Abstract
A highly conserved amino acid sequence, GVRAGGGIGD(4831), which may form part of the Ca(2+) release channel pore in RyR2, was subjected to Ala scanning or Ala to Val mutagenesis; function was then measured by expression in HEK-293 cells, followed by Ca(2+) photometry, high affinity [(3)H]ryanodine binding, and single-channel recording. All mutants except I4829A and I4829T (corresponding to the I4897T central core disease mutant in RyR1) displayed caffeine-induced Ca(2+) release in HEK-293 cells; only mutants G4826A, I4829V, and G4830A retained high affinity [(3)H]ryanodine binding; and single-channel function was found for all mutants tested, except for G4822A and A4825V. EC(50) values for caffeine-induced Ca(2+) release were increased for G4822A, R4824A, G4826A, G4828A, and D4831A; decreased for V4823A; and unchanged for A4825V, G4827A, I4829V, and G4830A. Ryanodine (10 microm), which did not stimulate Ca(2+) release in wild type (wt), did so in Ala mutants in amino acids 4823-4827. It inhibited the caffeine response in wt and most mutants, but enhanced the amplitude of caffeine-induced Ca(2+) release in mutant G4828A. It also restored caffeine-induced Ca(2+) release in mutants I4829A and I4829T. In single-channel recordings, mutants I4829V and G4830A retained normal conductance, whereas all others had decreased unitary channel conductances ranging from 27 to 540 picosiemens. Single-channel modulation was retained in G4826A, I4829V, and G4830A, but was lost in other mutants. In contrast to wt and G4826A, I4829V, and G4830A, in which divalent metals were preferentially conducted, mutants with loss of modulation had no selectivity of divalent cations over a monovalent cation. Analysis of Gly(4822) to Asp(4831) mutants in RyR2 supports the view that this highly conserved sequence constitutes part of the ion-conducting pore of the Ca(2+) release channel and plays a key role in ryanodine and caffeine binding and activation.
Highlights
A highly conserved amino acid sequence, GVRAGGGIGD4831, which may form part of the Ca2؉ release channel pore in RyR2, was subjected to Ala scanning or Ala to Val mutagenesis; function was measured by expression in HEK-293 cells, followed by Ca2؉ photometry, high affinity [3H]ryanodine binding, and single-channel recording
RyR2—In this study, most residues in the highly conserved sequence GVRAGGGIGD4831 in RyR2 were mutated to the small, non-polar residue Ala, Ala4825 was mutated to Val, and Ile4829, the residue equivalent to Ile4897 in RyR1, was mutated to Ala and Val and to Thr, a mutation that causes a clinically severe form of central core disease in humans [10]
Various mutations in amino acids 4822– 4831 of RyR2 Ca2ϩ release channels alter the sensitivity of channel opening to caffeine, the ability of the channel to bind [3H]ryanodine with high affinity, the ability of ryanodine to activate and inhibit channel function, the unitary conductance of single channels, the modulation of single channels by Ca2ϩ and other endogenous and exogenous agents, and ion permeability
Summary
Materials—Restriction endonucleases and other DNA modifying enzymes were from Stratagene, Roche Molecular Biochemicals, New England Biolabs, Promega, and Amersham Pharmacia Biotech; Fura-2 acetoxymethyl ester (AM) was from Molecular Probes; caffeine and protease inhibitors were from Sigma; [3H]ryanodine was from PerkinElmer Life Sciences; unlabeled ryanodine was from Calbiochem; CHAPS was from Bio-Rad; soybean phosphatidylcholine (PC) was from Northern Lipids. Extraction of Recombinant RyR2 and Mutant Proteins—Transfected HEK-293 cells grown in 100-mm Petri dishes were solubilized with 1% CHAPS, 5 mg/ml phosphatidylcholine, and a protease inhibitor mix (0.1 mM AEBSF, 1 mM benzamidine, 1 g/ml each of leupeptin, pepstatin, aprotinin, and E64) at 4 °C for 1 h, as described previously [30]. The resulting pellet was dissolved in 250 mM sucrose, 150 mM KCl, 25 mM HEPES, pH 7.1, for measurement of [3H]ryanodine binding, as described below, or used for purification of RyR2 and mutant proteins by sucrose gradient centrifugation, as described below. Purification of Expressed RyR2 and Mutant Proteins for Singlechannel Recording—The pellet obtained after centrifugation at 45,000 ϫ g (see above) was solubilized for 1 h in a buffer composed of 1 M NaCl, 1% CHAPS, 5 mg/ml PC, a mix of protease inhibitors (0.1 mM AEBSF, 1 mM benzamidine, 1 g/ml each of leupeptin, pepstatin, aprotinin, and E64) and 50 mM Hepes, pH 8.0. A value of p Յ 0.05 was considered to be statistically significant
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