MECHANISM INFLUENCING LIGAND-GATED CA2+ RELEASE CHANNELS

Project: Research project

Project Details

Description

The proposed studies will elucidate the structure and function of
cardiac, skeletal, and smooth muscle ligand-gated calcium release
channels (LGCRC) in isolate membranes and purified receptor
preparations. Particular emphasis is placed on defining the
mechanisms by which pertinent classes of pharmacological agents
and environmental toxicants alter normal channel function in vitro
and assesses their toxicological relevance in vivo. Analysis of
(3H)ryanodine receptor binding with concomitant spectrophotometric
assays of Ca2+ uptake and release from junctional vesicles with the
metallochromic Ca2+ indicator antipyrylazo III allows direct
inspection of receptor and Ca2+ channel function. Fluorescent and
photoaffinity probes will test the major premise that four distinct
effector domains modulate the gating behavior of LGCRC. The
principal hypotheses tested are: 1. (3H)ryanodine specifically binds to the Ca2+-induced open state
of LGCRC, and hence can directly assess modulation of LGCRC by
physiologically-relevant ligands or xenobiotics. Photoaffinity
labelling of the ryanoid-binding site reveals its position within
junctional foot oligomer. 2. The Ca2+ regulatory domain is primarily responsible for gating
LGCRC and unmasking the (3H)ryanodine-binding site. Lanthanides
compete for the Ca2+-binding sites while thiol-reactive heavy
metals and aryldisulfides specifically interact with critical
thiols associated with this domain and serve as functional and
structural probe. Fluorescent sulfhydryl reagents clarify the role
and position of this domain within the native LGCRC oligomer and
discriminate major differences among muscle types. 3. The xanthine domain allosterically influences the sensitivity
of LGCRC and the (3H)ryanodine binding site to activation by Ca2+-
. Antineoplastic anthracyclines specifically bind to this domain
causing potent sensitization to activation by Ca2+ which can be
antagonized by caffeine. 4. The adenine nucleotide domain enhances (3H)ryanodine receptor
occupancy and the intensity of the LGCRC response to Ca2+ and
functionally overlaps the xanthine domain. The combined use of physiological, biochemical, and toxicological
endpoints will yield important new information about LGCRC
structure and function and assess its involvement as a target for
pharmacological and toxicological agents and characterize
the mechanisms involved.
StatusFinished
Effective start/end date12/1/881/31/00

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Environmental Science(all)
  • Medicine(all)

Fingerprint Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.