Journal of Biological Chemistry
Volume 279, Issue 36, 3 September 2004, Pages 37566-37574
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Membrane Transport, Structure, Function, and Biogenesis
Role of the Sequence Surrounding Predicted Transmembrane Helix M4 in Membrane Association and Function of the Ca2+ Release Channel of Skeletal Muscle Sarcoplasmic Reticulum (Ryanodine Receptor Isoform 1)*

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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 Ca2+ 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, Δ4274–4535, deleted in the sequence surrounding both M3 and M4, restored robust, voltage-gated L-type Ca2+ currents and Ca2+ transients in dyspedic myotubes, demonstrating that this sequence is not required for either orthograde (DHPR activation of sarcoplasmic reticulum Ca2+ release) or retrograde (RyR1 increase in DHPR Ca2+ channel activity) signals of excitation-contraction coupling. Maximal amplitudes of L-currents and Ca2+ transients with Δ4274–4535 were larger than with wild-type RyR1, and voltage-gated sarcoplasmic reticulum Ca2+ 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 Ca2+ release channel opening.

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*

This work was supported by Canadian Institutes of Health Research Grant MT-3399 (to D. H. M.) and National Institutes of Health Grant AR44657 (to R. T. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.