We propose to study the ionic channels involved directly or indirectly in the process of excitation-contraction coupling in skeletal muscle. Inaccessibility of the transverse tubule (TT) and sarcoplasmic reticulum (SR) membranes have made it difficult or impossible until now to study the conductance pathways contained in these membranes by conventional electrophysiological techniques. In order to study the ionic channels, we will isolate and purify vesicles coming from surface membranes (SM), TT and SR. The membrane origin of the isolated vesicles will be checked by means of several procedures, such as protein and lipid content and composition, enzymatic activities and toxin-binding studies. The presence and density of ionic channels will be measured using specific toxins (TTX and analogues, scorpion toxins, dihydropyridines). We plan to study the single channel properties of SR and TT by incorporating these membranes into plannar bilayers, or in bilayers made on patch-clamp pipettes and/or liposomes. In particular, these methods will be used to study the selectivity, gating, pharmacological and regulation profile of TT calcium-activated potassium channel and of the calcium channel of SR. The SR calcium channel appears to be a possible candidate for the pathway of calcium release in vivo. The long-term goal of the proposal is the detection, characterization and reconstitution of the different conductance pathways contained in muscle membranes. Supporting data demonstrates the feasibility and possible generality of our approach.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM035981-01
Application #
3289523
Study Section
Physiology Study Section (PHY)
Project Start
1986-05-01
Project End
1989-04-30
Budget Start
1986-05-01
Budget End
1987-04-30
Support Year
1
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Center of Scientific Studies of Santiago
Department
Type
DUNS #
City
Santiago
State
Country
Chile
Zip Code
Naranjo, D; Latorre, R (1993) Ion conduction in substates of the batrachotoxin-modified Na+ channel from toad skeletal muscle. Biophys J 64:1038-50
Caviedes, P; Olivares, E; Salas, K et al. (1993) Calcium fluxes, ion currents and dihydropyridine receptors in a new immortal cell line from rat heart muscle. J Mol Cell Cardiol 25:829-45
Vergara, C; Ramirez, B; Behrens, M I (1993) Colchicine alters apamin receptors, electrical activity, and skeletal muscle relaxation. Muscle Nerve 16:935-40
Toro, L; Stefani, E; Latorre, R (1992) Internal blockade of a Ca(2+)-activated K+ channel by Shaker B inactivating ""ball"" peptide. Neuron 9:237-45
Labarca, P; Latorre, R (1992) Insertion of ion channels into planar lipid bilayers by vesicle fusion. Methods Enzymol 207:447-63
Behrens, M I; Vergara, C (1992) Increase of apamin receptors in skeletal muscle induced by colchicine: possible role in myotonia. Am J Physiol 263:C794-802
Correa, A M; Bezanilla, F; Latorre, R (1992) Gating kinetics of batrachotoxin-modified Na+ channels in the squid giant axon. Voltage and temperature effects. Biophys J 61:1332-52
Caviedes, R; Liberona, J L; Hidalgo, J et al. (1992) A human skeletal muscle cell line obtained from an adult donor. Biochim Biophys Acta 1134:247-55
Delgado, R; Latorre, R; Labarca, P (1992) K(+)-channel blockers restore synaptic plasticity in the neuromuscular junction of dunce, a Drosophila learning and memory mutant. Proc Biol Sci 250:181-5
Correa, A M; Latorre, R; Bezanilla, F (1991) Ion permeation in normal and batrachotoxin-modified Na+ channels in the squid giant axon. J Gen Physiol 97:605-25

Showing the most recent 10 out of 33 publications