Biomedical subjects

James D Fessenden

Publications and source records attributed to James D Fessenden.

6 recordsLinked to original sources

PubMed2006

Amino acid residues Gln4020 and Lys4021 of the ryanodine receptor type 1 are required for activation by 4-chloro-m-cresol.

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 (Gln(4020) and Lys(4021)) that, when mutated to their RyR3 counterparts (Leu(3873) and Gln(3874)), 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 Ca(2+) 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 Gln(4020)-Lys(4021) 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.

Allosteric Site↗

PubMed2006

Structural determinants of 4-chloro-m-cresol required for activation of ryanodine receptor type 1.

4-Chloro-m-cresol (4-CmC) is a clinically relevant activator of the intracellular Ca2+ release channel, the ryanodine receptor isoform 1 (RyR1). In this study, the chemical moieties on the 4-CmC molecule required for its activation of RyR1 were determined using structure-activity relationship analysis with a set of commercially available 4-CmC analogs. Separate compounds each lacking one of the three functional groups of 4-CmC (1-hydroxyl, 3-methyl, or 4-chloro) were poor activators of RyR1. Substitution of different chemical groups for the 1-hydroxyl of 4-CmC resulted in compounds that were poor activators of RyR1, suggesting that the hydroxyl group is preferred at this position. Substitution of hydrophobic groups at the 3-position enhanced bioactivity of the compound relative to 4-CmC, whereas substitution with hydrophilic groups abolished bioactivity. Likewise, 4-CmC analogs with hydrophobic groups substituted into the 4-position enhanced bioactivity, whereas hydrophilic or charged groups diminished bioactivity. 4-CmC analogs containing a single hydrophobic group at either the 3- or 4-position as well as 3,5-disubstituted or 3,4,5-trisubstituted phenols were also effective activators of RyR1. These results indicate that the 1-hydroxyl group of 4-CmC is required for activation of RyR1 and that hydrophobic groups at the 3,4- and 5-positions are preferred. These findings suggest that the 4-CmC binding site on RyR1 most likely consists of a hydrophilic region to interact with the 1-hydroxyl as well as a hydrophobic region(s) to interact with chemical groups at the 3- and/or 4-positions of 4-CmC.

Animals↗

PubMed2004

Conformational activation of Ca2+ entry by depolarization of skeletal myotubes.

Store-operated Ca(2+) entry (SOCE) occurs in diverse cell types in response to depletion of Ca(2+) within the endoplasmic/sarcoplasmic reticulum and functions both to refill these stores and to shape cytoplasmic Ca(2+) transients. Here we report that in addition to conventional SOCE, skeletal myotubes display a physiological mechanism that we term excitation-coupled Ca(2+) entry (ECCE). ECCE is rapidly initiated by membrane depolarization. Like excitation-contraction coupling, ECCE is absent in both dyspedic myotubes that lack the skeletal muscle-type ryanodine receptor 1 and dysgenic myotubes that lack the dihydropyridine receptor (DHPR), and is independent of the DHPR l-type Ca(2+) current. Unlike classic SOCE, ECCE does not depend on sarcoplasmic reticulum Ca(2+) release. Indeed, ECCE produces a large Ca(2+) entry in response to physiological stimuli that do not produce substantial store depletion and depends on interactions among three different Ca(2+) channels: the DHPR, ryanodine receptor 1, and a Ca(2+) entry channel with properties corresponding to those of store-operated Ca(2+) channels. ECCE may provide a fundamental means to rapidly maintain Ca(2+) stores and control important aspects of Ca(2+) signaling in both muscle and nonmuscle cells.

Animals↗

PubMed2004

Mutational analysis of putative calcium binding motifs within the skeletal ryanodine receptor isoform, RyR1.

