The transduction of action potential to muscle contraction (EC coupling) is an example of fast communication between cell membrane events and metabolic state. As in many systems, the central messenger of this coupling is calcium, which in muscle is released from the Sarcoplasmic Reticulum (SR) to activate contractile proteins. The release channels of the SR are controlled by changes in potential at the plasma and T-tubular membrane. The long term goal of this project is to understand this control. Since the last renewal of this grant, the identity of two key molecular players, the voltage sensor of the T membrane and the release channel of the SR, became known. The central problem of EC coupling -and of this proposal- can now be formulated as understanding the interaction between the dihydropyridine receptor or DHPr molecule (voltage sensor) and the ryanodine receptor or R Yr (release channel). A reductionist approach to this question will be used, consisting of studies of the complete system and its parts. Measurements in the complete system will include intramembrane charge movement (a manifestation of the voltage sensor) and Ca release flux in single skeletal muscle cells. A new voltage clamp technique will improve previous measurements. Understanding forward transmission will require the elucidation of a newly found backward transmission: Ca release feeds back on the voltage sensor. Charge movement, Ca release, and mechanisms of transmission will be unified in a comprehensive model of EC coupling. In this framework it will be possible to test quantitatively two possible mechanisms of forward transmission: mechanical, by direct or indirect contact between DHPr and RYr, or chemical, via a diffusible transmitter. To study the parts of the system separately, two preparations will be used: single channel recording with DHPrs and RYrs in bilayers and measurement of Ca channel gating currents in heart cells. A comparison of the properties of the system and its parts will identify properties that depend on the interaction, and help decide whether the transmission is mechanical. Promising preliminary work, in which an interaction was demonstrated in bilayers by putting the components together, will be continued and extended. The molecular mechanisms in E-C coupling are fundamentally similar to other phenomena of membrane biophysics, and the key molecular players are close isoforms of channel proteins found elsewhere. Thus, in addition to the obligatory relevance that the present studies should have to the understanding of muscle dysfunction and therapeutics, findings in Ec coupling should have a more general impact, on research of CNS problems, heart and smooth muscle.
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