It is proposed to study molecular aspects of gating and regulatory processes in voltage-dependent Na and K ion channels of rat myoballs, dorsal root ganglion cells and hippocampal neurons. The cells will be maintained in primary tissue culture. The patch clamp technique will be used to record from whole cell membranes and from small membrane patches containing only a few ion channels. For testing whether inactivation of Na channels is kinetically linked to their activation, small ensembles of Na channels will be stimulated with voltage protocols designed to test for effects of channel opening on the rate of channel inactivation. We will examine whether the opening/closing step of activation gating is slow with regard to other activation steps, as might be expected if channel opening involves a major change in configuration. The latter single-channel measurements will be done on Na channels whose inactivation has been chemically blocked or slowed. Inactivating and non-inactivating forms of K channel will be characterized at the single channel level; microscopic aspects of their gating kinetics will be compared to those found for Na channels. Regulatory effects of maintained membrane voltages will be examined in individual Na and K channels. Various ions (to be used for intracellular solutions), as well as chemicals with possible regulatory effects (cAMP, ATP, and thiamine derivatives) will be introduced into cells and their possible effects on the function of Na and K channels will be characterized. Conditions essential for survival of channels in excised membrane patches will be explored. We will test whether Na and K channels of our preparations, like K channels of myelinated nerve, undergo spontaneous changes of gating behaviour. The long range goal is to understand how Na and K channels in excitable membranes are controlled by changes of the membrane potential and, in the long term, by modulatory processes.
