A sequence motif, GXRXGGGXGD, located in the putative channel-forming domain, is conserved in all known ryanodine receptors and inositol 1,4,5-trisphosphate receptors. The functional significance of this conserved region was investigated by using site-directed mutagenesis together with functional assays consisting of Ca 2؉ Ryanodine receptors (RyRs) 1 are members of a superfamily of intracellular Ca 2ϩ channels that include the inositol 1,4,5-trisphosphate receptors (IP 3 Rs). These channels play an essential role in intracellular Ca 2ϩ signaling by virtue of releasing Ca 2ϩ from the lumen of sarco(endo)plasmic reticulum to the cytosol of muscle and non-muscle cells (1, 2).RyR is a homotetrameric structure composed of four identical subunits, each having ϳ5000 amino acids. Sequence analysis reveals that one-fifth of the COOH terminus of the molecule is likely to form the channel conducting pore. The remaining ϳ4000 amino acid residues apparently constitute the cytoplasmic "foot" domain (3-7). A truncated RyR in which the foot domain has been deleted has been shown to function as a Ca 2ϩ release channel. The truncated RyR channel was still regulated by Ca 2ϩ , was modified by ryanodine, and exhibited a single channel conductance similar to that of the full-length RyR (8). These studies indicate that the sites for Ca 2ϩ activation and ryanodine binding, and the ion conduction pathway are located within the COOH-terminal ϳ1000 amino acid residues. A glutamate residue located in the putative transmembrane sequence M2 has recently been identified as the Ca 2ϩ sensor of RyR (9). The locations of the ryanodine binding site and the pore-forming segment of RyR, however, have yet to be defined.RyRs and IP 3 Rs share some sequence homology, in particular in the COOH-terminal channel-forming domain (10). Considering their sequence homology and similar conduction properties (11-13), RyRs and IP 3 Rs are likely to share similar structural features in the channel pore. A hydrophobic region between the M5 and M6 transmembrane sequences of the mouse type 1 IP 3 R has been proposed to be the pore-forming region (14). The equivalent region in RyR, corresponding to the M9 transmembrane sequence proposed by Zorzato et al. (15), is also hydrophobic. Sequence alignment of these regions reveals a GXRXGGGXGD motif that can be found in all known RyRs and IP 3 Rs (Fig. 1). To investigate its role in RyR function, we have introduced point mutations into this highly conserved region and examined the functional consequences of these point mutations. Our data indicate that this region is critical for ryanodine binding and ion conduction and probably constitutes the pore-forming segment of RyRs. EXPERIMENTAL PROCEDURES Materials-Ryanodine was obtained from Calbiochem. [3 H]Ryanodine was from NEN Life Science Products. Monoclonal antibody 34C was a generous gift from Dr. John L. Sutko (16).Cloning of the Mouse Cardiac RyR cDNA-Total RNA from mouse heart tissue, isolated by the method of Chomczynski and Sacchi (17), was used to generate firs...

