Section of Molecular and Clinical Pharmacology (Nikolai M. Soldatov, Ph.D.) The long-term objective of our research is to pursue the study of the structure-functional relationships of human cardiac and vascular L-type Ca2+ channels, and to investigate the molecular correlates for the channel inactivation affected by alternative splicing, naturally occurring mutation in the pore region, and mobility of the C-terminal tail carrying calmodulin. 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+ sensor 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). Single-molecule fluorescence study have shown (Harms et al., 2001) that the mean number of the channels per cluster is 40. We extended our investigation of the molecular mechanisms of inactivation (Abernethy and Soldatov, 2002). 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 have identified a molecular determinant of slow voltage-dependent inactivation as a ring of hydrophobic residues in the inner pore region (Shi and Soldatov, 2002). Given the importance of the 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) using fluorescence resonance energy transfer as molecular ruler, to investigate the voltage-gated molecular rearrangements supporting the inactivation of the Ca2+ channels and the Ca2+ signaling function. (2) We will investigate the cardiovascular aspects in the developed transgenic animal models of human wild type and mutated Ca2+ channel. (3) Recently we have identified an the isoform of the Ca2+ channel specific for the aged (>54 years old) human aortic cells (unpublished) and characterized by lower sensitivity to dihydropyridine Ca2+ channel blockers (Soldatov et al., 1995). In addition, we obtained the genotyping data indicating that the A752T mutation of the channel impairing its inactivation occurs with the frequency of about 3% in humans. Using cell microdissection technique, RT-PCR and MS analysis, 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 the molecular properties of the 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 supporting transcription regulation as well as excitation-contraction coupling in human cardiac and vascular muscle cells and provide useful clues for molecular diagnostics and drug developments.
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