John Starkus - US grants

This proposal will utilize a particularly favorable axon preparation, the crayfish medial giant axon, to seek improved experimental resolution concerning a carefully chosen selection of key questions with respect to the physiological mechanisms of membrane excitability. Among the questions posed are: a) are the separate components of gating current generated by semi-independent particles of differing valence? b) are any of these gating current components directly generated by the opening and/or closing of channel gates? c) can kinetic components be similarly identified within sodium current transients? d) are fast and slow inactivation separate and parallel processes or are they integral components of a sequential conductance control system? e) can the kinetic components of sodium current be related to specific kinetic components of gating current? Answers will be sought through sophisticated kinetic analysis in addition to using pharmacological agents to modify normal axon behavior. Results will be used to generate a biophysically rigorous physiological model which can then be used to predict additional, experimentally testable, aspects of axon excitability. The health related aspects of this line of research may add to the understanding of neural excitability in man and help in designing new or safer local anesthetic and anti-arrythmic drugs. Results of this work will increase our basic understanding of other systems including: autonomic, central, peripheral and cardiovascular systems since these systems have similar basic mechanisms of ion conductance control.

The broad,long-term objective of this proposal is to develop a detailed understanding of structure-function relationships within the voltage- dependent sodium channel molecule. The Specific Aims are: 1) To clarify the functional significance of each of the four S4 segments within the tetrameric sodium channel molecule; 2) To clarify the interactions between activation and fast inactivation at the structural level; 3) To clarify the effects of "distant" sites on activity of the S4 segments within each domain. The experimental design involves detailed functional analysis of mutant channels expressed in Xenopus oocytes following site directed mutagenesis to alter specific regions of the channel molecule. The details of this design reflect our sophisticated approach to electrophysiological analysis of sodium channel mechanisms and our recent advances in this area. The methods used involve voltage-clamp analyses of channel properties (in which we have much expertise) and molecular biological methods which are relatively new to our laboratory. We have recruited a coinvestigator from this campus with extensive experience in site-directed mutagenesis and have developed collaborative agreements with other investigators to ensure appropriate support for this initiative. Our proposed work is strongly health-related since the sodium channel molecule is affected by many drugs and is the basis for the electrical excitability of cardiac muscle, skeletal muscle and nerve cells.

DESCRIPTION: The long-term objectives of this laboratory remain the delineatio of the structure/function relationships involved in the gating (opening and closing) of voltage-gated ion channels. Three major gating mechanisms have bee described in Shaker potassium channels: i. Activation/deactivation- the normal voltage sensitive opening and closing mechanism. ii. Fast (N-type) inactivatio which closes channels during short maintained depolarizations (tens of milliseconds duration). iii. Slow (C-type) inactivation which closes channels during longer periods of depolarization (seconds). Only the second of these mechanisms has been effectively characterized in previous work, as involving a tethered ball domain which blocks the channel by binding to an internal bindin site near the internal mouth of the permeation path. However, recent work from our group has established that slow, C-type, inactivation does not involve either closing or blocking of the permeation pathway in Shaker channels. This unusual inactivation mechanism results from a change of channel selectivity such that channels no longer conduct K+ ions. On the other hand, macroscopic Na+ ions can be recorded in K-free solutions even after full development of C-type inactivation. This proposal aims: A, to further characterize both the selectivity change and the properties of activation and deactivation gating in C-type inactivated channels and B, to us site-directed mutagenesis of pore domain residues to evaluate the molecular mechanisms involved in the change from normal to C-type selectivity. This work will be carried out using Xenopus oocyte-expressed Shaker channels. Ionic and gating currents will be evaluated using standard patch clamp methods This work is strongly health-related, in that C-type inactivation is the primary mechanism controlling the inactivation and recovery rates of the cardiac potassium channels responsible for both IKS and IKF currents. Defects in this mechanism are responsible for the LQT1 and LQT2 syndromes associated with potentially lethal cardiac arrhythmias.