) The long-term goal of my research is to understand how the dihydropyridine receptor (DHPR) functions as a channel and voltage sensor for excitation-contraction (EC) coupling. The DHPR is an L-type voltage gated calcium channel which regulates the flux of calcium across cell membranes in a variety of calcium-dependent processes. L-type channels are characterized by a low open-channel probability (Po) and have been shown to enter a potentiated state, defined by high Po and long openings, following large depolarizations and application of dihydropyridine (DHP) drugs. The immediate goal of this research is to determine the structural basis for L-type low Po and depolarization-induced potentiation in the cardiac DHPR. The results of this research will provide insight into the molecular mechanism of potentiation and allow us to understand how this mode of channel gating may be involved in regulating cardiac function. The following methods will be used to conduct the proposed research: Recombinant DNA techniques will be used with cardiac (L-type) and neuronal (non-L-type) channels to create several chimeric channels, each with an amino terminal green fluorescent protein (GFP) tag. Fresh cultures of dysgenic myotubes will be made weekly from mice homozygous for the mdg mutation. The chimeric channel cDNAs will be injected into the nuclei of dysgenic myotubes, which lack the functional alpha1 subunit of these channels. Expression of chimeric channels will be monitored via the GFP tag, which is visible under fluorescence 24-48 hours post-injection. Whole-cell patch clamp recording will be used to determine current-voltage relationships and gating charge, which will provide an indirect measurement of Po for each chimeric channel. The different chimeras will be used to identify regions critical for depolarization-induced potentiation and low Po.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31MH012512-03
Application #
6391734
Study Section
Special Emphasis Panel (ZRG1-MDCN-4 (01))
Program Officer
Goldschmidts, Walter L
Project Start
2001-09-30
Project End
Budget Start
2001-09-30
Budget End
2002-06-30
Support Year
3
Fiscal Year
2001
Total Cost
$17,303
Indirect Cost
Name
Colorado State University-Fort Collins
Department
Microbiology/Immun/Virology
Type
Schools of Veterinary Medicine
DUNS #
112617480
City
Fort Collins
State
CO
Country
United States
Zip Code
80523