Voltage-dependent inhibition of the sodium pump by external sodium: Species differences and possible role of the N-terminus of the ?-subunit

Abstract

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.

Does the colonic H,K-ATPase also act as an Na,K-ATPase?

“…Statistical significance: NS, not significant; ‫,ءءء‬ P Ͻ 0.001. -free, and ouabain-free medium, which allowed [Na] i increase to about 35 mM (17) or 80 mM (18). These discrepancies may be because the different experimental conditions.…”

Role of the Transmembrane Domain of FXYD7 in Structural and Functional Interactions with Na,K-ATPase

“…Extracellular Na ϩ binding is reflected in a voltage-dependent inhibition of the K ϩ -activated pump current by extracellular Na ϩ measured at 1 mM extracellular K ϩ (20). As shown in Fig.…”

A third Na + -binding site in the sodium pump

“…Extracellular Na ϩ binding can also be observed as a voltagedependent inhibition of the Na,K-pump activity by extracellular sodium (23), stimulation of the ADP-dependent Na ϩ ͞Na ϩ exchange (10), or stimulation of the backward running pump (12). We studied the inhibition of the K ϩ -activated current by 30-and 100-mM concentrations of Na ext ϩ over the Ϫ130 to ϩ30 mV membrane potential range and observed the known voltagedependent inhibition in the WT Na,K-pump and the E960A mutant, but a reduced and mostly voltage-independent inhibition by Na ext ϩ in the E961A mutant (Fig.…”