Section of Molecular and Clinical Pharmacology (Nikolai M. Soldatov, Ph.D.) The long-term objective of our research is to pursue the study of structure-functional alterations of human cardiac and vascular L-type Ca2+ channels due to alternative splicing, and to investigate the affected molecular correlates for the channel inactivation. We hypothesize that Ca2+-induced inactivation of the a1C channel is mediated by the interaction of identified Ca2+ sensors (Soldatov et al., 1998) with site(s) associated with the pore. The Ca2+ sensors appear to be differently targeted by calmodulin and permeating Ca2+ (Romanin et al., 2000) and contribute to the voltage- and Ca2+-dependent inactivation of the channel. The Ca2+ sensors are also critical for the run-down (Kepplinger et al., 2000a) and effects of modulatory proteins such as auxiliary b subunits (Soldatov et al., 1997), calmodulin (Romanin et al., 2000) and calpastatin (Kepplinger et al., 2000a). In addition, Ca2+ sensors were found to contribute to membrane targeting by a1C subunit and Ca2+ channels clusterization (Kepplinger et al., 2000b). Given the importance of the class C, L-type Ca2+ channel in cardiovascular physiology, we plan to extend our investigation of the human a1C splice variants and pursue the following specific aims: (1) Based on our data on human a1C,94 channel with inactivation impaired due to a naturally occurring mutation A752T at the cytoplasmic end of the transmembrane segment IIS6, we will investigate the nature and mechanism of slow inactivation. Using transgenic animal models, we will study whether this mutation is essential for the development of human pathologies linked to Ca2+ overload. (2) To identify the molecular correlates for the regulation of the a1C channel by Ca2+ sensors and calmodulin. (3) Using single-molecule fluorescent imaging and fluorescence correlation spectroscopy, we will characterize (in collaboration with Th. Schmidt, Univ. Leiden, The Netherlands) the clustering and molecular mobility of eYFP-labeled Ca2+ channel in live cell. (4) To investigate, using fluorescence resonance energy transfer, the state-dependent protein-protein interaction in the functional Ca2+ channels. (5) Recently we identified one of the isoforms of Ca2+ channel specific for aged human aortic cells and characterized by lower sensitivity to dihydropyridine Ca2+ channel blockers. We will further investigate the diversity of a1C transcripts generated in human cardiac and vascular cells and tissues in response to age, drugs, hormonal and pathological stimuli. We will examine whether alterations in molecular properties of a1C channels occur with age and as a result of cardiovascular diseases, including hypertension, cardiomyopathy, ischemia, arrhythmias and heart failure. Results of our study may give insights into the fundamental principles of Ca2+ signaling underlying excitation-contraction coupling in human cardiac and vascular muscle cells and provide useful clues for molecular diagnostics and drug developments.
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