HOW MANY AND WHERE ARE LOCATED THE MEMBRANE SPANNING SEGMENTS OF THE RYANODINE RECEPTOR/CALCIUM RELEASE CHANNEL?

Pierre Pouliquin^1,3, Susan Treves², Luca Moccagatta¹, Francesco Zorzato^1,2, 1 Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy, 2 Department of Anaesthesia and Research, Kantonsspital Basel, Basel, Switzerland, 3 Muscle Research Group, John Curtin School of Medical Research, Australian National University, Canberra ACT, Australia.

The calcium channels ryanodine receptor (RyR) and dihydropyridine receptor constitute the core of a supramacromolecular complex which performs the coupling between excitation and contraction in cardiac and skeletal muscles. Although functional characteristics of the RyR have been intensely investigated, there remain major gaps in our knowledge about the structure of the protein. Of particular importance are the transmembrane segments, which form the membrane-spanning domain of the protein, the pore, and dictate the cytoplasmic and luminal domains and the overall protein structure. Hydropathy profiles predict between 4 and 12 transmembrane segments; however there is remarkably little experimental evidence to support the various models.

We made EGFP fusion proteins covering the entire coding sequence of the rabbit skeletal muscle ryanodine receptor. Fusion proteins were expressed in mammalian cells, and microsomal fractions from transfected cells were isolated by differential centrifugation. Integral membrane fusion proteins were identified by Na₂CO₃ extration of microsomal membranes. Integral membrane proteins and soluble proteins were transferred onto nitrocellulose and stained with an anti-EGFP antibody. Our results indicate that 16 short fusion proteins from the rabbit skeletal muscle RyR formed integral membrane segments. To determine which of those integral membrane segments span the membrane, we developed a “sandwich approach”. New fusion proteins were made where all integral membrane segments carried two different tags at each end (EGFP and Flag, respectively). Microsomal fractions prepared from mammalian cells expressing the “sandwich” fusion proteins where exposed to trypsin. Crude and trypsin digested fusion proteins were transferred to nitrocellulose and stained either with an anti-EGFP antibody, or with an anti-Flag antibody. Our results indicate that the receptor could contain two membrane spanning domains, one located in the middle portion of the molecule, the other located at the COOH-terminal part of the molecule.