ION TRANSPORT MECHANISMS

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SUMMARY OF WORK:The ATP-dependent ion pumps generate transmembrane cation gradients that are essential for electrical excitation, solute transport, cell size and shape regulation and intracellular cation homeostasis. The Na+/K+-ATPase in mammalian cell membranes utilizes the energy available from ATP hydrolysis to transport 3 Na+ out of and 2 K+ into the cell during each catalytic cycle. The unequal stoichiometry of Na+ efflux and K+ influx results in an outward-flowing positive current that can be measured with sensitive electrical recording devices. Using the laser flash photolysis/artificial bilayer technique and quenched- flow mixing, we measured the kinetics of the E1P(Na3)to E2P(Na3) conformational transition. This reaction is presumed to be electrogenic (charge-translocating) because it is coupled to reorientation of the Na+ transport sites from the cytoplasmic to the extracellular surface. In two different Na+/K+-ATPase preparations (eel electric organ,pig kidney)the rate constant for the conformational transition evaluated from the quenched-flow studies coincided with the rate constant determined from the electrical measurements. Similar results were obtained using the potential-sensitive dye RH421 and stopped-flow mixing to measure the kinetics of the conformational transition. This suggests that the transition itself is electrogenic or that it controls the kinetics of charge translocation in a subsequent fast step (Na+ deocclusion?). Because K+-activated dephosphorylation of E2P follows the conformational transition, it is likely that Na+ dissociates from the enzyme (charge translocating step) before K+ binds (activating E2P hydrolysis). This behavior is consistent with the sequence of the cation binding and dissociation events predicted by the Albers-Post mechanism for the Na+/K+-ATPase and with evidence that Na+ and K+ share similar amino acid residues for cation binding in the alpha subunit. - Pumps, ATP, Sodium, Potassium