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 sidechain rearrangements at the site.P-type ATPases | pump currents | thermodynamics D uring each normal transport cycle of the Na þ ∕K þ -ATPase pump, three Na þ are exported from the cell in exchange for two K þ imported, a process driven by hydrolysis of one molecule of ATP. By establishing the Na þ and K þ gradients across cell membranes, the Na þ ∕K þ pump enables action potentials, synaptic signaling, and most solute transport in and out of cells. Two consequences of the unequal transport stoichiometry of Na þ and K þ are that steady pumping produces an outwardly directed current (1), proportional in magnitude to the turnover rate (2), which can be monitored electrically (3)(4)(5)(6)(7)(8), and that at least one step in the transport cycle must move charge through the membrane field (9). The latter implies that, under favorable conditions, charge relaxations following voltage jumps can be used to learn details about specific steps during the transport cycle (10-28).In the absence of internal and external K þ , the nominal absence of internal ADP and presence of an ATP regenerating system, the Na þ ∕K þ pump allows exchange of 3Na þ between their binding sites in a deeply occluded conformation and the external milieu (21, 29) ( Fig. 1A; encircled by dotted lines). Under these conditions there is no steady pump current observed at any voltage. Nevertheless, sudden voltage steps produce pre-steadystate currents that can be recorded (12, 13, 16-18, 21, 23, 26).These currents allow direct measurements of the rates of partial reactions of the pump cycle. Using giant axons from the squid Loligo pealeii, we have characterized th...

RECS1, a member of the TMBIM family, triggers lysosomal membrane permeabilization in cells undergoing lysosomal stress.

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and Gem/Kir (RGK) sub-family of small GTP-binding protein include e the most potent endogenous inhibitor of High-Voltage Activated (HVA) calcium channels. RGK proteins use several mechanisms to inhibit calcium current (I CA): i) they promote dynamin-dependent endocytocys; ii) the lower the Po of the channel: ii) and they voltage sensor or reduction of charge. All RGK proteins associate directly with Ca 2þ channel b subunits (Cavb), and the binding between CaVa1 / Cavb has been shown to be essential for their inhibitory action for CaV1.2 and Ca V 2.1 and Ca V 2.2. In this study, we investigated inhibition of Ca V 2.3 Ca 2þ channels by RGK proteins. We found that when Xenopus laevis oocytes expressing Cav2.3 channels were injected with purified Gem protein, but not Rem, calcium currents where significantly decreased. This reduction was accompanied by a right shift in the conductance-voltage (GV curve) relationship of the channel. Furthermore, Gem also decreases the number of channels in the plasma membrane, evidenced by a reduction in maximal charge movement after injection of purified protein. The kinetic and voltage dependence of the reduced charge movement was not affected and thus immobilization of a subset of voltage-sensor seems unlikely. Surprisingly both effects are not dependent on the binding between CaVa1 to Cavb since CaV2.3 were expressed alone. Thus unlike than other neuronal calcium channels, Gem inhibit Cav2.3 channels in a CaVb-independent manner and through two synergistic mechanisms: a increase in voltage for activation and a decrease in the number of channels.