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 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...

Mechanisms for achieving high speed and efficiency in biomolecular machines

“…This pump hydrolyzes ATP as input to export three Na + ions from the cell and import two K + ions uphill against their chemiosmotic gradients. This machine has such a good tradeoff because the output work is delocalized across the entire cycle and therefore the rate-limiting work step (the release of one of the Na + ions) is only weakly dependent on the total work of the machine (26). This pump effectively has many substeps, although we do not report an exact value since it is difficult to estimate the work contribution over the various transitions (the occlusion, transport, and release of each of the five ions).…”

Measuring Cation Transport by Na,K- and H,K-ATPase in <em>Xenopus</em> Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

“…Furthermore, we wanted to take advantage of the Xenopus oocyte expression system, which utilizes large single cells (about 1.0-1.5 mm diameter) that allow to achieve a remarkably low cell-to-cell variability regarding the protein expression level within a single batch. A simple calculation demonstrates the feasibility of the AAS assay: The detection limit (characteristic mass) for rubidium with the THGA-AAS technique is 10 pg or 1.2·10 -13 [1][2][3] , which results in an about 30% increase in the turnover rate upon a change from 20 °C to 22 °C), it is mandatory to carry out the flux measurements under precise temperature control (air conditioning) with well equilibrated buffer solutions. Furthermore, oocytes should be carefully selected regarding homogenous size for the expression of an ion transporter.…”

David Christopher Gadsby. 26 March 1947—9 March 2019

“…(His love of the sea propelled him to accompany the fishermen in open boats in the dark at 3 a.m. to catch squid; he'd deliver them to the lab by 7 a.m., and after a quick nap would join the team for the day's work.) The technical advantages of the Humboldt squid allowed the group to delve more deeply into pump mechanism: to quantify the thermodynamic landscape of the multiple states of Na + occupancy and conformations of the pump, and to detect the K + current transients of the 'return' half-reaction of the pump's full turnover cycle (16).…”