Project: Research project

Project Details


Voltage-gated Na+ channels play a central role in controlling excitability
in the heart and serve as receptors for local anesthetic antiarrhythmic
drugs. it is of considerable interest to cardiovascular biologists and
clinicians who treat patients with cardiac arrhythmias to consider how ion
channels work. There are two sets of essential features of ion channel
function; the ability to open and close in response to a biological
stimulus (gating), and the ability to permit ions to flow at high rates
through the pore with precise selectivity (permeation). The long-term goal
of this proposal is to understand, at the molecular level, ion permeation
through voltage-gated Na+ channels. Significant insights can be gained
from biophysical analysis of site-directed mutants of the channel protein.
The cardiac isoform of the Na+ channel is sensitive to blockade by group
IIB(Cd2+/Zn2+) divalent cations and insensitive to block by quanidinium
toxins such as tetrodotoxin (TTX) and saxitoxin (STX). The block by
divalent cations is mediated by a cysteine (cys) residue which is present
in the pore of cardiac but not skeletal muscle of nerve Na+ channels. Such
differences in isoform phenotype will be exploited to map the pore of the
channel by sequential replacement of amino acid in the permeation pathway
with cys and examination of Cd2+/Zn2+ and toxin blockade. These
experiments will allow us to define which amino acid residues contribute to
the pore and will provide information regarding the quaternary structure
and symmetry of this region of the protein. We will also investigate the
mechanism of the subconductance state produced by Zn2+ block of the TTX-
insensitive channel variants. Acidic residues in the permeation pathway
also influence permeation and blockade. We will study the mechanism by
which these negatively charged residues influence permeation by
neutralizing them and examining the conduction properties over a range of
ionic strengths and permeant ion concentrations. Finally we will
investigate local anesthetic blockade with a view to localizing the site(s)
of drug-channel interaction. At least one site of blockade by these drugs
is in the pore; we will isolate this mechanism by using membrane-impermeant
changed local anesthetics in combination with alteration of candidate
residues in the pore. The combination of recombinant DNA techniques and
high-resolution electrical recording promises to further our understanding
of the structure-function relationship of the channel pore and address
questions relating to ion transport and channel blockade on a more
mechanistic level.
Effective start/end date4/5/944/30/15


  • Cardiology and Cardiovascular Medicine
  • Medicine(all)
  • Cell Biology


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