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

VALSECCHI WM; FARAJ SE; CERF NT; ROSSI RC; MONTES MR

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

The gastric H,K-ATPase is an integral membrane protein responsible for gastric acid secretion. The enzyme catalyzes H+ transport at the expense of ATP hydrolysis through a mechanism 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 the formation of the E1P intermediate, which is then converted to E2P, releasing H+ at the opposite side of the membrane. The counter-transport ion, K+, binds from the lumen access and triggers E2P dephosphorylation leading to the occluded form, in which K+ is trapped within the membrane domain. The cycle completes with the transition to E1 and the release of K+ into the cell. The physiological dephosphorylation sequence proposed for this and other ATPases may be summarized as: E2-P (ground state) → E2·P (transition state) → E2Pi (product state) → E2 + Pi where each species should present different capacity to bind and occlude K+. Despite the great similarities between Na,K-ATPase and H,K-ATPase, in the gastric enzyme the isolation of K+‑occluded states has been challenging. It was shown that vanadate promotes 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 the intermediates during the E2‑P dephosphorylation sequence (Abe et al., 2010). To understand the coupling between the structures of the phosphorylated domain and the movement of the transmembrane segments, we analyzed the ability of the E2P intermediates to bind and occlude Rb+. Results show that the level of Rb+ occlusion increases from the E2P ground state (the species that first contacts with luminal K+) to the 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.