The application of molecular biology to electrophysiology has led to rapid progress during the last several years. Several voltage-gated cation channels have been cloned, and are composed of either large proteins with four repeated homologous subunits (Na+ and Ca2 + channels) or of smaller proteins which aggregate to form tetramers (K+ channels). Amino acid sequences show considerable structural homology among these channels; specifically, an area of conserved positively charged amino acids (found in each channel subunit and known as the S4 region) is thought to interact with the transmembrane electric field and to act as the voltage sensor which controls channel activation. Recent genetically-engineered mutations of this region by several laboratories support this hypothesis; a complete analysis of the function of S4 region has yet to be performed, however. Interactions between the subunits of the channel may play an important role in the voltage-gating process. This grant proposal describes the use of site-directed mutagenesis of the 54 region of a cloned delayed rectifier K+ channel (RCK1) to further elucidate the mechanisms of channel gating and the relationships between the channel subunits: a) less drastic mutations of the S4 residues which have previously formed nonfunctional channels will be engineered b) mutations will be restricted to a fraction of the subunits, and c) mutations will be made in acidic amino acids which may form ionic pairs with and stabilize the positively charged amino acids of the S4 region. Control and mutant channels will be expressed in-vitro in frog oocytes; gating properties will be determined by voltage-clamp, and compared to existing models of channel kinetics. Voltage-gated ion channels play key roles in pathophysiological and clinical pharmacology. A better understanding of the mechanisms of channel function may have profound clinical applications for the future.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
1K08HL002843-01
Application #
3083268
Study Section
Special Emphasis Panel (ZHL1-CCT-L (01))
Project Start
1993-09-01
Project End
1998-08-31
Budget Start
1993-09-01
Budget End
1994-08-31
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199
Jeron, A; Mitchell, G F; Zhou, J et al. (2000) Inducible polymorphic ventricular tachyarrhythmias in a transgenic mouse model with a long Q-T phenotype. Am J Physiol Heart Circ Physiol 278:H1891-8
Baker, L C; London, B; Choi, B R et al. (2000) Enhanced dispersion of repolarization and refractoriness in transgenic mouse hearts promotes reentrant ventricular tachycardia. Circ Res 86:396-407
London, B; Wang, D W; Hill, J A et al. (1998) The transient outward current in mice lacking the potassium channel gene Kv1.4. J Physiol 509 ( Pt 1):171-82
London, B; Trudeau, M C; Newton, K P et al. (1997) Two isoforms of the mouse ether-a-go-go-related gene coassemble to form channels with properties similar to the rapidly activating component of the cardiac delayed rectifier K+ current. Circ Res 81:870-8
Breitbart, R E; London, B; Nguyen, H T et al. (1995) Recent advances in the Laboratory of Molecular and Cellular Cardiology. Ann Thorac Surg 60:S509-12