Datensatz

Zusammenfassung

Charge translocation by the NaK-ATPase from shark rectal gland was measured by adsorption of proteoliposomes to a planar lipid membrane. The proteoliposomes were prepared by reconstitution of purified NaK-ATPase into liposomes consisting of E. coli lipids. The protein was activated by applying an ATP concentration jump produced by photolysis of a protected derivative of ATP, caged ATP. K⁺ titrations were used to study the effect of K⁺ on the charge translocation kinetics of the protein. The time-dependent currents obtained after activation of the enzyme with caged ATP were analyzed with a simplified Albers-Post model (E₁ ^k1-- E₁ATP ^k2-- E₂P ^k3-- E₁) 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 k₃ on the K⁺ concentration at the extracellular side of the protein, indicating the K⁺ activated dephosphorylation reaction. In contrast, k₁ and k₂ remained constant. The K⁺ dependence of the rate k₃ could be well described with a K⁺ binding model with two equivalent binding sites (E₂P + 2K⁺ <==> E₂P(K) + K⁺ <==> E2 P(2K)) followed by a rate limiting reaction (E₂P(2K) --> E₁(2K)). The half saturating K⁺ concentration K_3,0.5 and the microscopic dissociation constant K₃ for the K⁺ dependence of k₃ were 4.5mM and 1.9mM respectively. At saturating K⁺ concentration the rate constant k₃ was approximately 100 s^-1. The relative amount of net charge transported during the Nav and the K⁺ dependent reactions could be determined from the experiments. Our results suggest electroneutral K⁺ translocation and do not support electrogenic K⁺ binding in an extracellular access channel. This is compatible with a model where 2 negative charges are cotransported with 3Na⁺ and 2K⁺ ions. Error analysis gives an upper limit of 20% charge transported during K⁺ translocation or during electrogenic K⁺ binding in a presumptive access channel compared to Na⁺ translocation.