The importance of voltage-dependent calcium channels in cellular processes is indisputable. Their pivotal role in controlling such functions as muscle contraction, neurosecretion, and membrane permeability has made calcium channels a natural target for both extracellular and intracellular modulatory agents. Despite the variety of modulators that have been identified and the prevalence of the modulation, the fundamental mechanisms responsible for controlling calcium channel function have not been adequately described. Impediments to our understanding have derived, at least in part, from the inadequacy of early classification schemes to effectively discriminate between the different calcium channel subtypes and a reluctance to treat calcium channel modulation as a potentially inhomogeneous process. Experiments in this application will investigate the mechanisms underlying transmitter-induced inhibition of one type of high voltage-activated calcium channels (N-type) in embryonic chick sensory neurons. Preliminary results presented in the proposal demonstrate that the modulation produced by GABA can be separated into two components (not previously recognized in the calcium channel field). These can be distinguished from one another on the basis of their transmitter concentration-dependence, time course, and voltage-dependence. We will test the hypothesis that the two components are produced by separate mechanisms. Experiments are proposed to dissect and characterize the components biophysically (Aim 1) and to investigate whether they involve the activation of different GTP-binding proteins (Aim 2) and/or protein kinase C (Aim 3). Differences in the underlying mechanisms and/or the relative contributions of these two distinct components may explain a number of discrepancies surrounding the modulation that have been reported in the calcium channel literature. Experiments in Aim 4 will determine whether this is, in fact, the case comparing selected aspects of the modulation in two different, commonly-studied preparations..
In Aim 5, we will test the extent of conservation of the mechanisms that underlie N-type calcium channel modulation by determining whether they also characterize the modulation of other high voltage-activated calcium channel types. In these experiments, we will examine the inhibition of non-N-type channels in chromaffin cells. As specific calcium-dependent cellular processes are likely to be triggered by particular calcium channel subtypes, an in-depth understanding of the varied mechanisms by which the subtypes are controlled win lead to the development of methods that can differentially target (and alter) individual cellular functions. Such an achievement may, ultimately, allow sophisticated clinical interventions into a wide variety of pathological conditions, particularly in the cardiovascular, neurological, and endocrine systems.
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