K-DEPENDENCE OF ELECTROGENIC TRANSPORT BY THE NAK-ATPASE()

Charge translocation by the NaK-ATPase from shark rectal eland was mea sured by adsorption of proteoliposomes to a planar lipid membrane. Til e proteoliposomes were prepared by reconstitution of purified NaK-ATPa se into liposomes consisting of E. coli lipids. The protein was activa ted by applying an ATP concentration jump produced by photolysis of a protected derivative of ATP, caged ATP. K+ titrations were used to stu dy the effect of K+ on the charge translocation kinetics of the protei n. The time-dependent currents obtained after activation of the enzyme with caged ATP were analyzed with a simplified Albers-Post model E-1 (k(1)) under right arrow E-1 ATP (k(2)) under right arrow E2P (k(3)) u nder right arrow E-1), taking into account the capacitive coupling of the protein to the measuring system. The results of the K+ titrations show a strong dependence of the rate constant k(3) on the K+ concentra tion at the extracellular side of the protein, indicating the K+ activ ated dephosphorylation reaction. In contrast, k(1) and k(2) remained c onstant. The K+ dependence of the rate k(3) could be well described wi th a K+ binding model with two equivalent binding sites (E2P + 2K(+) r eversible arrow E2P(K) + K+ reversible arrow E2P(2K)) followed by a ra te limiting reaction (E2P(2K) --> E-1(2K)). The half saturating K+ con centration K-3,K-0.5 and the microscopic dissociation constant K-3 for the K+ dependence of k(3) were 4.5 mM and 1.9 mM respectively. At sat urating K+ concentration the rate constant k(3) was approximately 100 s(-1). The relative amount of net charge transported during the Na+ an d the K+ dependent reactions could be determined from the experiments. Our results suggest electroneutral K+ translocation and do not suppor t electrogenic K+ binding in an extracellular access channel. This is compatible with a model where 2 negative charges are cotransported wit h 3Na(+) and 2K(+) ions. Error analysis gives an upper limit of 20% ch arge transported during K+ translocation or during electrogenic K+ bin ding in a presumptive access channel compared to Na+ translocation. (C ) 1998 Elsevier Science B.V.