The release of Ca from the sarcoplasmic reticulum in heart is activated by Ca. This has the potential to be an inherently self-regenerating process, yet it is well controlled and graded relative to the level of activating Ca. It has been proposed by the investigator that single RyR channel adaptation may serve as a negative feedback mechanism that counters or stabilizes the intrinsic positive feedback of the Ca-induced Ca release mechanism. This project will utilize single channel approaches to address two Aims. The first will test the hypothesis that adaptation of single RyR channels does serve to stabilize the CICR process in heart. Rapid changes in free Ca generated by laser flash photolysis of caged compounds will be utilized to define the activation and deactivation kinetics of cardiac RyR channels reconstituted into planar bilayers. These data will be used to differentiate deactivation from slower regulatory mechanisms, such as adaptation and/or inactivation. The properties of these slower mechanisms will be analyzed and used to test several existing models of RyR adaptation.
The second Aim will test the hypothesis that brief trigger Ca signals less than 1 msec in duration are adequate to activate the RyR channel and that activation of a single RyR channel is sufficient to produce a Ca spark. Photolysis of caged Ca will be used to generate brief trigger Ca stimuli in the vicinity of single cardiac RyR channels reconstituted in bilayers to define the kinetic limits of an effective trigger Ca signal. To define the basis of the Ca spark, Ca translocation through RyR channels in bilayers will be imaged using a confocal microscope. How single RyR channel gating governs the spatial and temporal features of the spark will be defined. Based on these data, a model that deconvolves Ca spark-like fluorescence signals and accurately predicts the underlying channel gating behavior will be developed and tested in cells. In a final series of studies, the investigator will attempt to define the properties of an effective trigger Ca signal.
Showing the most recent 10 out of 44 publications
