Datensatz

Zusammenfassung

Currents generated by the Na⁺/K⁺ ATPase were measured under voltage clamp in oocytes of Xenopus laevis. The dependence of pump current on external [Na⁺] was investigated for the endogenous Xenopus pump as well as for wild-type and mutated pumps of electroplax of Torpedo californica expressed in the oocytes. The mutants had alpha-subunits truncated before position Lys²⁸ (alpha delta K28) or Thr²⁹ (alpha delta T29) of the N-terminus. The currents generated by all variants of pump molecules in the presence of 5 mM K⁺ show voltage-dependent inhibition by external [Na⁺]. The apparent K₁ values increase with membrane depolarisation, and the potential dependence can be described by the movement of effective charges in the electrical potential gradient across the membrane. Taking into account Na⁺-K⁺ competition for external binding to the E₂P form, apparent K₁ values and effective charges for the interaction of the Na⁺ ions with the E₂P form can be estimated. For the Xenopus pump the effective charge amounts to 1.1 of an elementary charge and the K₁ value at 0 mV to 44 mM. For the wild-type Torpedo pump, the analysis yields values of 0.73 of an elementary charge and 133 mM, respectively. Truncation at the N-terminus removing a lysine-rich cluster of the alpha-subunit of the Torpedo pump leads to an increase of the effective charge and decrease of the K₁ value. For alpha delta K28, values of 0.83 of an elementary charge and 117 mM are obtained, respectively. If Lys²⁸ is included in the truncation (alpha delta T29), the effective charge increases to 1.5 of an elementary charge and the apparent K₁ value is reduced to 107 mM. The K₁ values for pump inhibition by external Na⁺, calculated by taking into account Na⁺-K⁺ competition, are smaller than the K_/12 values determined in the presence of 5 mM [K⁺]. The difference is more pronounced for those pump variants that have higher K_m values. The variations of the parameters describing inhibition by external [Na⁺] are qualitatively similar to those described for the stimulation of the pumps by external [K⁺] in the absence of extracellular [Na⁺]. The observations may be explained by an access channel within the membrane dielectric that has to be passed by the external Na⁺ and K⁺ ions to reach or leave their binding sites. The potential-dependent access and/or the interaction with the binding sites shows species differences and is affected by cytoplasmic lysine residues in the N-terminus.