The functional relevance of putative Ca(2+) binding motifs previously identified with Ca(2+) overlay binding analysis within the skeletal muscle ryanodine receptor isoform (RyR1) was examined using mutational analysis. EF hands between amino acid positions 4081 and 4092 (EF1) and 4116 and 4127 (EF2) were scrambled singly or in combination within the full-length rabbit RyR1 cDNA. These cDNAs were expressed in 1B5 RyR-deficient myotubes and channel function assessed using Ca(2+)-imaging techniques, [(3)H]ryanodine binding measurements, and single channel experiments. In intact myotubes, these mutations did not affect functional responses to either depolarization or RyR agonists (caffeine, 4-chloro-m-cresol) compared with wtRyR1. However, in [(3)H]ryanodine binding measurements, both Ca(2+) activation and inhibition of the EF1 mutant was significantly altered compared with wtRyR1. No high affinity [(3)H]ryanodine binding was observed in membranes expressing the EF2 mutation, although in single channel measurements, the EF2-disrupted channel could be activated by micromolar Ca(2+) concentrations. In addition, micromolar levels of ryanodine placed these channels into the classical half-conductance state, thus indicating that occupancy of high affinity ryanodine binding sites is not required for ryanodine-induced subconductance states in RyR1. Disruption of three additional putative RyR1 calcium binding motifs located between amino acid positions 4254 and 4265 (EF3), 4407 and 4418 (EF4), or 4490 and 4502 (EF5) either singly or in combination (EF3-5) did not affect functional responses in 1B5 myotubes except that the EC(50) for caffeine activation for the EF3 construct was significantly increased compared with wtRyR1. However, in [(3)H]ryanodine binding experiments, the Ca(2+)-dependent activation and inactivation of mutated RyRs containing EF3, EF4, or EF5 was unaffected when compared with wtRyR1.

Amino Acid Motifs↗

PubMed2004

Evidence for conformational coupling between two calcium channels.

Ryanodine receptor 1 (RyR1, the sarcoplasmic reticulum Ca(2+) release channel) and alpha(1S)dihydropyridine receptor (DHPR, the surface membrane voltage sensor) of skeletal muscle belong to separate membrane systems but are functionally and structurally linked. Four alpha(1S)DHPRs associated with the four identical subunits of a RyR form a tetrad. We treated skeletal muscle cell lines with ryanodine, at concentrations that block RyRs, and determined whether this treatment affects the distance between DHPRs in the tetrad. We find a substantial ( approximately 2-nm) shift in the alpha(1S)DHPR positions, indicating that ryanodine induces large conformational changes in the RyR1 cytoplasmic domain and that the alpha(1S)DHPR-RyR complex acts as a unit.

Animals↗

PubMed2003

Identification of a key determinant of ryanodine receptor type 1 required for activation by 4-chloro-m-cresol.

4-Chloro-m-cresol (4-CmC) is a potent and specific activator of the intracellular Ca2+ release channel, the ryanodine receptor (RyR). We have previously shown that RyR1 expressed in dyspedic 1B5 myotubes is activated by 4-CmC, whereas RyR3 is not (Fessenden, J. D., Wang, Y., Moore, R. A., Chen, S. R. W., Allen, P. D., and Pessah, I. N. (2000) Biophys. J. 79, 2509-2525). To identify region(s) on RyR1 that are responsible for mediating activation by 4-CmC, we expressed RyR1-RyR3 chimeric proteins in dyspedic 1B5 myotubes and then measured 4-CmC-induced increases in intracellular Ca2+. Substitution of the C-terminal third of RyR1 into RyR3 imparted 4-CmC sensitivity to the resulting chimera, thus suggesting that determinants required for activation by 4-CmC are located in this region. We subdivided the C-terminal third of RyR1 into smaller segments and identified two overlapping regions of RyR1 (amino acids 3769-4180 and 4007-4382) that each imparted 4-CmC sensitivity to RyR3. Substitution of the 173 amino acids of RyR1 common to these two chimeras (amino acids 4007-4180) also weakly restored 4-CmC sensitivity in the resulting chimera. To confirm these findings, we created a complementary set of chimeras containing RyR3 substitutions in RyR1. Substitution of the RyR3 C terminus into RyR1 disrupted 4-CmC sensitivity in the resulting chimera. In addition, substitution of the corresponding RyR3 sequence into positions 4007-4180 of RyR1 disrupted 4-CmC sensitivity. Taken together, these results suggest that essential determinants required for activation of RyR1 by 4-CmC reside within a 173-amino acid region between residues 4007 and 4180.

Animals↗