Depolarization of a skeletal muscle fiber causes the release of Ca2+ from the SR. The release of Ca2+ occurs via ryanodine receptor Ca2+ channels located in the SR, gated by voltage sensor in adjacent t-tubular membranes. The mechanism by which the voltage sensors control the activation of ryanodine receptors remains a central question.
The aim of the present proposal is to study the activation of individual ryanodine receptor channels at the level of single triads in voltage-clamped frog skeletal muscle fibers with intact control of SR Ca2+ release by voltage sensors. The proposed experiments will be carried out using confocal line-scan imaging of the fluorescence of Ca2+-sensitive indicator dyes inside the fiber. This approach has been used by the investigator, as well as by another lab to measure the Ca2+ arising from single SR Ca2+ release channels or from small clusters of interacting channels. The proposed experiments have five Specific Aims.
In Aim 1, the investigator will study the average amplitude and the spatio-temporal waveforms of local Ca2+ release events in single, voltage-clamped frog skeletal muscle fibers and relate these characteristics to the gating properties of the SR Ca2+ release channels.
The second aim addresses the voltage dependence of local Ca2+ release events. Ca2+ release events will be recorded after small depolarizations and under conditions that affect DHPR inactivation so that large depolarizations will produce relative few individual Ca2+ release events. These studies will also investigate the frequency of occurrence and the properties of Ca2+ release events that occur spontaneously under conditions that should inactivate the voltage sensors.
The third aim will investigate the effects of agents on both the voltage-activated and the spontaneous Ca2+ release events. These will include, caffeine, procaine, cADPR, diminished Mg2+ levels, and the ability of Ca2+ to induce further Ca2+ release (CICR).
Aim 4 will investigate if single or unitary Ca2+ release events and multiple amplitude release events are related in a causal way or if they are independent.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR044197-02
Application #
2442857
Study Section
Physiology Study Section (PHY)
Project Start
1996-07-20
Project End
2001-06-30
Budget Start
1997-07-01
Budget End
1998-06-30
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Maryland Baltimore
Department
Biochemistry
Type
Schools of Medicine
DUNS #
003255213
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Lee, Dong I; Klein, Michael G; Zhu, Weizhong et al. (2009) Activation of (Na+ + K+)-ATPase modulates cardiac L-type Ca2+ channel function. Mol Pharmacol 75:774-81
McKinney, Leslie C; Butler, Thomas; Mullen, Shawn P et al. (2006) Characterization of ryanodine receptor-mediated calcium release in human B cells: relevance to diagnostic testing for malignant hyperthermia. Anesthesiology 104:1191-201
Lacampagne, A; Klein, M G; Ward, C W et al. (2000) Two mechanisms for termination of individual Ca2+ sparks in skeletal muscle. Proc Natl Acad Sci U S A 97:7823-8
Lacampagne, A; Ward, C W; Klein, M G et al. (1999) Time course of individual Ca2+ sparks in frog skeletal muscle recorded at high time resolution. J Gen Physiol 113:187-98
Klein, M G; Lacampagne, A; Schneider, M F (1999) A repetitive mode of activation of discrete Ca2+ release events (Ca2+ sparks) in frog skeletal muscle fibres. J Physiol 515 ( Pt 2):391-411
Lacampagne, A; Klein, M G; Schneider, M F (1998) Modulation of the frequency of spontaneous sarcoplasmic reticulum Ca2+ release events (Ca2+ sparks) by myoplasmic [Mg2+] in frog skeletal muscle. J Gen Physiol 111:207-24
Klein, M G; Lacampagne, A; Schneider, M F (1997) Voltage dependence of the pattern and frequency of discrete Ca2+ release events after brief repriming in frog skeletal muscle. Proc Natl Acad Sci U S A 94:11061-6
Lacampagne, A; Lederer, W J; Schneider, M F et al. (1996) Repriming and activation alter the frequency of stereotyped discrete Ca2+ release events in frog skeletal muscle. J Physiol 497 ( Pt 3):581-8