Homer protein increases activation of Ca2+ sparks in permeabilized skeletal muscle.
Members of the Homer family of proteins are known to form multimeric complexes capable of cross-linking plasma membrane channels (e.g. metabotropic glutamate receptor) and intracellular Ca2+ release channels (e.g. inositol trisphosphate receptor) in neurons, which potentiates Ca2+ release. Recent work has demonstrated direct interaction of Homer proteins with type 1 and type 2 ryanodine receptor (RyR) isoforms. Moreover, Homer proteins have been shown to modulate RyR-dependent Ca2+ release in isolated channels as well as in whole cell preparations. We now show that long and short forms of Homer H1 (H1c and H1-EVH1) are potent activators of Ca2+ release via RyR in skeletal muscle fibers (e.g. Ca2+ sparks) and potent modulators of ryanodine binding to membranes enriched with RyR, with H1c being significantly more potent than H1-EVH1. Homer did not significantly alter the spatio-temporal properties of the sparks, demonstrating that Homer increases the rate of opening of RyRs, with no change in the overall RyR channel open time and amount of Ca2+ released during a spark. No changes in Ca2+ spark frequency or properties were observed using a full-length H1c with mutation in the EVH1 binding domain (H1c-G89N). One novel finding with each Homer agonist (H1c and H1-EVH1) was that in combination their actions on [3H]ryanodine binding was additive, an effect also observed for these Homer agonists in the Ca2+ spark studies. Finally, in Ca2+ spark studies, excess H1c-G89N prevented the effects of H1c in a dominant negative manner. Taken together our results suggest that the EVH1 domain is critical for the agonist behavior on Ca2+ sparks and ryanodine binding, and that the coiled-coil domain, present in long but not short form Homer, confers an increase in agonist potential apparently through the multimeric association of Homer ligand.
Ward
Christopher W
CW
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Feng
Wei
W
Tu
Jiangchen
J
Pessah
Isaac N
IN
Worley
Paul K
PK
Schneider
Martin F
MF
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005799:Genes, Dominant; D005822:Genetic Vectors; D000072233:Homer Scaffolding Proteins; D008024:Ligands; D018482:Muscle, Skeletal / Q000378:metabolism*; D009154:Mutation; D009479:Neuropeptides / Q000378:metabolism / Q000502:physiology*; D016366:Open Reading Frames; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011894:Rana pipiens; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013997:Time Factors
D002352:Carrier Proteins; D000072233:Homer Scaffolding Proteins; D008024:Ligands; D009479:Neuropeptides; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002118:Calcium