Search

The predicted TM10 transmembrane sequence, [sup 4844]IIFDITFFFFVIVILLAIIQGLII[sup 4867], has been proposed to be the pore inner helix of the ryanodine receptor (RyR) and to play a crucial role in channel activation and gating, as with the inner helix of bacterial potassium channels. However, experimental evidence for the involvement of the TM10 sequence in RyR channel activation and gating is lacking. In the present study, we have systematically investigated the effects of mutations of each residue within the 24-amino acid TM10 sequence of the mouse cardiac ryanodine receptor (RyR2) on channel activation by caffeine and Ca[sup 2+]. Intracellular Ca[sup 2+] release measurements in human embryonic kidney 293 cells expressing the RyR2 wild type and TM10 mutants revealed that several mutations in the TM10 sequence either abolished caffeine response or markedly reduced the sensitivity of the RyR2 channel to activation by caffeine. By assessing the Ca[sup 2+] dependence of [³H]ryanodine binding to RyR2 wild type and TM10 mutants we also found that mutations in the TM10 sequence altered the sensitivity of the channel to activation by Ca[sup 2+] and enhanced the basal activity of [³H]ryanodine binding. Furthermore, single I4862A mutant channels exhibited considerable channel openings and altered gating at very low concentrations of Ca[sup 2+]. Our data indicate that the TM10 sequence constitutes an essential determinant for channel activation and gating, in keeping with the proposed role of TM10 as an inner helix of RyR. Our results also shed insight into the orientation of the TM10 helix within the RyR channel pore. [ABSTRACT FROM AUTHOR]

Despite the pivotal role of ryanodine in ryanodine receptor (RyR) research, the molecular basis of ryanodine-RyR interaction remains largely undefined. We investigated the role of the proposed transmembrane helix TM10 in ryanodine interaction and channel function. Each amino acid residue within the TM10 sequence, [sup 4844]IIFDITFFFFVIVILLAIIQGLII[sup 4867], of the mouse RyR2 was mutated to either alanine or glycine. Mutants were expressed in human embryonic kidney 293 cells, and their properties were assessed. Mutations D4847A, F4850A, F4851A, L4858A, L4859A, and I4866A severely curtailed the release of intracellular Ca[sup 2+] in human embryonic kidney 293 cells in response to extracellular caffeine and diminished [³H]ryanodine binding to cell lysates. Mutations F4846A, T4849A, I4855A, V4856A, and Q4863A eliminated or markedly reduced [³H]ryanodine binding, but cells expressing these mutants responded to extracellular caffeine by releasing stored Ca[sup 2+]. Interestingly these two groups of mutants, each with similar properties, are largely located on opposite sides of the predicted TM10 helix. Single channel analyses revealed that mutation Q4863A dramatically altered the kinetics and apparent affinity of ryanodine interaction with single RyR2 channels and abolished the effect of ryanodol, an analogue of ryanodine, whereas the single channel conductance of the Q4863A mutant and its responses to caffeine, ATP, and Mg[sup 2+] were comparable to those of the wild type channels. Furthermore the effect of ryanodine on single Q4863A mutant channels was influenced by the transmembrane holding potential. Together these results suggest that the TM10 sequence and in particular the Q4863 residue constitute an important determinant of ryanodine interaction. [ABSTRACT FROM AUTHOR]

Examines the location of divergent region 3 (DR3) in the three-dimensional structure of ryanodine receptors. Excitation-contraction coupling; Channel regulation by calcium and magnesium; Structural basis of DR3 function.

Tests the hypothesis that smooth muscle and peripheral tissues express a unique isoform of the type 3 calcium[sup 2+] release channel, ryanodine receptor (RyR3), as a result of alternative splicing. Experimental procedures; Pharmacological and functional heterogeneity of RyR3; Spliced variants of RyR3.

Investigates the significance of the isoleucine-proline motif in FK506-binding protein (FKBP12.6) interaction with ryanodine receptor. Assessment of the ability of the mutants to bind GST-FKBP12.6; Role of the binding of FKPB12.6 to the NH[sub 2]-terminal domain in stabilizing the conformation in the region.

• The N-methyl-d-glucamine (NMDG) slicing method better preserves neuronal activity. • Limiting RyR2 open time prevented neuronal hyperactivity of CA1 neurons in slices. • R-carvedilol prevented and rescued neuronal hyperactivity of CA1 neurons in slices. Neuronal hyperactivity is an early, common manifestation of Alzheimer's disease (AD), and is believed to drive AD progression. Neuronal hyperactivity in the form of baseline activity (or spontaneous Ca2+ transients) has consistently been demonstrated in mouse models of AD using two-photon in vivo Ca2+ imaging of cortical or hippocampal neurons in anesthetized animals. Notably, these AD-related spontaneous Ca2+ transients were hardly detected in acute hippocampal slices, probably due to neuronal damage during brain slicing. To better preserve neuronal activity, we employed the N-methyl-D-glucamine (NMDG) protective brain slicing protocol. We performed confocal in vitro Ca2+ imaging of hippocampal CA1 neurons in optimized hippocampal slices. Consistent with previous in vivo studies, our in vitro studies using optimized brain slices also showed that limiting the open duration of the ryanodine receptor 2 (RyR2) by the RyR2 mutation E4872Q or by the R-carvedilol enantiomer prevented and rescued neuronal hyperactivity of hippocampal CA1 neurons from 5xFAD mice. Thus, genetically and pharmacologically limiting RyR2 open time prevented and rescued AD-related neuronal hyperactivity in vitro in optimized brain slices in the absence of anesthetics' influence. Our data also suggest that the NMDG protective brain slicing preparation offers an alternative means to study neuronal hyperactivity of various cell types in different brain regions, especially in regions that are not readily accessible to two-photon in vivo Ca2+ imaging. [ABSTRACT FROM AUTHOR]