Project Details


Although considerable primary sequence data have been developed for
voltage-dependent sodium channels by this laboratory and others, and a
number of detailed models have been proposed for channel tertiary
structure, experimental data have been reported that relates directly to
the three-dimensional conformation of the channel in the membrane. Given
the difficulties encountered in obtaining x-ray crystallographic
information for membrane proteins such as the sodium channel, it is likely
that high-resolution structural data will still be some years in coming.
In the meantime, studies relating structure to function require the best
possible analysis of channel three-dimensional structure in order to
proceed. Because of the indirect nature of most structural approaches
short of crystallography, a number of different techniques will have to be
brought to bear. Building on our recent biochemical studies of the skeletal muscle sodium
channel, and our cloning of the primary sequence for the rat TTX-sensitive
and TTX-resistant isoforms of the sodium channel and their human homologs,
we propose here to continue a multidisciplinary approach to muscle sodium
channel structure, and to begin an analysis of the relationship between
this structure and its function. We will probe the organization of the
sodium channel in the membrane with studies that focus on the membrane
topography of specific segments of the primary sequence, on the
organization of the large extramembrane regions in the channel structure,
and on the structure of the compactly folded internal repeat domains.
Specifically, we will complete our analysis of the location of sites of
post-translational modification in the channel primary sequence. We will
extend our detailed analysis of the location and kinetics of
protease-sensitive regions in the channel structure to include a
topographical analysis of these regions, and an analysis of their response
to alterations in membrane potential or toxin binding. We will extend our
use of monoclonal antibodies to probe the interaction and location of the
extramembranous domains. We will use antibodies to interhelical loops or
to foreign epitopes inserted into interhelical loops to begin an analysis
of the folding pattern within the repeat domains. Finally, in collaboration with Dr. Richard Horn, we will begin an analysis
of structure function relationships in the muscle channel that takes
advantage of the characteristic differences in toxin binding, single
channel conductance, and kinetics between the muscle TTX-sensitive and
TTX-resistant channels. This analysis will take specific advantage of the
availability of expressible full-length clones for the two rat skeletal
muscle sodium channel isoforms. Chimeras will be constructed between the
two clones and the partitioning of characteristic properties investigated.
Initial leads will be pursued with site-directed mutagenesis. Special
attention will be paid to the S5-S6 interhelical loop regions in light of
their potential role in toxin binding, and their markedly different
treatment in key models of channel structure.
Effective start/end date12/1/8111/30/96


  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $250,950.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $351,918.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health


  • Medicine(all)
  • Neuroscience(all)

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