SGER: Molecular Basis of Modulation of Neuronal Sodium Channels

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Principal Investigator: Satin, JonathanAbstractVoltage-gated sodium channels are key molecules for initiating and shaping neuronal excitability. It is known that protein kinase C modulates cellular excitability in part via attenuation of fast Na current, but there is an incomplete understanding of the molecular mechanism for effects of modulation on Na channel function. In particular, little is known regarding modulation of slow Na current components. The reason for this incomplete understanding is that two factors have complicated the study of Na channel modulation: 1) an inadequate understanding of the interaction between channel kinetics and channel modulation, and 2) the expression of numerous Na channel isoforms in many native preparations. My long-term research goal is elucidate structure-function relationships of voltage-gated ion channels. Heterologously expressed, cloned voltage-gated Na channels serve as an ideal model system to determine molecular structure-function constraints. The objectives of this proposal are to test our molecular structure-function hypothesis for the interaction of channel domains, and to evaluate conditions that promote slow to fast gating transitions. These studies are motivated by our preliminary data showing that protein kinase C mediated phosphorylation of the Na channel leads to slowing of activation gating, and shifting of the fraction of fast gating channels. In the first aim we will perform site-directed mutagenesis of the rat brain ha Na channel to define the site of action of PK-C. Replacement of the PK-C target serine residue with a cysteine residue will allow us to probe structural hypothesis by the use of chemical modifiers. Chemical modifiers in the methanethiosulfonate class (MTS) will allow us to add positive or negative charges, and to create reversible cross-linkers with known molecular dimensions. In the second aim we will test our hypothesis that PK-C mediated phosphorylation of the serine on the inactivation domain contributes to slow -- fast mode switching. We will also test the effects of concentration of kinase. Finally, we will use a second neuronal isoform that may exhibit additional slow gating components. Our hypothesis suggests that channels with different slow gating may respond differently to modulation by PK-C activators. The latter experimental design will stress comparison of two different isoforms of neuronal Na channels expressed in an identical expression system. We will utilize molecular biological and electrophysiological techniques to understand molecular structure/function correlates for fast and slow inactivation, and modal gating. This proposal will advance our knowledge of the biophysical basis of membrane electrical properties, and their regulation by intracellular second messengers.