The Na/K pump generates the transmembrane electrochemical gradients of Na and K ions that underlie electrical signaling and secondary coupled transport, and is the receptor for digoxin, the widely prescribed cardiotonic steroid that specifically inhibits the Na/K pump. Inherited Na/K pump mutations are now linked to rapid-onset dystonia-parkinsonism and familial hemiplegic migraines. Our long term goal remains to understand in detail how the Na/K pump works, i.e. what the ion translocation pathways and associated gates look like, and how their orchestrated interaction transports first 3 Na and then 2 K ions in opposite directions across the cell membrane. We view the Na/K pump as a specialized ion channel with cytoplasmic and extracellular gates whose movements are tightly coupled so that both gates are never normally open at the same time (unlike the gates of ion channels). Accordingly, we investigate the Na/K pump by applying powerful electrophysiological methods that have proven successful in learning how ion channels work. Thus, the stationary current that results from the unequal transport of Na and K ions sensitively assays turnover rate during steady cycling, and charge relaxations following voltage jumps monitor conformational transitions that rate limit certain partial reactions. Together, these signals have shed light on the molecular mechanism of ion transport by the Na/K pump. Now, to further investigate the ion transport mechanism, we combine these recording methods with three new tools: (a) homology models of the Na/K pump alpha subunit based on recent high-resolution crystal structures of key conformations of the related SR Ca pump; (b) site-specific mutagenesis based on those structural models, and expression in Xenopus oocytes, of ouabain-resistant mutant Xenopus Na/K pumps, some bearing novel cysteine residues introduced to test their accessibility to small sulfhydryl-specific reagents; (c) the marine toxin, palytoxin, which disrupts the coupling between the Na/K pump's two gates, transforming it into an ion channel gated by the pump's physiological ligands, Na and K ions and nucleotides.
The specific aims are to use these tools: (1) to determine the location, structure, and physico-chemical characteristics of the ion-translocation pathway (or pathways) traversed by the transported Na and K ions, (2) to determine the location and structure of the Na/K pump's two principal gates, and (3) to examine the mechanisms controlling opening and closing of the gates. ? ? ?
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