Voltage sensitive Ca channels play a pivotal role in the control of a wide spectrum of biological activities. In the present proposal we will investigate the molecular mechanism of Ca channel inactivation, or spontaneous closing. Its physiological relevance extends from sharpening the temporal precision of Ca signals to control of activity-dependent interactions of Ca channels with other proteins. Inactivation is accompanied by changes of the intramembrane charge movement reflecting conformational transitions in the channel molecule. We have devised a way to monitor inactivation of Ca channels by recording the intramembrane charge movement generated only in inactivated channels. The power of this method is that the kinetics of inactivation can be studied (and compared) both with and without ionic current flow. With this technique, we have shown that Ca ions permeating through neuronal N-type Ca channels modify their intramembrane charge movement. Our main idea is that both Ca- and voltage-dependent components of inactivation of different types of Ca channels are different facets of the same molecular mechanism, which induces the same final inactivating states and involves Ca binding to a yet unknown region of the channel. It is widely believed that Ca-dependent inactivation is caused solely by binding of Ca ions to a site, which is located on, or near, the intracellular carboxyl terminus of the main pore-forming subunit of the channel molecule. Instead, our data indicate that, to close the channel during inactivation, Ca may act at a different site, which is likely to be in the permeation pathway. We will expand the studies of the interaction between Ca and voltage sensing moieties of cardiac L-type and neuronal N-type Ca channels expressed in mammalian cell lines in order to answer the following questions: 1. Does Ca current promote inactivation of gating currents in L-type channels? 2. Do Ca and voltage induce the same inactivating transitions? 3. Where do Ca ions bind to inactivate Ca channels during depolarization? 4. What is the nature of gating current changes that precede inactivation of Ca channels? Results from this work - and from work in other laboratories - will be interpreted in terms of a biophysical model explaining how the interaction between permeable cations and voltage sensing parts of Ca channels controls inactivation gating. An ultimate achievement of the present proposal will be to guide the future detailed structure/function studies of molecular aspects of inactivation.
