Voltage-dependent inhibition of the sodium pump by external sodium: species differences and possible role of the N-terminus of the alpha-subunit.
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 KI 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 KI 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 KI 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 KI 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 KI value is reduced to 107 mM. The KI values for pump inhibition by external Na+, calculated by taking into account Na(+)-K+ competition, are smaller than the K1/2 values determined in the presence of 5 mM [K+]. The difference is more pronounced for those pump variants that have higher Km 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.
Max-Planck-Institut für Biophysik, Frankfurt/Main, Germany.
L A
LA
Vasilets
T
T
Ohta
S
S
Noguchi
M
M
Kawamura
W
W
Schwarz
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D004563:Electrochemistry; D005110:Extracellular Space / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D009154:Mutation; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects / Q000502:physiology*; D013045:Species Specificity; D014101:Torpedo; D014982:Xenopus laevis
D046911:Macromolecular Substances; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium