title: Role of the sequence surrounding predicted transmembrane helix M4 in membrane association and function of the Ca(2+) release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor isoform 1).
abstract: The role of the sequence surrounding M4 in ryanodine receptors (RyR) in membrane association and function was investigated. This sequence contains a basic, 19-amino acid M3/M4 loop, a hydrophobic 44-49 amino acid sequence designated M4 (or M4a/M4b), and a hydrophilic M4/M5 loop. Enhanced green fluorescent protein (EGFP) was inserted into RyR1 and truncated just after the basic sequence, just after M4, within the M4/M5 loop, just before M5 and just after M5. The A52 epitope was inserted into RyR2 and truncated just after M4a. Analysis of these constructs ruled out a M3/M4 transmembrane hairpin and narrowed the region of membrane association to M4a/M4b. EGFP inserted between M4a and M4b in full-length RyR2 was altered conformationally, losing fluorescence and gaining trypsin sensitivity. Although it was accessible to an antibody from the cytosolic side, tryptic fragments were membrane-bound. The expressed protein containing EGFP retained caffeine-induced Ca(2+) release channel function. These results suggest that M4a/M4b either forms a transmembrane hairpin or associates in an unorthodox fashion with the cytosolic leaflet of the membrane, possibly involving the basic M3/M4 loop. The expression of a mutant RyR1, Delta4274-4535, deleted in the sequence surrounding both M3 and M4, restored robust, voltage-gated L-type Ca(2+) currents and Ca(2+) transients in dyspedic myotubes, demonstrating that this sequence is not required for either orthograde (DHPR activation of sarcoplasmic reticulum Ca(2+) release) or retrograde (RyR1 increase in DHPR Ca(2+) channel activity) signals of excitation-contraction coupling. Maximal amplitudes of L-currents and Ca(2+) transients with Delta4274-4535 were larger than with wild-type RyR1, and voltage-gated sarcoplasmic reticulum Ca(2+) release was more sensitive to activation by sarcolemmal voltage sensors. Thus, this region may act as a negative regulatory module that increases the energy barrier for Ca(2+) release channel opening.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
authors.forename: Guo Guang
authors.initials: GG
authors.lastname: Du
authors.forename: Guillermo
authors.initials: G
authors.lastname: Avila
authors.forename: Parveen
authors.initials: P
authors.lastname: Sharma
authors.forename: Vijay K
authors.initials: VK
authors.lastname: Khanna
authors.forename: Robert T
authors.initials: RT
authors.lastname: Dirksen
authors.forename: David H
authors.initials: DH
authors.lastname: MacLennan
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D017931:DNA Primers; D019837:Ryanodine Receptor Calcium Release Channel