The contribution of hydrophobic residues in the pore-forming region of the ryanodine receptor channel to block by large tetraalkylammonium cations and Shaker B inactivation peptides.

Although no high-resolution structural information is available for the ryanodine receptor (RyR) channel pore-forming region (PFR), molecular modeling has revealed broad structural similarities between this region and the equivalent region of K(+) channels. This study predicts that, as is the case in K(+) channels, RyR has a cytosolic vestibule lined with predominantly hydrophobic residues of transmembrane helices (TM10). In K(+) channels, this vestibule is the binding site for blocking tetraalkylammonium (TAA) cations and Shaker B inactivation peptides (ShBPs), which are stabilized by hydrophobic interactions involving specific residues of the lining helices. We have tested the hypothesis that the cytosolic vestibule of RyR fulfils a similar role and that TAAs and ShBPs are stabilized by hydrophobic interactions with residues of TM10. Both TAAs and ShBPs block RyR from the cytosolic side of the channel. By varying the composition of TAAs and ShBPs, we demonstrate that the affinity of both species is determined by their hydrophobicity, with variations reflecting alterations in the dissociation rate of the bound blockers. We investigated the role of TM10 residues of RyR by monitoring block by TAAs and ShBPs in channels in which the hydrophobicity of individual TM10 residues was lowered by alanine substitution. Although substitutions changed the kinetics of TAA interaction, they produced no significant changes in ShBP kinetics, indicating the absence of specific hydrophobic sites of interactions between RyR and these peptides. Our investigations (a) provide significant new information on both the mechanisms and structural components of the RyR PFR involved in block by TAAs and ShBPs, (b) highlight important differences in the mechanisms and structures determining TAA and ShBP block in RyR and K(+) channels, and (c) demonstrate that although the PFRs of these channels contain analogous structural components, significant differences in structure determine the distinct ion-handling properties of the two species of channel.

Pharmacological regulators of intracellular calcium release channels.

Intracellular Ca(2+) release channels, such as inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs), facilitate the release of Ca(2+) from intracellular storage organelles in response to extracellular and intracellular stimuli. Consequently, these large, tetrameric proteins play a central role in Ca(2+) signalling and Ca(2+) homeostasis in virtually all cells. Recent data suggests that intracellular Ca(2+) release channels may also have an important pathophysiological function in certain disease states, including cardiac arrhythmias and heart failure. As a result, there has been much interest in the identification and characterization of novel, selective regulators of these channels. In this article, we review the wide array of pharmacological agents that interact directly with intracellular Ca(2+) release channels and describe the mechanisms underlying their ability to modify channel function.

Ryanodine receptor interaction with the SNARE-associated protein snapin.

The ryanodine receptor (RyR) is a widely expressed intracellular calcium (Ca(2+))-release channel regulating processes such as muscle contraction and neurotransmission. Snapin, a ubiquitously expressed SNARE-associated protein, has been implicated in neurotransmission. Here, we report the identification of snapin as a novel RyR2-interacting protein. Snapin binds to a 170-residue predicted ryanodine receptor cytosolic loop (RyR2 residues 4596-4765), containing a hydrophobic segment required for snapin interaction. Ryanodine receptor binding of snapin is not isoform specific and is conserved in homologous RyR1 and RyR3 fragments. Consistent with peptide fragment studies, snapin interacts with the native ryanodine receptor from skeletal muscle, heart and brain. The snapin-RyR1 association appears to sensitise the channel to Ca(2+) activation in [(3)H]ryanodine-binding studies. Deletion analysis indicates that the ryanodine receptor interacts with the snapin C-terminus, the same region as the SNAP25-binding site. Competition experiments with native ryanodine receptor and SNAP25 suggest that these two proteins share an overlapping binding site on snapin. Thus, regulation of the association between ryanodine receptor and snapin might constitute part of the elusive molecular mechanism by which ryanodine-sensitive Ca(2+) stores modulate neurosecretion.

A model of the putative pore region of the cardiac ryanodine receptor channel.

Using the bacterial K+ channel KcsA as a template, we constructed models of the pore region of the cardiac ryanodine receptor channel (RyR2) monomer and tetramer. Physicochemical characteristics of the RyR2 model monomer were compared with the template, including homology, predicted secondary structure, surface area, hydrophobicity, and electrostatic potential. Values were comparable with those of KcsA. Monomers of the RyR2 model were minimized and assembled into a tetramer that was, in turn, minimized. The assembled tetramer adopts a structure equivalent to that of KcsA with a central pore. Characteristics of the RyR2 model tetramer were compared with the KcsA template, including average empirical energy, strain energy, solvation free energy, solvent accessibility, and hydrophobic, polar, acid, and base moments. Again, values for the model and template were comparable. The pores of KcsA and RyR2 have a common motif with a hydrophobic channel that becomes polar at both entrances. Quantitative comparisons indicate that the assembled structure provides a plausible model for the pore of RyR2. Movement of Ca2+, K+, and tetraethylammonium (TEA+) through the model RyR2 pore were simulated with explicit solvation. These simulations suggest that the model RyR2 pore is permeable to Ca2+ and K+ with rates of translocation greater for K+. In contrast, simulations indicate that tetraethylammonium blocks movement of metal cations.

A novel and rapid approach to isolating functional ryanodine receptors.

Conventional methods of isolating and reconstituting ryanodine receptors (RyRs) from native membranes into proteoliposomes take a minimum of 2 days to complete. We have developed an alternative strategy that can be used to isolate and reconstitute functional RyRs in just 3 h with a similar degree of purification. RyRs isolated by this method display characteristic functional behaviour as assessed by radioligand binding and single channel analyses.