William A. Sather - US grants

voltage gated channel; calcium channel; striated muscles; myocardium; protein structure function; protein sequence; pertussis toxin; gene mutation; point mutation; calcium; calcium channel blockers; chemical binding; molecular site; glutamates; hydrogen; lithium; complementary DNA; magnesium; Xenopus oocyte; Xenopus; site directed mutagenesis; voltage /patch clamp;

DESCRIPTION: 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.

[unreadable] DESCRIPTION (provided by applicant): Dendritic computation, activity-dependent gene expression, synaptic plasticity, learning and memory are neuronal processes that are based in part upon Ca2+ influx through CaV1.2 voltage-gated L-type Ca2+ channels. The activity of CaV1.2 channels is modulated by their phosphorylation state. The scaffolding protein AKAP79 anchors both a regulating kinase (protein kinase A, PKA) and a phosphatase (calcineurin, CaN) to the channel. In hippocampal pyramidal neurons, norepinephrine binding to beta adrenergic receptors initiates a cascade that leads to cAMP production and PKA activation. Phosphorylation of CaV1.2 enhances channel activity, resulting in increased Ca2+ influx. The elevated Ca2+ near the intracellular mouth of the channel activates the CaN phosphatase, which acts to reverse channel phosphorylation and consequently down-modulate channel activity and reduce Ca2+ influx. We plan to obtain from single living cells on a microscope stage dynamic measurements of fluorescence resonance energy transfer (FRET) between the various partners in this signaling complex, in an effort to uncover intra- and intermolecular movements that underlie channel modulation. We will correlate in time these movements with changes in CaV1.2 current that we will simultaneously measure using whole-cell patch-clamp recording. To obtain FRET measurements, we will fuse fluorescent proteins (cyan CFP, yellow YFP) to various pairs of signaling partners (CaV1.2, AKAP79, PKA regulatory and catalytic subunits, CaN and its Ca2+-dependent activator calmodulin). These fluorescent fusion constructs will be transfected into HEK293 cells or cultured rat hippocampal neurons. We will use a ratiometric fluorescence approach (CFP intensity/YFP intensity) to measure dynamic changes in FRET between signaling partners in single live cells. The goal of the research plan is to gain substantial insight into the mechanics, at the molecular level, of modulation of CaV1.2 channel function. In addition, the dynamics of signaling within molecular complexes, such as the one proposed for study here, helps support higher order function in neurons, such as computation, and thus the general significance of the work is broad. [unreadable] Relevance to Public Health. Calcium channels help control activity of nerve cells, beating of heart cells, contraction of smooth muscle cells that are wrapped around blood vessels, and secretion of insulin from the pancreas. Calcium channels are therefore targets of drugs used to protect against nerve cell death following stroke, to treat cardiac arrhythmias and angina, and to combat high blood pressure. A mutation in the calcium channel studied here has been identified as the cause of Timothy syndrome in humans, symptoms of which are cognitive impairment and potentially fatal disturbance of heart rhythm. [unreadable] [unreadable]