We have investigated the molecular basis for ryanodine receptor (RyR) activation by Ca 2؉ by using sitedirected mutagenesis together with functional assays consisting of Ca 2؉ release measurements and single channel recordings in planar lipid bilayers. We report here that a single substitution of alanine for glutamate at position 3885 (located in the putative transmembrane sequence M2 of the type 3 RyR) reduces the Ca 2؉ sensitivity, as measured by single channel activation, by more than 10,000-fold, without apparent changes in channel conductance and in modulation by other ligands (e.g. ATP and ryanodine). Co-expression of the wild type and mutant RyR proteins results in the synthesis of single channels that have intermediate Ca 2؉sensitivities. These results suggest that the glutamates at position 3885 of each monomer may act in a coordinated way to form the Ca 2؉ sensor in the tetrameric structure corresponding to RyR.Ryanodine receptors (RyRs) 1 are a family of Ca 2ϩ channels which mediate intracellular Ca 2ϩ release that is essential for a variety of cellular functions including muscle contraction, egg fertilization, and synaptic transmission (1, 2). Three RyR isoforms (RyR1, RyR2, and RyR3) have been identified in mammalian tissues; all three are activated by Ca 2ϩ (3)(4)(5)(6)(7)(8). Activation of RyR by Ca 2ϩ is the mechanism underlying Ca 2ϩ -induced Ca 2ϩ release from the sarco(endo)plasmic reticulum (9 -11).Of the many ligands known to modulate the activity of RyR, Ca 2ϩ is the essential regulator. Most other ligands exert their effect on RyR activity by influencing the Ca 2ϩ sensitivity of 12). Alterations in the Ca 2ϩ sensitivity of RyR have been implicated in at least one disease, malignant hyperthermia (13). Thus, understanding the molecular mechanism that controls the Ca 2ϩ sensitivity is fundamental to the understanding of RyR regulation and intracellular Ca 2ϩ signaling. RyR activation by Ca 2ϩ is thought to be mediated by high affinity Ca 2ϩ binding sites in the protein (14), but the molecular identity of these Ca 2ϩ activation sites, the Ca 2ϩ sensor, has yet to be defined. It has been shown that negatively charged residues within a transmembrane sequence are often involved in binding and translocation of cations across the membrane (15-17). Analysis of the amino acid sequences of RyRs reveals that of the 12 predicted transmembrane sequences of RyR (18), four (M1, M2, M7, and M10) contain negatively charged amino acid residues that are conserved in all known RyR isoforms ( Fig. 1A) (19 -26). To investigate their roles in RyR function, we have mutated these negatively charged residues in the rabbit type 3 RyR. The functional consequence of one of these point mutations, a glutamate-to-alanine mutation at position 3885 (E3885A) located in the M2 transmembrane sequence (Fig. 1B), was assessed. Our results demonstrate that glutamate 3885 plays an essential role in determining the Ca 2ϩ sensitivity and provide important new insights into the Ca 2ϩ -sensing mechanism of RyR. EXPERIMENTAL PROCEDUR...

This review presents the basic principles, protocols and examples of using the machine learning approaches to investigate the bioactivity of natural products.

CD19 is a coreceptor on B cells that enhances the increase in cytoplasmic calcium and ERK2 activation when coligated with the B cell Ag receptor. Constructs containing point mutations and truncations were expressed in Daudi human B lymphoblastoid cells to systematically determine the requirement for individual CD19 cytoplasmic tyrosines in these responses. Evidence for activity was found for Y330, Y360, and Y421 as well as that previously published for Y391. Precipitates formed with phosphopeptides consisting of CD19 sequences flanking these residues were used to screen for cytoplasmic proteins that mediate signaling. Phosphopeptide Y330 precipitated Grb2 and Sos, whereas phosphopeptides Y391 and Y421 both precipitated Vav and phospholipase C-γ2. These molecules also were found associated with native CD19. In mapping studies with altered constructs, CD19 Y330 and/or Y360 were necessary for binding Grb2 and Sos. Vav associated with CD19 constitutively in unstimulated cells by a tyrosine-independent mechanism requiring the portion of CD19 encoded by exons 9–12. After B cell Ag receptor stimulation, Vav association was tyrosine-dependent, but binding was influenced by multiple residues. However, when maximally phosphorylated by pervanadate, Y391 and, to a lesser extent, Y421 were sufficient. CD19 Y391 was also both necessary and sufficient for binding phospholipase C-γ2. Thus, different tyrosines along the CD19 cytoplasmic domain provide scaffolding for the formation of complexes of different signaling molecules.

Fungal drug resistance is a major health threat, and reports of clinical resistance worldwide are becoming increasingly common. In a research program to discover new molecules to help overcome this problem, 14 new lanostane-type triterpenoids, gibbosicolids A−G (2−8) and gibbosic acids I−O (9−15), were isolated from the fruiting bodies of Ganoderma gibbosum, along with seven known triterpenoid derivatives. These compounds featured high levels of oxidation, epimerization, and γ-lactonization. Structures were elucidated by comprehensive spectroscopic analyses and HRMS data. Absolute configurations were assigned based on quantum chemical calculations, including calculated chemical shift with DP4+ analysis, coupling constants, and electronic circular dichroism (ECD) methods. Results show that the calculated NMR with DP4+ analysis could not reliably establish the overall spatial configuration of molecules possessing independent and free-rotational stereoclusters. All these compounds significantly increased the sensitivity of fluconazole (FLC)-resistant C. albicans to FLC. Compounds 2, 5, 9, 12, 16, 17, and 21 exhibited strong antifungal activity against FLC-resistant C. albicans when combined with FLC, with MIC 50 values ranging from 3.8 to 8.8 μg/mL.