アイテム詳細

要旨

Wild-type and mutants with alpha-subunits truncated at the N terminus of Na⁺/K⁺ pumps of Torpedo electroplax were expressed in Xenopus oocytes by injection of cRNAs encoding for one of the alpha-subunits and for the beta-subunit. Currents generated by the pump were investigated under voltage clamp in Na⁺-free solution, a condition where stimulation by external [K⁺] is the only voltage-dependent and rate-determining step in the pump cycle (Rakowski, R. F., Vasilets, L. A., LaTona, J., and Schwarz, W. (1991) J. Membr. Biol. 121, 177-187). Voltage dependence of the apparent K_m value for pump stimulation and of maximum transport activity was investigated. Truncation of the intracellular N-terminal end of the alpha-subunit at the trypsin-accessible site (alpha delta K37, leaving Lys³⁷) leads to nearly complete inhibition of pump current at physiological potentials, whereas ouabain binding capacity is retained indicating an essential involvement of the N-terminal end in the process of ion translocation. Truncation at the N-terminal end leaving Lys²⁸ (alpha delta K28) or Thr²⁹ (alpha delta T29) leads to removal of 6 or 7 lysine residues, respectively, and has no effect on maximum transport activity. On the other hand, the mutated pumps with alpha delta K²⁸ or alpha delta T²⁹ exhibit more pronounced voltage dependences for stimulation of pump current by external [K⁺] compared with the wild-type Torpedo pump. In particular, a pronounced increase in voltage dependence of the apparent affinity of pump stimulation is obtained by the removal of the Lys²⁸. The results support the view that the lysine-rich region in the N-terminal end affects the cation binding to the pump molecule and that Lys²⁸ is important.