Role of the transmembrane and extracytoplasmic domain of β subunits in subunit assembly, intracellular transport, and functional expression of Na,K-pumps

Abstract

The ubiquitous Na,K- and the gastric H,K-pumps are heterodimeric plasma membrane proteins composed of an α and a β subunit. The H,K-ATPase β subunit (βHK) can partially act as a surrogate for the Na,K-ATPase β subunit (βNK) in the formation of functional Na,K-pumps (Horisberger et al., 1991. J. Biol. Chem. 257:10338-10343). We have examined the role of the transmembrane and/or the ectodomain of βNK in (a) its ER retention in the absence of concomitant synthesis of Na,K-ATPase α subunits (αNK) and (b) the functional expression of Na,K-pumps at the cell surface and their activation by external K+. We have constructed chimeric proteins between Xenopus βNK and rabbit βHK by exchanging their NH2-terminal plus transmembrane domain with their COOH-terminal ectodomain (βNK/HK, βHK/NK). We have expressed these constructs with or without coexpression of αNK in the Xenopus oocyte. In the absence of αNK, Xenopus βNK and all chimera that contained the ectodomain of βNK were retained in the ER while βHK and all chimera with the ectodomain of βHK could leave the ER suggesting that ER retention of unassembled Xenopus βNK is mediated by a retention signal in the ectodomain. When coexpressed with αNK, only βNK and βNK/HK chimera assembled efficiently with αNK leading to similar high expression of functional Na,K-pumps at the cell surface that exhibited, however, a different apparent K+ affinity. βHK or chimera with the transmembrane domain of βHK assembled less efficiently with αNK leading to lower expression of functional Na,K-pumps with a different apparent K+ affinity. The data indicate that the transmembrane domain of βNK is important for efficient assembly with αNK and that both the transmembrane and the ectodomain of β subunits play a role in modulating the transport activity of Na,K-pumps.