸的效果。我们发现，用带正电荷的[(2 - 三甲基铵) - 乙基]甲硫基磺酸盐（MTSET）或3(三乙铵)丙基]甲硫基磺酸盐（MTS - PTrEA）对轴突进行细胞内而非细胞外灌注，会不可逆地降低钠离子电流（INa）和门控电流（Ig）。钠通道的修饰速率取决于修饰剂的浓度和跨膜电压。超极化膜电位（例如，-110 mV）可保护通道免受MTS - PTrEA的修饰。除了降低INa和Ig的幅度外，MTS - PTrEA还改变了它们的动力学，使得剩余的INa似乎不会失活，而Ig变得更尖锐且更快地下降到基线。用MTS - PTrEA修饰通道后，Ig的形状和幅度似乎被“电荷固定”，就好像修饰后的通道失活了一样。当用链霉蛋白酶对轴突进行内部灌注以消除失活时，MTS - PTrEA不会影响INa或Ig。此外，我们发现修饰后的钠通道的稳态失活曲线变得更浅，并明显向超极化电位移动。在MTS - PTrEA修饰的通道中，激活、失活或开放状态失活的速率没有改变。不带电荷的巯基试剂甲硫基甲烷磺酸盐（MMTS）既不影响INa也不影响Ig，但可防止MTS - PTrEA或MTSET对钠通道的不可逆影响。有人提出，带正电荷的甲硫基磺酸盐MTS - PTrEA和MTSET修饰了枪乌贼钠通道中一个天然的内部半胱氨酸，这样做会促进从关闭状态的失活，导致电荷固定和INa的降低。
