Robert F. Rakowski, Ph.D.

     The objective of the research conducted in my laboratory is to obtain a molecular level understanding of the mechanism of ion translocation by electrogenic transport pumps. We have made significant progress and have identified several reaction steps in the transport cycle of the Na⁺/K⁺ pump that translocate charge and, therefore, are voltage dependent. Surprisingly, rather than finding that charge translocation is associated with the major inside-facing to outside-facing conformational change of the enzyme, we find that the principal voltage dependent step in the pump cycle is associated with the binding of extracellular Na⁺. This highly asymmetrical voltage dependence can be accounted for by the hypothesis that there is an external access channel (ion-well) that Na⁺ ions must enter in order to reach their binding sites. We are investigating the ability of this hypothesis to explain steady-state and transient behavior of Na⁺/K⁺ pump current in voltage-clamped Xenopus oocytes and squid giant axons.

     Investigation of the mechanism of charge translocation by the Na⁺/K⁺ pump in Xenopus oocytes.

     1. Steady-state Na⁺/K⁺ pump current-voltage relationship in oocytes. A two-microelectrode voltage-clamp technique is used to measure ouabain-sensitive currents in Xenopus oocytes under various ionic conditions. A strong prediction of the access channel model is that changes in external [Na⁺] should be kinetically equivalent to changes in membrane potential. That is, hyperpolarization should increase the effective [Na⁺] within the access channel and compensate for lowering external [Na⁺]. We observe shifts in the mid-point voltage of the steady-state pump current-voltage relationship that are consistent with an access channel depth of 2/3 of the electrical field across the membrane.

     2. Pre-steady-state transient measurements in Xenopus oocytes. We are using the cut-open oocyte voltage clamp technique to investigate pre-steady-state transient currents in Xenopus oocytes. This technique has the advantages of clamp speed and the ability to control the intracellular as well as extracellular solution composition. Under experimental conditions that promote electroneutral Na⁺/Na⁺ exchange, we have been able to directly demonstrate that the rate coefficient for external Na⁺ binding to the Na⁺/K⁺ pump is voltage dependent while the unbinding rate coefficient is relatively voltage-insensitive. Work is in progress to investigate the behavior of transient pump currents in various mutant forms of the enzyme.

     Investigation of the mechanism of charge translocation by the Na⁺/K⁺ pump in internally-dialyzed voltage-clamped squid giant axons.

     During the summer months studies are conducted at the Marine Biological Laboratory in Woods Hole, MA in collaboration with Drs. Paul De Weer (University of Pennsylvania) and David Gadsby (Rockefeller University) on the mechanism of charge translocation by the Na⁺/K⁺ pump in squid giant axons. This line of investigation has led to several major advances in the understanding of events during Na⁺/K⁺ pumping. We have demonstrated that the pump is electrogenic when it operates in reverse, that it operates at a constant 3Na⁺/2K+ stoichiometry independent of membrane voltage, that both the forward and reverse modes of operation of the pump are voltage- and external Na⁺-sensitive, and that electroneutral Na⁺/Na⁺ exchange by the pump is voltage dependent.

     These investigations are unique in that they permit the simultaneous measurement of electrogenic pump current and unidirectional radiotracer fluxes. This has enabled us to measure the voltage dependence of electrically silent steady-state Na⁺/Na⁺ exchange by the Na⁺/K⁺ pump. Despite the fact that the overall Na⁺/Na⁺ exchange process is electroneutral, it is voltage dependent. The characteristics of this voltage dependence provide strong evidence for the existence of an external access channel in which Na⁺ ions are bound.

Selected References:

• Holmgren, M., J. Wagg, F. Bezanilla, R.F. Rakowski, P. De Weer and D.C. Gadsby. Three distinct sequential steps in extracellular release of three Na⁺ ions by the Na,K-ATPase. Nature 403:898-901 (2000).

• Sagar, A. and R.F. Rakowski. Access channel model for the voltage dependence of the forward-running Na⁺/K⁺ pump. Journal of General Physiology 103:869-894 (1994).

• Holmgren, M. and R.F. Rakowski. Pre-steady-state transient currents mediated by the Na/K pump in internally-perfused Xenopus oocytes. Biophysical Journal 66:912-922 (1994).

• Gadsby, D.C., R.F. Rakowski and P. De Weer. Extracellular access to the Na,K pump: pathway similar to ion channel. Science, 260:100-103 (1993).

• Rakowski, R.F., D.C. Gadsby and P. De Weer. Stoichiometry and voltage dependence of the sodium pump in voltage-clamped, internally dialyzed squid giant axon. Journal of General Physiology 93:903-941(1989).