Bi-directional coupling of sarcolemmal and SR Ca2+ channels in skeletal muscle: Structural and functional dissection of the interaction

Research project P 13831 Bi-directional coupling in skeletal muscle Manfred GRABNER 28.06.1999 Coordinated muscle contractions require the controlled release of Ca2+ from intracellular Ca2+ stores. In skeletal muscle, intracellular Ca2+ release is governed by a tightly coupled, likely allosteric interaction between two distinct Ca2+ channels, the voltage-gated L-type Ca2+ channel or dihydropyridine receptor (skDHPR) of the sarcolemma and the Ca2+ release channel or ryanodine receptor (RyR-1) of the sarcoplasmic reticulum (SR). Depolarization-induced conformational changes of the skDBPR are communicated to the RyR-1, thereby inducing C2a+ release from SR stores. In addition to this orthograde signaling or excitationcontraction (EC) coupling, RyR- 1 generates a retrograde signal that enhances the Ca2+ channel activity of the DBPR. We have recently investigated that one common sequence motif in the intracellular II-III loop of skDHPR seems to be essential for this interchannel crosstalk. Strong evidences point to a protein-protein interaction, but the molecular mechanisms of "bi-directional coupling" in their entirety are far from to be fully understood. The first aim of the proposed project will be the fine-mapping of the so far identified 46 residues interaction motif on the skDHPR`s II-III loop to find the minimal motif(s) using the ancestral and therefore very heterologous H-111 loop from a Musca domestica L-type Ca2+ channel as the background sequence. Chimeras will be expressed in the dysgenic (lack of functional skDHPRs) skeletal muscle cell line GLT and analyzed for coupling by patch clamp (RyR->DBPR) and by microfluorimetric; C2+ measurements (DBPR->RyR). Especially in the fight of the recent investigation that a point mutation on the skDHPR`s intracellular III-IV loop can produce malignant hyperthermia (MH; an anomalous high release of Ca2+ through RyR-1, triggered by volatile anesthetics), cytoplasmic regions additional to the II-III loop will be studied for their putative contribution to the DBPR<->RyR interaction by chimerization and alanine scanning techniques. Special attention will be paid to the biophysical characterization of MH mutant R1086H and to the role of this mutation for bi-directional coupling. After the interaction motif(s) is/are mapped to the minimal sequence we will test if peptides corresponding to thishese motif(s) can compete for binding to the interaction motifs on RyR-1 and therefore decrease bi-directional coupling. Block of interaction would be an additional, strong evidence for the assumed DHPR<->RyR protein- protein interaction. To elucidate if bi-directional coupling is a direct or an indirect (intermediary protein-mediated) allosteric interaction, peptides corresponding to the minimal interaction motif(s) should be used as "bait" against an artificial random peptide library in a phage display library screen. The observation that a skeletal 11-HI loop enables an otherwise cardiac DHPR to ortho- and retrograde interaction suggests a targeting mechanism of the skeletal 11-Ul loop towards a skeletal-type (physical) contact to RyR-1, thereby enforcing 4 skDHPRs to arranged in a quatrefoil formation (tetrads). Chimeras generated during the course of the mapping analyses should be studied by freeze-fracture electron microscopy (via an international cooperation with C. Franzini-Armstrong) to address the question if skeletal-type DHPR<->RyR interaction is correlated with the association of the DHPRs to tetrads and if this tetrad formation is determined by the same motif(s) of the skeletal 11-111 loop responsible for bi-directional coupling.

The central aim of this project was the molecular dissection of the interaction of two Ca2+ channels that represent the major structures of initiating muscle contraction by using methods of molecular biology, electrophysiology, immunocytochemistry and freeze-fracture electron microscopy. Coordinated muscle contractions require the controlled release of Ca2+ ions from intracellular Ca2+ stores. In skeletal muscle, intracellular Ca2+ release is governed by a protein-protein interaction between two distinct Ca2+ channels, the voltage-gated L-type Ca2+ channel or dihydropyridine receptor (DHPR) of the plasma surface membrane and the Ca2+ release channel or ryanodine receptor (RyR1) of the sarcoplasmic reticulum (SR). Depolarization-induced conformational changes of the DHPR are communicated to the RyR1, thereby inducing Ca2+ release from SR stores, which in turn activates the proteins of sarcomere, ultimately resulting in contraction of the muscle fiber. In addition to this orthograde signaling or excitation-contraction (EC) coupling, RyR1 generates a retrograde signal that enhances the Ca2+ channel activity of the DHPR. This interchannel crosstalk, termed bidirectional coupling, is mediated by an intracellular domain of the DHPR, the intracellular II-III loop. In our work we succeeded to establish structure-function relationships of the two most essential requirements for this bidirectional DHPR<=> RyR interplay. These requirements are, i) the tight colocalization of both channels as a prerequisite of a physical interaction, and ii) a specific molecular domain in the II-III loop enabling the DHPR to allosterically interact with the RyR1. Ad i) The chimeric approach allowed us to identify a short sequence of 55 residues in the C-terminus of the DHPR to be responsible for targeting the DHPR into close proximity of the RyR in the triad junctions of the muscle cell. In addition, we learned from freeze-fracture electron microscopy that the skeletal DHPR II-III loop is a major determinant for the subsequent fine localization of 4 DHPRs into a quatrefoil arrangement (Tetrad) exactly opposite of the homotetrameric RyR1. Ad ii) Using chimeras and point mutants we were able to pin down four skeletal muscle specific residues within a 15 residue minimal skeletal sequence of the DHPR II-III loop to be essential for skeletal-type EC coupling. Our results point to a striking structure-function correlation between EC coupling properties of the individual chimeras or point mutants and the predicted secondary structure (Cardiac/ a-helical and skeletal/random coil) of the II-III loop interaction domain. Thus our results provide evidence that the secondary structure of the DHPR-RyR1 interaction domain in the DHPR II-III loop might determine the tissue specific mode of EC coupling. Accomplishing a better understanding of the mechanisms of skeletal muscle EC coupling will contribute to overcome one day severe muscle channelopathies linked to the DHPR-RyR interaction like malignant hyperthermia (MH) or central core disease (CCD).