Transmembrane Ca channels have an important dual function: they generate electrical signals which are used in many cells for conduction of impulses and for the control of rhythmical electrical activity, but Ca channels also deliver a specific chemical messenger to the cell in the form of Ca ions, whose message is decoded by intracellular Ca binding effector proteins. Ca channels therefore play a fundamental role in the regulation of key cellular processes in most excitable cells. In heart cells, Ca channels initiate contraction, help determine heart rate by contributing to the pacemaker depolarization, promote slow conduction in nodal regions, and support the action potential plateau, thus determining the duration of electrical excitability. In partially depolarized regions of the heart, Ca channels can initiate slowly conducted action potentials and promote arrhythmic activity. The L-type Ca channel is the pharmacological receptor for some of the most widely used drugs (Ca channel blockers) in the management of coronary heart disease, hypertension and cardiac arrhythmias. This grant application proposes to investigate the elementary functional properties of Ca channels. Patch clamp recordings will be used to study the openings and closings of single Ca channel molecules in intact cardiac cells and sympathetic neurons. The kinetics of individual openings and closings, and of transitions between longer lasting gating modes will be analyzed quantitatively in order to better understand the mechanisms and stimuli which govern the activity of Ca channels in cells. The amplitude of the unitary current flowing through single open Ca channels under various experimental conditions will be measured to learn more about the process of ion permeation and selectivity, a process to which the channels owe their capability of passing Ca ions at a high rate and yet to remain selectively permeant to Ca ions even in the presence of high concentrations of competing ions. Separate experiments on a K-channel for which we have the cloned cDNA will attempt to correlate functional domains with structurally and functionally conserved domains of voltage gated ion channels relevant for both K- and Ca channels. An antigenic epitope will be introduced into various domains of the channel sequence by in-vitro mutagenesis in an effort to locate key segments of the primary structure with respect to the plasma membrane. Mutations of positive and negative charges in the presumed membrane spanning regions of the channel will be used to establish the relative location and functional interactions between residues conserved in all voltage dependent ion channels. From the proposed combination of functional studies at the single channel level and manipulation of the channel structure by site-directed mutagenesis we hope to gain new insight into the molecular mechanisms which determine the function of these important regulators of cellular excitability.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL037124-07
Application #
3352678
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Project Start
1986-07-01
Project End
1993-06-30
Budget Start
1992-07-02
Budget End
1993-06-30
Support Year
7
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Nakazawa, K (1994) Modulation of the inhibitory action of ATP on acetylcholine-activated current by protein phosphorylation in rat sympathetic neurons. Pflugers Arch 427:129-35
Nakazawa, K (1994) ATP-activated current and its interaction with acetylcholine-activated current in rat sympathetic neurons. J Neurosci 14:740-50
Kuo, C C; Hess, P (1993) Ion permeation through the L-type Ca2+ channel in rat phaeochromocytoma cells: two sets of ion binding sites in the pore. J Physiol 466:629-55
Kuo, C C; Hess, P (1993) Characterization of the high-affinity Ca2+ binding sites in the L-type Ca2+ channel pore in rat phaeochromocytoma cells. J Physiol 466:657-82
Kuo, C C; Hess, P (1993) Block of the L-type Ca2+ channel pore by external and internal Mg2+ in rat phaeochromocytoma cells. J Physiol 466:683-706
Nakazawa, K; Inoue, K (1993) ATP- and acetylcholine-activated channels co-existing in cell-free membrane patches from rat sympathetic neuron. Neurosci Lett 163:97-100
Kuo, C C; Hess, P (1992) A functional view of the entrances of L-type Ca2+ channels: estimates of the size and surface potential at the pore mouths. Neuron 9:515-26
Pietrobon, D; Prod'hom, B; Hess, P (1989) Interactions of protons with single open L-type calcium channels. pH dependence of proton-induced current fluctuations with Cs+, K+, and Na+ as permeant ions. J Gen Physiol 94:1-21
Prod'hom, B; Pietrobon, D; Hess, P (1989) Interactions of protons with single open L-type calcium channels. Location of protonation site and dependence of proton-induced current fluctuations on concentration and species of permeant ion. J Gen Physiol 94:23-42
Hess, P; Prod'Hom, B; Pietrobon, D (1989) Mechanisms of interaction of permeant ions and protons with dihydropyridine-sensitive calcium channels. Ann N Y Acad Sci 560:80-93

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