Testing for Direct Interactions Between the DHPR and the RyR1 Cytoplasmic Foot

Abstract

In skeletal muscle, RyR1 (5,037 residues) releases calcium from the sarcoplasmic reticulum (SR) in response to an orthograde signal from the DHPR in the plasma membrane (PM), and transmits a retrograde signal which increases the L-type calcium current via the DHPR (which contains CaV1.1 as its principal subunit). Previously, we tested the behavior of a RyR1 construct (YFP-RyR11:4300), which encodes the cytoplasmic domain of RyR1 (foot) but lacks the C-terminal domains which form the ion pore for SR calcium release and which anchor RyR1 in the SR membrane. We found that YFP-RyR11:4300 targets to PM-SR junctions in dyspedic myotubes (null for endogenous RyR1) and transmits the retrograde signal to the DHPRs present in those myotubes. We have now begun to examine whether these interactions between the RyR1 foot and DHPR can occur in the absence of other proteins which are present in PM-SR junctions of muscle cells. Toward this end, we used tsA201 cells to co-express CFP-RyR11:4300, YFP-CaV1.1 (or CaV1.1/CaV1.2 chimeras) and the DHPR auxiliary subunit β1a. Most of the CaV constructs appeared to be retained in a peri-nuculear compartment (CaV1.1, and chimeras in which three CaV1.1 repeats were replaced by the corresponding CaV1.2 repeats). PM-targeting was observed for a construct composed of CaV1.2 with the cytoplasmic II-III loop replaced by that of CaV1.1. However, we have not yet observed co-localization of CFP-RyR11:4300 with any of the CaV constructs. We are currently testing whether the presence of additional, junctional proteins will result in co-localization of RyR11:4300 and CaV. Supported by grants NIH AR055104 and MDA 277475 to K.G.B.