The long term goal of this project remains a detailed understanding in physicochemical terms, of the transport mechanism of the Na/K pump. This is important because the Na/K pump maintains the electrochemical gradients for Na and K ions that underlie electrical signalling, essential coupled transport processes, and cell volume regulation; the Na/K pump is also thought to mediate the therapeutic action of the cardiotonic steroid and specific inhibitor of the pump, digoxin, still one of the most widely prescribed cardiac drugs. Charge translocation by the pump during the ion transport cycle, or during its partial reactions, is a fundamental feature of pump activity and not only provides a readily accessible, reproducible, and exquisitely sensitive signal for assaying turnover rates and rates of conformational transitions, but also sheds light on the mechanisms of ion transport.
The specific aims are (i) to further characterize the charge translocating steps in the transport cycle by pursuing quantitative analysis of the dependence of pump charge movements on membrane potential, [Na]o, [K]o, and temperature, and (ii) to address the questions of whether, under what conditions, and by which mechanisms, pump activity may be modulated by cellular regulatory processes like protein kinase-mediated phosphorylation of the pump (or closely associated regulatory molecule). Steady-state, and voltage jump induced transient (pre-steady-state), pump currents are measured in entire guinea-pig ventricular myocytes, in giant excised patches of myocyte membrane, or in squid giant axons (in which unidirectional tracer fluxes are also measured under voltage clam), all preparations with a relatively high pump site density (about 1200 um-2). Myocytes and axons are voltage clamped and internally dialyzed with solutions that can be exchanged during recordings. Access to the cytoplasmic surface of the membrane is further improved in excised inside-out patches, a particular advantage for investigating modulation of the Na/K pump. Recent improvements in hardware and software now permit ultra high-speed measurements of pump-mediated charge movements in squid axons and in giant patches, allowing resolution of processes with relaxation rates as fast as 10-5 s-1, some 3 orders of magnitude faster than the Na/K pump's maximum turnover rate. Explicit kinetic models of the Na/K transport mechanism are developed to account rigorously and economically for experimental observations: fits to the data are used to refine the models, and then simulations based on them explore predicted pump behavior and suggest new experiments.
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