Voltage-gated Ca channels convert electrical signals into intracellular Ca signals that are paramount for a variety of cellular processes, including neurotransmitter release, muscle contraction and gene expression. Ca channels trigger these processes by opening in response to electrical stimulation, thus allowing Ca ions to flow into the cell where they act as signaling molecules that are detected by Ca binding proteins. L-type Ca channels, one subset of the Ca channel superfamily, are the target proteins for a number of drugs including the dihydropyridines (DHPs). DHPs are an important class of drugs, used extensively to treat angina, hypertension and stroke. DHPs modulate Ca channel gating, but the molecular details that link DHP binding to changes in Ca channel gating are not known. We have found that the DHP receptor site and a Ca binding site in the pore exhibit cooperativity and are allosterically coupled. Ca binding to the pore promotes the energetic coupling between two amino acid residues located in the pore and DHP antagonists disrupt this coupling. This finding is intriguing given that gating for several types of ion channels is linked to structural rearrangements in their respective pore regions. We hypothesize that conformational changes that occur upon DHP binding are transduced to the pore where gating is altered. By studying the allosteric interactions between Ca and DHP binding, we intend to identify important molecular interactions that link DHP binding to changes in channel gating. We are integrating several techniques, including whole-cell patch-clamp electrophysiology, kinetic modeling and radioligand binding to develop a deep understanding of how DHPs modulate the gating behavior of L-type Ca channels. Results from these studies are expected to facilitate the rational design of new classes of drugs that modulate the gating behavior of Ca channels. This application is divided into four Specific Aims: 1. Identify interacting amino acid pairs in the Ca channel pore. 2. Identify changes in gating that are dependent on Ca binding to the channel pore. 3. Clarify the relationship between Ca and DHP binding and channel block. 4. Develop an allosteric-kinetic scheme that links DHP- and Ca- binding to Ca channel gating.
