The C-terminal cavity of the Na,K-ATPase analyzed by docking and electrophysiology

Abstract

The Na,K-ATPase is essential to all animals, since it maintains the electrochemical gradients that energize the plasma membrane. Naturally occurring inhibitors of the pump from plants have been used pharmaceutically in cardiac treatment for centuries. The inhibitors block the pump by binding on its extracellular side and thereby locking it. To explore the possibilities for designing an alternative way of targeting the pump function, we have examined the structural requirements for binding to a pocket that accommodates the two C-terminal residues, YY, in the crystal structures of the pump. To cover the sample space of two residues, we first performed docking studies with the 400 possible dipeptides. For validation of the in silico predictions, pumps with 13 dipeptide sequences replacing the C-terminal YY were expressed in Xenopus laevis oocytes and examined with electrophysiology. Our data show a significant correlation between the docking scores from two different methods and the experimentally determined sodium affinities, which strengthens the previous hypothesis that sodium binding is coupled to docking of the C-terminus. From the dipeptides that dock the best and better than wild-type YY, it may therefore be possible to develop specific drugs targeting a previously unexplored binding pocket in the sodium pump.

Tuning of the Na,K-ATPase by the beta subunit

“…4 ). Thus, we find that the β isoforms can influence structural elements in the α subunit that are known to be important for the kinetic properties of Na + binding 29 45 46 .…”

“…Data was recorded and analysed with pClamp 10.4 (Molecular Devices) and Graph Pad Prism 6 (Graph Pad Software) 46 .…”

ATP1A2 Mutations in Migraine: Seeing through the Facets of an Ion Pump onto the Neurobiology of Disease

“…The inward current was found to be augmented by mutations at the Na + ,K + -ATPase's C-terminus (Yaragatupalli et al, 2009 ; Meier et al, 2010 ; Poulsen et al, 2010 ; Vedovato and Gadsby, 2010 ; Paulsen et al, 2012 ), and sometimes further enhanced with increasing extracellular [Na + ], which conforms with an earlier proposal that Na + ions might flow along what has been referred to as the Na + ,K + -ATPase “leak” pathway, a process that is promoted by extracellular protons (Vasilyev et al, 2004 ). The studied C-terminal mutations frequently correlate with drastic decreases in the apparent affinity for extracellular and intracellular Na + (Toustrup-Jensen et al, 2009 , 2014 ), because C-terminal mutations interfere with Na + binding site III.…”

“…This has prompted investigations of the functional importance of C-terminal residues by studying deletions, mutations of the terminal tyrosines, or C-terminal extensions. These studies showed that alterations in the C-terminal sequence entail drastic decreases in Na + affinity and enhance the propensity of the mutant pumps to permit inward proton leak (Yaragatupalli et al, 2009 ; Meier et al, 2010 ; Poulsen et al, 2010 ; Vedovato and Gadsby, 2010 , 2014 ; Paulsen et al, 2012 ). This has given rise to the concept that the C-terminal pathway occupied by the terminal tyrosines defines a mandatory access route for intracellular protons to the Na + binding site III to bring about stoichiometric 3Na + /2K + transport, in which Arg-937 and Asp-923 play a critical role (Poulsen et al, 2010 ).…”

Estudo bioquímico comparativo da (Na+ , K+)-ATPase branquial de Macrobrachium amazonicum (Heller, 1862) habitante de regiões continentais

“…Os resíduos Tir1017, Tir768, Arg935 e Lis768 favorecem a fosforilação de E1 e, consequentemente também influenciam a interação da enzima com o Na . O motivo KETYY na extremidade C-terminal é conservado e neste local se localiza o terceiro sítio de ligação do Na + onde os resíduos de Tir desta sequência são essenciais para a oclusão e a afinidade deste íon (Meier et al, 2010;Poulsen et al, 2010b;2012;Vedovato & Gadsby, 2010;Paulsen et al, 2013).…”