Rb+ occlusion in dephosphorylation intermediates of the gastric H,K‑ATPase catalytic cycle

WANDA M. VALSECCHI; SANTIAGO E. FARAJ; NICOLE T. CERF; ROLANDO C. ROSSI; MÓNICA R. MONTES

San Luis

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

Sociedad Argentina de Biofisica

The gastric H,K-ATPase is an integral membrane protein responsible for gastric acidsecretion. The enzyme catalyzes H+ transport at the expense of ATP hydrolysis through amechanism traditionally described by the E1-E2 reaction cycle. In the E1 conformation,H+ are bound to the cytoplasmic side of the pump, leading to phosphorylation and theformation of the E1P intermediate, which is then converted to E2P, releasing H+ at theopposite side of the membrane. The counter-transport ion, K+, binds from the lumenaccess and triggers E2P dephosphorylation leading to the occluded form, in which K+ istrapped within the membrane domain. The cycle completes with the transition to E1 andthe release of K+ into the cell. The physiological dephosphorylation sequence proposedfor this and other ATPases may be summarized as:E2-P (ground state) → E2·P (transition state) → E2Pi (product state) → E2 + Piwhere each species should present different capacity to bind and occlude K+. Despite thegreat similarities between Na,K-ATPase and H,K-ATPase, in the gastric enzyme theisolation of K+‑occluded states has been challenging. It was shown that vanadatepromotes the occlusion of Rb+, a K+ congener (Rabon et al., 1982; Montes et al., 2011),and that vanadate and metal fluoride complexes induce structures that represent theintermediates during the E2‑P dephosphorylation sequence (Abe et al., 2010). Tounderstand the coupling between the structures of the phosphorylated domain and themovement of the transmembrane segments, we analyzed the ability of the E2Pintermediates to bind and occlude Rb+. Results show that the level of Rb+ occlusionincreases from the E2P ground state (the species that first contacts with luminal K+) tothe E2P product state. These observations are in accordance with the shift from an openluminal to a close-luminal conformation during the transition from E2P to E2.