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.
Highlights
In our first analysis of the transmembrane (TM)1 sequences in the ryanodine receptor isoform 1 (RyR1), we predicted that five TM hairpin loops were present in the C terminus of RyR1, among which M3 was formed by amino acids 4277– 4300 and M4 was formed by amino acids 4342– 4362 [1]
Exploration of Membrane Association through the M4 Region of RyR1—As in a previous study of overall RyR1 topology [8], we initiated analysis of the topology of the M4 region by determining the orientation of enhanced green fluorescent protein (EGFP) fused to the C terminus of truncated RyR1 sequences
Analysis by confocal microscopy showed that soluble proteins such as EGFP and RyR1 truncated at amino acid 4300 could be washed out of HEK-293 or MEF cells if the cells were permeabilized with saponin before fixation
Summary
In our first analysis of the transmembrane (TM)1 sequences in the ryanodine receptor isoform 1 (RyR1), we predicted that five TM hairpin loops were present in the C terminus of RyR1, among which M3 was formed by amino acids 4277– 4300 and M4 was formed by amino acids 4342– 4362 [1]. An RyR2 construct that contained an EGFP insertion between M4a and M4b formed an active channel, and an RyR1 construct in which the M3/M4 region was deleted retained both orthograde and retrograde Ca2ϩ release channel function.
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