Structural and functional conservation of key domains in InsP3 and ryanodine receptors.

Inositol-1,4,5-trisphosphate receptors (InsP(3)Rs) and ryanodine receptors (RyRs) are tetrameric intracellular Ca(2+) channels. In each of these receptor families, the pore, which is formed by carboxy-terminal transmembrane domains, is regulated by signals that are detected by large cytosolic structures. InsP(3)R gating is initiated by InsP(3) binding to the InsP(3)-binding core (IBC, residues 224-604 of InsP(3)R1) and it requires the suppressor domain (SD, residues 1-223 of InsP(3)R1). Here we present structures of the amino-terminal region (NT, residues 1-604) of rat InsP(3)R1 with (3.6 Å) and without (3.0 Å) InsP(3) bound. The arrangement of the three NT domains, SD, IBC-ß and IBC-α, identifies two discrete interfaces (α and ß) between the IBC and SD. Similar interfaces occur between equivalent domains (A, B and C) in RyR1 (ref. 9). The orientations of the three domains when docked into a tetrameric structure of InsP(3)R and of the ABC domains docked into RyR are remarkably similar. The importance of the α-interface for activation of InsP(3)R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations. Binding of InsP(3) causes partial closure of the clam-like IBC, disrupting the ß-interface and pulling the SD towards the IBC. This reorients an exposed SD loop ('hotspot' (HS) loop) that is essential for InsP(3)R activation. The loop is conserved in RyR and includes mutations that are associated with malignant hyperthermia and central core disease. The HS loop interacts with an adjacent NT, suggesting that activation re-arranges inter-subunit interactions. The A domain of RyR functionally replaced the SD in full-length InsP(3)R, and an InsP(3)R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by InsP(3) and blocked by ryanodine. Activation mechanisms are conserved between InsP(3)R and RyR. Allosteric modulation of two similar domain interfaces within an N-terminal subunit reorients the first domain (SD or A domain), allowing it, through interactions of the second domain of an adjacent subunit (IBC-ß or B domain), to gate the pore.

Selective inhibition of histamine-evoked Ca2+ signals by compartmentalized cAMP in human bronchial airway smooth muscle cells.

Intracellular Ca2+ and cAMP typically cause opposing effects on airway smooth muscle contraction. Receptors that stimulate these pathways are therapeutic targets in asthma and chronic obstructive pulmonary disease. However, the interactions between different G protein-coupled receptors (GPCRs) that evoke cAMP and Ca2+ signals in human bronchial airway smooth muscle cells (hBASMCs) are poorly understood. We measured Ca2+ signals in cultures of fluo-4-loaded hBASMCs alongside measurements of intracellular cAMP using mass spectrometry or [3H]-adenine labeling. Interactions between the signaling pathways were examined using selective ligands of GPCRs, and inhibitors of Ca2+ and cAMP signaling pathways. Histamine stimulated Ca2+ release through inositol 1,4,5-trisphosphate (IP3) receptors in hBASMCs. ß2-adrenoceptors, through cAMP and protein kinase A (PKA), substantially inhibited histamine-evoked Ca2+ signals. Responses to other Ca2+-mobilizing stimuli were unaffected by cAMP (carbachol and bradykinin) or minimally affected (lysophosphatidic acid). Prostaglandin E2 (PGE2), through EP2 and EP4 receptors, stimulated formation of cAMP and inhibited histamine-evoked Ca2+ signals. There was no consistent relationship between the inhibition of Ca2+ signals and the amounts of intracellular cAMP produced by different stimuli. We conclude that ß-adrenoceptors, EP2 and EP4 receptors, through cAMP and PKA, selectively inhibit Ca2+ signals evoked by histamine in hBASMCs, suggesting that PKA inhibits an early step in H1 receptor signaling. Local delivery of cAMP within hyperactive signaling junctions mediates the inhibition.

The pharmacological rationale for combining muscarinic receptor antagonists and ß-adrenoceptor agonists in the treatment of airway and bladder disease.

Muscarinic receptor antagonists and ß-adrenoceptor agonists are used in the treatment of obstructive airway disease and overactive bladder syndrome. Here we review the pharmacological rationale for their combination. Muscarinic receptors and ß-adrenoceptors are physiological antagonists for smooth muscle tone in airways and bladder. Muscarinic agonism may attenuate ß-adrenoceptor-mediated relaxation more than other contractile stimuli. Chronic treatment with one drug class may regulate expression of the target receptor but also that of the opposing receptor. Prejunctional ß2-adrenoceptors can enhance neuronal acetylcholine release. Moreover, at least in the airways, muscarinic receptors and ß-adrenoceptors are expressed in different locations, indicating that only a combined modulation of both systems may cause dilatation along the entire bronchial tree. While all of these factors contribute to a rationale for a combination of muscarinic receptor antagonists and ß-adrenoceptor agonists, the full value of such combination as compared to monotherapy can only be determined in clinical studies.

Intracellular Ca(2+) channels - a growing community.

The Ca(2+) signals that control almost every cellular activity are generated by regulating Ca(2+) transport, usually via Ca(2+)-permeable channels, across the plasma membrane or the membranes of intracellular organelles. The most widespread and best understood of the intracellular Ca(2+) channels are inositol trisphosphate receptors (IP(3)R) and ryanodine receptors, most of which are expressed in the endoplasmic or sarcoplasmic reticulum. However, accumulating evidence suggests physiological roles for many additional Ca(2+) channels in both ER and other intracellular organelles. Interactions between these channels, whether mediated by Ca(2+) itself or interactions between proteins, is a recurrent feature of the Ca(2+) signals evoked by physiological stimuli. We focus on two specific examples, clustering of IP(3)Rs and NAADP (nicotinic acid dinucleotide phosphate)-evoked Ca(2+) release from endo-lysosomes, to illustrate the diversity of Ca(2+) channels and the interplay between them.