title: Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid.
abstract: The Na(+)/K(+) pump is a nearly ubiquitous membrane protein in animal cells that uses the free energy of ATP hydrolysis to alternatively export 3Na(+) from the cell and import 2K(+) per cycle. This exchange of ions produces a steady-state outwardly directed current, which is proportional in magnitude to the turnover rate. Under certain ionic conditions, a sudden voltage jump generates temporally distinct transient currents mediated by the Na(+)/K(+) pump that represent the kinetics of extracellular Na(+) binding/release and Na(+) occlusion/deocclusion transitions. For many years, these events have escaped a proper thermodynamic treatment due to the relatively small electrical signal. Here, taking the advantages offered by the large diameter of the axons from the squid Dosidicus gigas, we have been able to separate the kinetic components of the transient currents in an extended temperature range and thus characterize the energetic landscape of the pump cycle and those transitions associated with the extracellular release of the first Na(+) from the deeply occluded state. Occlusion/deocclusion transition involves large changes in enthalpy and entropy as the ion is exposed to the external milieu for release. Binding/unbinding is substantially less costly, yet larger than predicted for the energetic cost of an ion diffusing through a permeation pathway, which suggests that ion binding/unbinding must involve amino acid side-chain rearrangements at the site.
authors.affiliation: Laboratorio de Fisiologia Celular, Facultad de Ciencias, Universidad de Chile, Montemar 2366103, Chile.
authors.forename: Juan P
authors.initials: JP
authors.lastname: Castillo
authors.forename: Daniela
authors.initials: D
authors.lastname: De Giorgis
authors.forename: Daniel
authors.initials: D
authors.lastname: Basilio
authors.forename: David C
authors.initials: DC
authors.lastname: Gadsby
authors.forename: Joshua J C
authors.initials: JJ
authors.lastname: Rosenthal
authors.forename: Ramon
authors.initials: R
authors.lastname: Latorre
authors.forename: Miguel
authors.initials: M
authors.lastname: Holmgren
authors.forename: Francisco
authors.initials: F
authors.lastname: Bezanilla
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001369:Axons / Q000502:physiology*; D049832:Decapodiformes; D004058:Diffusion; D004594:Electrophysiology / Q000379:methods; D006868:Hydrolysis; D007477:Ions; D007700:Kinetics; D011485:Protein Binding; D012964:Sodium / Q000737:chemistry*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics
chemical_list: D007477:Ions; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase