This research will be done primarily in Chile at the Universidad de Chile, Santiago, in collaboration with Dr. Enrique Jaimovich as an extension of grant PO1AR44750-05. Our hypothesis is that the conformational changes of the DHPR during depolarisation activate a signaling pathway that in turn activates PLC. The present research is directed to establish how the DHPR of skeletal muscle cells is involved in the generation of the slow calcium signal in skeletal muscle, with a particular emphasis in establishing the domain or segment of the alpha1s-DHPR protein that initiates PLC activation. The DHPR is considered to be the sensor of the membrane potential in the T tubule during skeletal muscle E-C coupling, regulating the opening of the RyR. In this approach we are looking for an alterative function of the DHPR, in addition to its role as the voltage sensor, activating other signalling pathways related to adaptive responses of the cell. In this line of thinking, the PLC dependent-slow calcium signal, described by us previously is probably the """"""""top of the iceberg"""""""" in a complex web of signalling pathways induced by membrane depolarisation that modulate cell function. The determination of segments or domains of the DHPR involved in PLC activation may be the key needed to identify physiological partners of the DHPR upstream of PLC activation.
Our Specific Aims are:
Aim 1. Determine the signaling pathways by which depolarization of the DHPR activates PLC and subsequent slow calcium signal development Aim 2. To determine whether or not IP 3 generation and the slow calcium signal, by L-type calcium channels is isoform specific.
Aim 3. To identify the region in the alpha1s-DHPR subunit that drives PLC activation. Using this approach we expect to discover the regions of the alpha1s segment that are involved in PLC activation on either a """"""""gain of function"""""""" or if necessary a """"""""loss of function"""""""" experiment. The relationships between this and the parent proposal are: 1. The different research and methodological scope of both laboratories results in a synergistic interaction in the study of the physiological role of the DHPR as voltage sensor and 2. The data gained will conceptually increase our knowledge of signal transduction that results from depolarisation stimuli in muscle cells.
