K+ Translocation by the Giant Axon of the Humboldt Squid Na+/K ATPase

Abstract

The Na+/K+ pump is a membrane protein which plays a fundamental role in maintaining the Na+ and K+ electrochemical gradients in animal cells. When internal and external Na+ is absent the pump can only undergo K+ translocation reactions. At equilibrium, the distribution of the different protein conformations depends on the rate constants of each step leading to K+ binding and unbinding. If some of these rate constants are voltage-dependent, sudden changes in membrane electric potential will shift the binding-unbinding equilibrium. In those translocation reactions, K+ has to travel a fraction of the membrane electric field generating a transient current signal. Here, K+ pump currents were measured under voltage clamp conditions using the giant axon of the Humboldt squid, which due to its large diameter (1 -1.5 mm) allows the detection of these charge movements. By using H2DTG, a reversible inhibitor of the squid Na+/K+ pump, we were able to obtain H2DTG-sensitive transient currents in response to voltage jumps in K+/K+ conditions. Kinetics of these transient currents shows two main components, that in contrast to their Na+ counterpart, appeared to be uncoupled. The origin of the fast component appears to be the movement of ions along an access channel that it is always open, suggesting that the gate that occlude K ions is deep in the permeation pathway. On the other hand, charge displacement distribution and rate constants of the slow component show a clear dependence on the K+ external concentration revealing that the entrance of the K+ to the Na+/K+ pump from the external side is a voltage-dependent step. Supported by FIRCA grant R03 TW008351 and U54GM087519, GM030376, NS64259, HL36783 and the Intramural Program of the NINDS/NIH and FONDECYT 1110430.