Abstract
The potential role in Ca2+ release channel function of highly conserved, polar, and small amino acids in predicted transmembrane sequences in the rabbit skeletal muscle ryanodine receptor (RyR1) was investigated through mutagenesis. Acidic amino acids Asp3987, Glu4032, Asp4815, Asp4917, Asp4938, and Asp4969 and amidated residues Asn4034, Asn4037, Asn4574, Asn4805, Asn4806, and Gln4933, and Gly4033 were mutated to Ala, and Ala3988 was mutated to Val. When expressed in HEK-293 cells and challenged with either caffeine or 4-chloro-m-cresol, mutants E4032A, N4806A, D4815A, and D4917A did not respond, indicating that Ca2+ release channel function was impaired. None of these mutants exhibited specific binding of [3H]ryanodine. Mutants N4805A and Q4933A showed a diminished response to both caffeine and 4-chloro-m-cresol, but [3H]ryanodine binding was not altered. Other mutant responses and the responses of mutants E4032D, N4806Q or D, D4815N or E, and D4938N or E were unaltered when compared with RyR1. However, mutants E4032Q, D4917N or E, and Q4933N or E displayed neither caffeine nor 4-chloro-m-cresol response nor [3H]ryanodine binding. Sedimentation assays indicated that the nonfunctional mutants did contain tetrameric complexes, implying that defects in the assembly of a functional channel did not occur with specific mutations in transmembrane sequences. These results support the view that amino acids Glu4032 (M2), Asn4806 (M7), Asp4815 (M7), Asp4917 (M10), and Gln4933 (M10) are involved in channel function and regulation.
Highlights
The number of transmembrane sequences in the COOHterminal region is still undefined
Glu4032, Asp4815, and Asp4917 are absolutely conserved (Fig. 1), suggesting that these amino acids might play an important role in structure/function relationships in Ca2ϩ release channels
We measured [3H]ryanodine binding by wild type and mutant RyR1 proteins, and sedimentation was used to assess the oligomeric status of the expressed receptors
Summary
Materials—Pfu polymerase, restriction endonucleases, and other DNA-modifying enzymes were purchased from Stratagene, Boehringer Mannheim, New England Biolabs, Promega, and Amersham Pharmacia Biotech. Fluorescence Measurements—A microfluorimetry system (Photon Technologies Inc.) was used to monitor the fluorescence changes before and after addition of caffeine or 4-chloro-m-cresol to the surface of HEK-293 cells that had been transfected with wild type or mutant RyR1 cDNA, as described previously [25]. The resulting supernatant from cells or microsomes was placed on the top of a 7.5–25% (w/v) linear sucrose gradient solution containing 50 mM Tris-Hepes, pH 7.4, 0.3 M NaCl, 0.1 mM CaCl2, 0.3% CHAPS, 0.15% L-phosphatidylcholine, and protease inhibitor mix and was centrifuged at 28,000 rpm in a Beckman SW 40 rotor for 18 –20 h at 4 °C. Enzyme-linked Immunoabsorbent Assay—Immunoreactivity in the sucrose density gradient fractions was determined by direct enzymelinked immunoabsorbent assay using monoclonal antibody 34C [24], as described previously [31]. Protein Assay—Protein concentration was determined by dye binding using bovine serum albumin as a standard [32]
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