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- W2480494291 abstract "The Na/K pump hydrolyzes one ATP molecule to export 3 Na+ and import 2 K+ across the plasma membrane. Its reaction cycle involves a set of reversible conformational changes between phosphorylated and dephosphorylated states alternately opening ion binding sites externally (E2) or internally (E1), depending on ion (Na+, K+) and ligand (ATP, ADP, PO4, etc.) binding. Under whole-cell voltage clamp, with conditions that promote E2-P (high Na+i and ATP) and sub-saturating [Na+]o and [K+]o, the pump passively imports protons generating an inward current (IH). To elucidate the partial reactions involved in this non-canonical transport mode, we evaluated the effects of intracellular application of transported ions and ligands on giant inside-out patches excised from Xenopus oocytes heterologously expressing Na/K pumps. Without Na+ o or K+o in the pipette, IH activation by ATP in the presence of K+i was ∼1/8th of that observed in Na+i. ADP did not activate IH in Na+i, but activated it like ATP in K+i. The [Na+i]-dependence at saturating [ATP] showed K0.5, Na = 2 mM and the [ATP]-dependence at saturating [Na+i] yielded K0.5, ATP = 55 μM. Comparatively, the voltage-dependent transient currents caused by external binding/release of Na+o, required Na+i and ATP with K0.5, Na = 1 mM and K0.5, ATP = 0.4 μM, respectively. These data indicate that IH requires low affinity binding of ATP to externally open E2 conformations and that the Na+- and K+-free E2 state allowing proton flow can be reached from E1 either through forward pump phosphorylation in the presence of Na+i or backward, through binding of intracellular K, even in the absence of PO4. The smaller current with Ki solutions must reflect the lower probability of external K release from the unphosphorylated E2 compared to E2P. NSF MCB-1515434 and NIH GM-061583." @default.
- W2480494291 created "2016-08-23" @default.
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- W2480494291 date "2016-02-01" @default.
- W2480494291 modified "2023-09-26" @default.
- W2480494291 title "Enzymatic Requirements for Non-Canonical Proton Import by Na/K Pumps" @default.
- W2480494291 doi "https://doi.org/10.1016/j.bpj.2015.11.3367" @default.
- W2480494291 hasPublicationYear "2016" @default.
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