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 recently identified discrete Ca2+ sensors (Soldatov et al., 1998) with site(s) associated with the pore. The Ca2+ sensors may differentially contribute to the Ca2+-induced inactivation of the channel because they are selectively targeted by permeating vs. cytoplasmic Ca2+ due to specific arrangement vis-a-vis the pore. Ca2+ sensors may in addition have different affinity to Ca2+, which may determine a dependence of inactivation rate on the speed of increase of [Ca2+] in the vicinity of Ca2+ sensors. Finally, Ca2+ sensors may also be critical for the run-down and effects of modulatory proteins (auxiliary subunits, calmodulin, calpastatin). 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) To investigate whether the determinants for Ca2+-induced inactivation of the a1C channel are differentially targeted by pore-permeating and cytoplasmically-released Ca2+. (2) To identify the molecular correlates for the regulation of the a1C channel by Ca2+ sensors and their participation in local intracellular Ca2+ signaling. (3) We will characterize (in collaboration with C. Romanin, University of Linz, Austria) the molecular correlates for run-down of L-type Ca2+ channels and involvement of Ca2+ sensors. (4) Using the a1C,94 channel, which lacks inactivation due to a naturally occurring mutation A752T at the cytoplasmic end of the transmembrane segment IIS6, we will investigate whether this and corresponding residues in other similar motifs serve as critical structural determinant(s) for inactivation and ion selectivity of the channel. We will study whether this mutation is essential for the development of human pathologies linked to Ca2+ overloading, such as brain ischemia or Alzheimer disease. (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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