Molecular (patha)physiology of L-type Ca2+ channels

Calcium entry through voltage-dependent L-type Ca2+ channels is an important determinant of vascular smooth muscle tone. Normal physiologic function of L-type Ca2+ channels is governed by the interaction of regulatory enzymes such as protein kinases as well as of intracellular regulatory factors such as H+ and redox-active species with subunits of the Ca2+ channel complex. These regulatory mechanisms are based on a reversible chemical modification of Ca2+ channel proteins. Disturbances in the cross-talk between channel proteins, regulatory proteins and cellular regulatory factors is one potential origin of vascular diseases such as hypertension and atherosclerosis. The proposed project aims at clarifying the molecular basis of L-type Ca2+ channel regulation using an approach which combines biophysical, biochemical and molecular biological methods. The project will investigate Ca2+ channel regulation by protein kinase C, intracellular redox state and intracellular pH. Particular attention will be paid to the role the cytoplasmic beta subunit of the channel as a target of regulatory modulation. In course of this project, we will study the gating behavior of native L-type Ca2+ channels in vascular smooth muscle cells and of recombinant L-type Ca2+ channels expressed in an heterologous expression system (human embryonic kidney cells). The regulatory environment of these channels will be controlled by exploiting the technique of Ca2+ channel recording in cell free membranes and by co- and overexpression strategies. Site directed mutagenesis will be used in attempts to identify the molecular structures which serve as chemical switches in Ca2+ channel proteins, and the regulatory role of the beta subunit will be studied for the first time at the level of single channels using a high resolution fluorescence microscopy technique. These experiments are expected to contribute to a better understanding of the role of individual signal transduction mechanisms in cellular regulation of L-type Ca2+ channels in vascular smooth muscle. The results of this projects may help to identify mechanisms of pathophysiologic relevance and thereby contribute to the development of novel strategies for the therapy of cardiovascular diseases.

Molecular mechanisms that regulate voltage-gated Ca2+ channels, and the role of these regulatory principles in pathoplysiology, are still incompletely understood. This project was focused on different, pathophysiologically relevant aspects of class C (CaV1.2) channel regulation which are considered crucial for control of the cardiovasuclar functions. Our results open the view on novel concepts in the control of Ca2+ channel functions and provide evidence for new regulatory principles: 1) Regulation based on reversible protein-protein interactions within the Ca2+ channel complex. 2) A pivotal role of a C-terminal domain of the central channel subunit as a determinant of subcellular localization, gating and conductance. 3) Reversible S-nitrosation of protein residues as a mechanism of Ca2+ channel regulation. 4) Control of channel functions by phosphorylation-independent effects of lipid mediators. 5) Control of CaV1.2 channel functions via a tight functional coupling to nonselective cation channels of the Trp family. Notably, our work includes the first demonstration of a close interaction between two distinct membrane channels which have so far been considered to function rather independently of each other. Moreover, evidence is provided for local Ca2+ signalling as a key mechanism of functional coupling between these two ion channels. The identified molecular mechanisms of Ca2+ channel regulation are expected to prepare the ground for development of novel strategies that enable a better therapy of cardiovascular disorders.