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Abstract:
Phosphorylation of the catalytic subunit of the Na+,K+-ATPase by protein kinases plays an important role in regulation of cation transport. The most extensively studied protein kinase, PKC, exhibits its functional effects on cellular and molecular levels, particularly by (1) changing the number of the active Na+,K+-ATPase molecules in the plasma membranes by activation of endocytosis, and by (2) phosphorylation of the α subunit and direct modulation of transport activity (reviewed in Vasilets,1 for example). Which of the two mechanisms dominates depends on cell type and on phosphorylation conditions. Often direct regulation of the transport cycle may be overlooked because of the complexity of the PKC effects during activation of intracellular signaling. In addition, the transient character of the PKC effects restricts detailed investigation of transport properties of phosphorylated Na+,K+-ATPase. To investigate effects of PKC-mediated phosphorylation on transport cycle, we constructed mutants that mimic the continuously phosphorylated state at Ser-23. This was achieved by substitution of Ser-23 of the rat α1 subunit by negatively charged Glu or Asp. Previously, we demonstrated that the acidic replacement mimics PKC-mediated inhibition of cation transport by the Na+,K+-ATPase.2 In addition, substitution of Ser-23 by Glu increased cellular expression of the α subunit, as judged from immunoblots of oocyte homogenates. However, the possibility could not be excluded that cellular expression of the modified α subunits differs from that in the surface membrane. To understand whether targeting of functionally active pumps in the surface membrane is also enhanced due to modification at Ser-23, we performed voltage-clamp analysis of rat α1 wild-type (WT) and S23E- and S23D-mutant Na+,K+ pumps expressed in Xenopus oocytes. Figure 1A shows transient currents generated by the expressed pump variants in Na+/Na+ exchange mode in response to selected potential steps. Figure 1B shows voltage dependencies of the transiently moved charges obtained by integration of transient currents. From the voltage dependence of the transient currents in Na+/Na+ exchange mode, the effective charge zeffe moved by a single pump molecule and the total charge transported by the entire population of the ATPase in the cell membrane Qtot have been determined. The number of functioning α1/β complexes in the oocyte membrane has been determined from the ratio N=Qtot/zeffe, and was 2.6 × 1010 for the WT and 4.8 × 1010 and 3 × 1010 for S23E and S23D mutants, respectively (increase by factors 1.9 and 1.1). Western blot analysis showed that the total number of expressed S23E and S23D pumps was by factors of 2.3 and 1.3 higher than the number of WT pumps. Thus calculation of the number of active pump complexes from charge movement and from Western blot analysis yields quantitatively similar results with respect to an increase in expression of the phosphorylation mutants. The transport rate determined from the ratio of steady-state currents to total charge k=Iss/Qtot was 24.6 s−1 for the WT and was reduced to 10 s-1 and 19 s−1 for the S23E and S23D mutants, respectively.
