Peter Hess - US grants

Calcium channels play a crucial role in the control of cellular function. In the heart, they initiate contraction and help determine pacemaker rhythm and conduction in the nodal regions. In vascular smooth muscle they regulate contraction and therefore control vascular tone. One Ca channel type is the target for the widely used Ca anatagonist drugs and its gating is modulated by neurotransmitters and hormones. The interest in Ca channels is further enhanced by the fact that as Ca selective transport proteins, they may share many properties with other important Ca binding molecules. We propose to combine functional measurements at the level of individual channel molecules with biochemical attempts to purify the channel protein. Ca channels from cardiac and vascular smooth muscle will be investigated. Single channel recording techniques will be used for a quantitative description of the biophysical channel properties. Patch clamp measurements on intact cells will be compared with recordings from cardiac Ca channels incorporated into lipid bilayers, an approach which gives ideal control over the ionic and lipid environment of the channel. Reconstitution of the L-type cardiac Ca channel in lipid bilayers will also be used to follow the functional integrity of the channel protein through biochemical solubilization and purification. These techniques will help answer some fundamental questions about Ca channel function: What are the parameters determing an ion's rate of Ca channel entry? How many ion binding sites does the channel have? Can it be occupied by more than one ion at a time? Do ions interact within the pore? Is the channel pore functionally symmetrical? How does the composition of the membrane lipid affect channel function? What are the differences between cardiac and smooth muscle Ca channel types? In an attempt to relate channel function to structure, we will investigate the role of sugar residues for channel function and will try to create proteolytic lesions with specific functional consequences. The functional consequences of Ca channel phosphorylation will be investigated in detail. We will ask the following questions: Is phosphorylation necessary and sufficient to make a Ca channel available for activation by depolarization? What are the rates of phosphorylation and dephosphorylation? What protein kinase systems are involved? Are the regulatory systems different in heart and vascular smooth muscle?

Transmembrane Ca channels have an important dual function: they generate electrical signals which are used in many cells for conduction of impulses and for the control of rhythmical electrical activity, but Ca channels also deliver a specific chemical messenger to the cell in the form of Ca ions, whose message is decoded by intracellular Ca binding effector proteins. Ca channels therefore play a fundamental role in the regulation of key cellular processes in most excitable cells. In heart cells, Ca channels initiate contraction, help determine heart rate by contributing to the pacemaker depolarization, promote slow conduction in nodal regions, and support the action potential plateau, thus determining the duration of electrical excitability. In partially depolarized regions of the heart, Ca channels can initiate slowly conducted action potentials and promote arrhythmic activity. The L-type Ca channel is the pharmacological receptor for some of the most widely used drugs (Ca channel blockers) in the management of coronary heart disease, hypertension and cardiac arrhythmias. This grant application proposes to investigate the elementary functional properties of Ca channels. Patch clamp recordings will be used to study the openings and closings of single Ca channel molecules in intact cardiac cells and sympathetic neurons. The kinetics of individual openings and closings, and of transitions between longer lasting gating modes will be analyzed quantitatively in order to better understand the mechanisms and stimuli which govern the activity of Ca channels in cells. The amplitude of the unitary current flowing through single open Ca channels under various experimental conditions will be measured to learn more about the process of ion permeation and selectivity, a process to which the channels owe their capability of passing Ca ions at a high rate and yet to remain selectively permeant to Ca ions even in the presence of high concentrations of competing ions. Separate experiments on a K-channel for which we have the cloned cDNA will attempt to correlate functional domains with structurally and functionally conserved domains of voltage gated ion channels relevant for both K- and Ca channels. An antigenic epitope will be introduced into various domains of the channel sequence by in-vitro mutagenesis in an effort to locate key segments of the primary structure with respect to the plasma membrane. Mutations of positive and negative charges in the presumed membrane spanning regions of the channel will be used to establish the relative location and functional interactions between residues conserved in all voltage dependent ion channels. From the proposed combination of functional studies at the single channel level and manipulation of the channel structure by site-directed mutagenesis we hope to gain new insight into the molecular mechanisms which determine the function of these important regulators of cellular excitability.