[The experiments that are planned in this proposal are designed to provide insight into the molecular processes underlying 1,4 dihydropyridine (DHP) Ca2+ channel antagonist regulation of cardiovascular L-type Ca2 channel function with a particular emphasis on the distinct modulatory properties of ionized 1,4-DHP derivatives. Recent work in other laboratories has identified specific residues on the sixth membrane spanning segment of domain IV (IVS6) of the L-type Ca2+ channel alpha1c subunit that are key to modulatory actions of neutral DHP compounds, and has shown that Ca2+ binding to physically distinct pore region glutamate residues in each alpha1c domain allosterically modifies neutral drug interactions in this laboratory, studies of native cardiac L-type channels have revealed unique functional properties that distinguish charged from neutral DHP derivatives, and preliminary data summarized in this application suggest that these differences in activity are not simply due to restrictions in access to a common DHP receptor site. The purpose of the work proposed in the present application is to determine the structural basis of charged DHP actions and to use a combination of neutral and charged drugs to distinguish between allosteric and direct ionic interactions. The planned experiments will employ custom-synthesized 1,4 DHP derivatives in which a test head group (charged or neutral) is separated from an active DHP moiety by a hydrocarbon spacer chain of variable length in combination with path clamp studies of recombinant wild type (WT) and mutant-type Ca2+ channels to study the interrelationship between channel protein and modulating drug structure and function. There are three specific aims of this proposal. The first is to study the modulation of recombinant WT L- type Ca2+ channels by charged DHPs of variable spacer chain lengths in order to define the role of spacer chain length in changed drug interactions and to establish the background necessary for structural studies of aims 2 and 3.
The second aim i s to study in detail the calcium-dependence of changed and neutral DHP modulation of recombinant Ca2+ channels and to distinguish between high and low affinity Ca2+- dependent interactions.
The third aim of the project is to use site- directed mutagenesis of pore region glutamates and specific residues in IVS6 of the alpha 1c L-channel subunit to determine the structural basis of charged DHP interactions. This work will provide new insight into the molecular architecture of functionally key residues of the calcium channel alpha1c subunit, and mechanistic data that will account, for the first time, for differences between the modes of action of changed and neutral DHP derivatives which most likely underlie unique therapeutic advantages of long-lasting tertiary DHP calcium channel antagonists.]
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