The long term goal of this proposal is to gain insight on how voltage dependent Ca2+ channels function in molecular terms by performing structure-function studies of cloned Ca2+ channels. The main focus is to study the movement of the voltage sensor (gating currents) and its coupling to channel opening and closing, and the mechanism(s) and the molecular domains for the regulatory actions of coexpressed beta subunits, of Ca2+ influx and of phosphorylation. These studies can be performed because the success in measuring gating currents of cloned Ca2+ channels expressed in frog oocytes. This is due to: 1) a large functional expression, 2) the fast response of the cut-open oocyte vaseline gap technique, and 3) the success of the giant-macropatch technique (20-30 mum) for noise analysis. The main questions are; 1. How are the open and closed states of the channel related to charge movement? 2. What are the number of effective charges per channel and the unitary charge element? 3. Can the coupling between the voltage sensor and the pore be probed with mutagenesis to define molecular domains for the charge movement directly associated to channel opening? 4. Is the inactivation process associated with charge immobilization? 5. Which are the mechanisms by which beta regulatory subunits and phosphorylation facilitate channel opening? 6. Can we identify critical phosphorylating sites for channel function? 7. Are these regulatory actions altering specific components of the gating currents or are they altering the final coupling between the charge movement and channel opening? The Specific Aims are: 1. To investigate the properties of Ca2+ channel charge movement to gain insight on the molecular mechanism(s) and the domains involved in the coupling between the movement of the voltage sensor and channel opening; 2. To define the charged residues and the structural determinants in the alpha1 pore subunit responsible for the voltage dependence of Ca2+ channel activation; 3. To study the mechanism(s) of Ca2+-dependent inactivation in the cardiac alpha1C channel, and of voltage- dependent inactivation in the neuronal alpha1E channel; 4. To define the mechanism(s) and molecular domains for the interactions between the Ca2+ channel alpha1 pore forming subunits and the regulatory beta subunits; and 5. To define in alpha1 subunits the role of phosphorylation in the coupling of the voltage sensor with channel opening. These structure- function studies of Ca2+ channels should serve as the basis for understanding the actions of many therapeutical agents anf for the development of new drugs.
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