Intracellular calcium controls a variety of cellular functions such as contraction, secretion, proliferation, and gene expression. One of the major pathways of calcium influx into cardiac myocytes is through T- and L-type voltage-gated Ca2+ channels. T-type, or low voltage-activated (LVA) channels, open after small depolarizations of the membrane. T-type currents have been found in atrial myocytes and pacemaker cells, where they are involved in determining pacemaker activity. Numerous drugs block L-type channels, however, there are no selective blockers of T-type channels. One of the long-term objectives of this research is to provide an assay system that will allow the development of T-type channel blockers. Calcium channels are multisubunit complexes composed of a large, ion-conducting subunit, alpha1, and several accessory subunits(alpha 2delta, beta and gamma). Six genes encoding alpha1 subunits have been cloned. Cloning and expression of channels has allowed their electrophysiological and pharmacological classification. These studies established that all of these alpha1s are high voltage-activated channels.
The aim of this project is to clone the cardiac low voltage-activated T- type Ca2+ channel. Exciting preliminary studies have identified a novel Ca2+ channel gene expressed in heart. The hypothesis is that this gene encodes a T-type channel.
The specific aims of this project are to: 1) clone the full-length cDNA, 2) suppress the expression of native T-type channels in rat neonatal ventricular myocytes, 3) determine the tissue specific expression of this gene (Northerns and in situ hybridizations, 4) express the cDNA in heterologous expression systems and measure the biophysical properties of the channel (single channel conductance, voltage-dependence), and 5) determine the pharmacological properties of the expressed current. The research design uses recombinant DNA techniques to clone T-type channels from human heart tissue and express the cloned channels in both Xenopus laevis oocytes and HEK-293 cells. Electrophysiological methods are used to study the expressed channel at both the single channel and whole cell level. The hypothesis will then be tested by comparing the properties of the cloned channel to that observed from T-type channels in isolated cells.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL058728-03
Application #
6030843
Study Section
Physiology Study Section (PHY)
Project Start
1997-07-01
Project End
1999-08-31
Budget Start
1999-07-01
Budget End
1999-08-31
Support Year
3
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Loyola University Chicago
Department
Physiology
Type
Schools of Medicine
DUNS #
791277940
City
Maywood
State
IL
Country
United States
Zip Code
60153
Perez-Reyes, E (2006) Molecular characterization of T-type calcium channels. Cell Calcium 40:89-96
Perez-Reyes, Edward (2003) Molecular physiology of low-voltage-activated t-type calcium channels. Physiol Rev 83:117-61
Martin, R L; Lee, J H; Cribbs, L L et al. (2000) Mibefradil block of cloned T-type calcium channels. J Pharmacol Exp Ther 295:302-8
Cribbs, L L; Gomora, J C; Daud, A N et al. (2000) Molecular cloning and functional expression of Ca(v)3.1c, a T-type calcium channel from human brain. FEBS Lett 466:54-8
Todorovic, S M; Perez-Reyes, E; Lingle, C J (2000) Anticonvulsants but not general anesthetics have differential blocking effects on different T-type current variants. Mol Pharmacol 58:98-108
Serrano, J R; Perez-Reyes, E; Jones, S W (1999) State-dependent inactivation of the alpha1G T-type calcium channel. J Gen Physiol 114:185-201
Klockner, U; Lee, J H; Cribbs, L L et al. (1999) Comparison of the Ca2 + currents induced by expression of three cloned alpha1 subunits, alpha1G, alpha1H and alpha1I, of low-voltage-activated T-type Ca2 + channels. Eur J Neurosci 11:4171-8
Lee, J H; Gomora, J C; Cribbs, L L et al. (1999) Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H. Biophys J 77:3034-42