title: Inhibition of K+ transport through Na+, K+-ATPase by capsazepine: role of membrane span 10 of the α-subunit in the modulation of ion gating.
abstract: Capsazepine (CPZ) inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity. In this study we have investigated the functional effects of CPZ on Na+,K+-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na+,K+-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na+,K+-ATPase current in the presence of extracellular K+. In contrast, CPZ stimulated pump current in the absence of extracellular K+. Similar conclusions were attained using HEK293 cells loaded with the Na+ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na+,K+-ATPase indicated that CPZ stabilizes ion interaction with the K+ sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na+ congener at the Na+ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na+,K+-ATPase. CPZ increased oxonol VI fluorescence in the absence of K+, reflecting increased Na+ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K+, likely indicating opening of an intracellular pathway selective for K+. As revealed by the recent crystal structure of the E1.AlF4-.ADP.3Na+ form of the pig kidney Na+,K+-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K+ sites in Na+,K+-ATPase.
authors.affiliation: Department of Biomedicine, University of Aarhus, DK-8000 Aarhus C, Denmark.
authors.forename: Yasser A
authors.initials: YA
authors.lastname: Mahmmoud
authors.affiliation: Cardiovascular Division, King's College London, The Rayne Institute, St. Thomas' Hospital, London, United Kingdom.
authors.forename: Michael
authors.initials: M
authors.lastname: Shattock
authors.affiliation: Department of Biomedicine, University of Aarhus, DK-8000 Aarhus C, Denmark.
authors.forename: Flemming
authors.initials: F
authors.lastname: Cornelius
authors.affiliation: Cardiovascular Division, King's College London, The Rayne Institute, St. Thomas' Hospital, London, United Kingdom.
authors.forename: Davor
authors.initials: D
authors.lastname: Pavlovic
mesh_terms: D009597:4-Nitrophenylphosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002211:Capsaicin / Q000031:analogs & derivatives* / Q000494:pharmacology; D020134:Catalytic Domain; D002462:Cell Membrane / Q000187:drug effects* / Q000201:enzymology; D019286:Endopeptidase K / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects*; D007555:Isoxazoles / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D059748:Proteolysis / Q000187:drug effects; D051381:Rats; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D014357:Trypsin / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D007555:Isoxazoles; D014638:Vanadates; C031184:oxonol VI; D012964:Sodium; D009597:4-Nitrophenylphosphatase; D014357:Trypsin; D019286:Endopeptidase K; D000254:Sodium-Potassium-Exchanging ATPase; C071423:capsazepine; D011188:Potassium; D002211:Capsaicin