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J. Biol. Chem., Vol. 281, Issue 30, 21022-21031, July 28, 2006

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Amino Acid Residues Gln⁴⁰²⁰ and Lys⁴⁰²¹ of the Ryanodine Receptor Type 1 Are Required for Activation by 4-Chloro-m-cresol^*

James D. Fessenden¹, Wei Feng, Isaac N. Pessah^¶, and Paul D. Allen

From the Department of Anesthesia Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115 and the Department of Veterinary Medicine-Molecular Biosciences, ^¶Center for Children's Environmental Health, Davis, California 95616

The ryanodine receptor type 1 (RyR1) and type 2 (RyR2), but not type 3 (RyR3), are efficiently activated by 4-chloro-m-cresol (4-CmC). We previously showed that a 173-amino acid segment of RyR1 (residues 4007-4180) is required for channel activation by 4-CmC (Fessenden, J. D., Perez, C. F., Goth, S., Pessah, I. N., and Allen, P. D. (2003) J. Biol. Chem. 278, 28727-28735). In the present study, we used site-directed mutagenesis to identify individual amino acid(s) within this region that mediate 4-CmC activation. In RyR1, substitution of 11 amino acids conserved between RyR1 and RyR2, but divergent in RyR3, with their RyR3 counterparts reduced 4-CmC sensitivity to the same degree as substitution of the entire 173-amino acid segment. Further analysis of various RyR1 mutants containing successively smaller numbers of these mutations identified 2 amino acid residues (Gln⁴⁰²⁰ and Lys⁴⁰²¹) that, when mutated to their RyR3 counterparts (Leu³⁸⁷³ and Gln³⁸⁷⁴), abolished 4-CmC activation of RyR1. Mutation of either of these residues alone did not abolish 4-CmC sensitivity, although Q4020L partially reduced 4-CmC-induced Ca²⁺ transients. In addition, mutation of the corresponding residues in RyR3 to their RyR1 counterparts (L3873Q/Q3874K) imparted 4-CmC sensitivity to RyR3. Recordings of single RyR1 channels indicated that 4-CmC applied to either the luminal or cytoplasmic side activated the channel with equal potency. Secondary structure modeling in the vicinity of the Gln⁴⁰²⁰-Lys⁴⁰²¹ dipeptide suggests that the region contains a surface-exposed region adjacent to a hydrophobic segment, indicating that both hydrophilic and hydrophobic regions of RyR1 are necessary for 4-CmC binding to the channel and/or to translate allosteric 4-CmC binding into channel activation.

Received for publication, January 23, 2006 , and in revised form, May 30, 2006.

^* This work was supported by National Institutes of Health Grants R01-AR43640 and 1F32 HL67572-01. 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.

¹ To whom correspondence should be addressed: Boston Biomedical Research Institute, 64 Grove St., Watertown, MA 02472. Tel.: 617-658-7886; Fax: 617-971-1761; E-mail: fessenden{at}bbri.org.

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