Voltage-gated Ca2+ channels are the principal link between electrical signals in nerve cells and intracellular Ca2+ signaling pathways that allow nerve cells to, for example, release neurotransmitter or alter their gene expression. To accomplish these tasks, voltage-gated Ca2+ channels open in response to an action potential and allow exclusively Ca2+ to travel through the channel's highly selective pore into the cellular interior. Malfunction of neuronal voltage-gated Ca2+ channels has serious health consequences for humans, including the genetic diseases spinocerebellar ataxia type 6, familial hemiplegic migraine, and episodic ataxia type-2. The goal of the proposed research is to understand the structural basis of selective ion flux through Ca2+ channels. In pursuit of this broad goal, we plan to carry out three Specific Aims: (1) determine the topography of the pore in an L-type Ca2+ channel; (2) localize Ca2+ channel gate(s) by testing for state-dependent accessibility of sulfhydryl-modifiers; and (3) measure the electrostatic potential profile in the pore of an L-type Ca2+ channel. In all of these studies we will measure the accessibility to sulfhydryl-modifying agents of cysteine-substituted mutant forms of the a1c L-type Ca2+ channel. If the sulfhydryl-bearing side chain of a substituted cysteine residue is exposed in the lumen of the pore, then covalent attachment of a sulfhydryl-modifying reagent may result in obstruction of permeant ion flow through the pore. Using the resulting persistent block as an index, we will determine which residues of putative pore-lining sequences (S5, P-loop, S6 segment) in fact line the pore. We will determine the dimensions of several parts of the ion-conducting pore (external and internal vestibules, ion selectivity filter) using sulfhydryl-modifiers of various sizes. We will use open/closed/inactivated state-dependent accessibility to localize the gate(s) of the Ca2+ channel. An important parameter of selective ion transport is the intrinsic electrostatic potential in the pore, and this will be determined from measurements of modification rate for differently charged sulfhydryl modifiers. In all experiments, block of current will be measured for voltage-clamped, heterologously expressed Ca2+ channels.
