Kirill Kiselyov

Functional interaction between InsP3 receptors and store-operated Htrp3 channels

Calcium ions are released from intracellular stores in response to agonist-stimulated production of inositol 1,4,5-trisphosphate (InsP3), a second messenger generated at the cell membrane. Depletion of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+ across the plasma membrane,. The influx of Ca2+ can be recorded as store-operated channels (SOC) in the plasma membrane or as a current known as the Ca2+-release-activated current (Icrac). A critical question in cell signalling is how SOC and Icrac sense and respond to Ca2+-store depletion: in one model, a messenger molecule is generated that activates Ca2+ entry in response to store depletion,; in an alternative model, InsP3 receptors in the stores are coupled to SOC and Icrac. The mammalian Htrp3 protein forms a well defined store-operated channel, and so provides a suitable system for studying the effect of Ca2+-store depletion on SOC and Icrac. We show here that Htrp3 channels stably expressed in HEK293 cells are in a tight functional interaction with the InsP3 receptors. Htrp3 channels present in the same plasma membrane patch can be activated by Ca2+ mobilization in intact cells and by InsP3 in excised patches. This activation of Htrp3 by InsP3 is lost on extensive washing of excised patches but is restored by addition of native or recombinant InsP3-bound InsP3 receptors. Our results provide evidence for the coupling hypothesis, in which InsP3 receptors activated by InsP3 interact with SOC and regulate Icrac.

Aberrant CFTR-dependent HCO3- transport in mutations associated with cystic fibrosis

Cystic fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Initially, Cl- conductance in the sweat duct was discovered to be impaired in CF, a finding that has been extended to all CFTR-expressing cells. Subsequent cloning of the gene showed that CFTR functions as a cyclic-AMP-regulated Cl- channel; and some CF-causing mutations inhibit CFTR Cl- channel activity. The identification of additional CF-causing mutants with normal Cl- channel activity indicates, however, that other CFTR-dependent processes contribute to the disease. Indeed, CFTR regulates other transporters, including Cl--coupled HCO-3 transport. Alkaline fluids are secreted by normal tissues, whereas acidic fluids are secreted by mutant CFTR-expressing tissues, indicating the importance of this activity. HCO-3 and pH affect mucin viscosity and bacterial binding. We have examined Cl--coupled HCO-3 transport by CFTR mutants that retain substantial or normal Cl- channel activity. Here we show that mutants reported to be associated with CF with pancreatic insufficiency do not support HCO-3 transport, and those associated with pancreatic sufficiency show reduced HCO-3 transport. Our findings demonstrate the importance of HCO-3 transport in the function of secretory epithelia and in CF.

The N-Terminal Domain of the IP3 Receptor Gates Store-Operated hTrp3 Channels

In the present work, we studied the interaction and effect of several IP3 receptor (IP3R) constructs on the gating of the store-operated (SOC) hTrp3 channel. Full-length IP3R coupled to silent hTrp3 channels in intact cells but did not activate them until stores were depleted of Ca2+. By contrast, constructs containing the IP3-binding domain activated silent hTrp3 channels in unstimulated cells and restored gating of hTrp3 by IP3 in excised plasma membrane patches. We conclude that the N-terminal domain of the IP3R functions as a gate and is sufficient for activation of SOCs. The sensing and transduction domains of the IP3R are required to maintain SOCs in an inactive state.

Gating of Store-Operated Channels by Conformational Coupling to Ryanodine Receptors

We report here that RyRs interact with and gate the store-operated hTrp3 and Icrac channels. This gating contributes to activation of hTrp3 and Icrac by agonists. Coupling of hTrp3 to IP3Rs or RyRs in the same cells was found to be mutually exclusive. Biochemical and functional evidence suggest that mutually exclusive coupling reflects clustering and segregation of hTrp3-IP3R and hTrp3-RyR complexes in plasma membrane microdomains. Gating of CCE by RyRs indicates that gating by conformational coupling is not unique to skeletal muscle but is a general mechanism for communication between events in the plasma and endoplasmic reticulum membranes.

Signalling specificity in GPCR-dependent Ca2+ signalling

Cells use signalling networks to translate with high fidelity extracellular signals into specific cellular functions. Signalling networks are often composed of multiple signalling pathways that act in concert to regulate a particular cellular function. In the centre of the networks are the receptors that receive and transduce the signals. A versatile family of receptors that detect a remarkable variety of signals are the G protein-coupled receptors (GPCRs). Virtually all cells express several GPCRs that use the same biochemical machinery to transduce their signals. Considering the specificity and fidelity of signal transduction, a central question in cell signalling is how signalling specificity is achieved, in particular among GPCRs that use the same biochemical machinery. Ca(2+) signalling is particularly suitable to address such questions, since [Ca(2+)](i) can be recorded with excellent spatial and temporal resolutions in living cells and tissues and now in living animals. Ca(2+) is a unique second messenger in that both biochemical and biophysical components form the Ca(2+) signalling complex to regulate its concentration. Both components act in concert to generate repetitive [Ca(2+)](i) oscillations that can be either localized or in the form of global, propagating Ca(2+) waves. Most of the key proteins that form Ca(2+) signalling complexes are known and their activities are reasonably well understood on the biochemical and biophysical levels. We review here the information gained from studying Ca(2+) signalling by GPCRs to gain further understanding of the mechanisms used to generate cellular signalling specificity.

Regulation of Ca2+-release-activated Ca2+ current (Icrac) by ryanodine receptors in inositol 1,4,5-trisphosphate-receptor-deficient DT40 cells.

Persistence of capacitative Ca(2+) influx in inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-deficient DT40 cells (DT40(IP(3)R-/-)) raises the question of whether gating of Ca(2+)-release activated Ca(2+) current (I(crac)) by conformational coupling to Ca(2+)-release channels is a general mechanism of gating of these channels. In the present work we examined the properties and mechanism of activation of I(crac) Ca(2+) current in wild-type and DT40(IP(3)R-/-) cells. In both cell types passive depletion of internal Ca(2+) stores by infusion of EGTA activated a Ca(2+) current with similar characteristics and time course. The current was highly Ca(2+)-selective and showed strong inward rectification, all typical of I(crac). The activator of ryanodine receptor (RyR), cADP-ribose (cADPR), facilitated activation of I(crac), and the inhibitors of the RyRs, 8-N-cADPR, ryanodine and Ruthenium Red, all inhibited I(crac) activation in DT40(IP(3)R-/-) cells, even after complete depletion of intracellular Ca(2+) stores by ionomycin. Wild-type and DT40(IP(3)R-/-) cells express RyR isoforms 1 and 3. RyR levels were adapted in DT40(IP(3)R-/-) cells to a lower RyR3/RyR1 ratio than in wild-type cells. These results suggest that IP(3)Rs and RyRs can efficiently gate I(crac) in DT40 cells and explain the persistence of I(crac) gating by internal stores in the absence of IP(3)Rs.

Regulation of the Miniature Plasma Membrane Ca2+ Channel I min by Inositol 1,4,5-Trisphosphate Receptors

I min is a plasma membrane-located, Ca2+-selective channel that is activated by store depletion and regulated by inositol 1,4,5-trisphosphate (IP3). In the present work we examined the coupling betweenI min and IP3 receptors in excised plasma membrane patches from A431 cells. I minwas recorded in cell-attached mode and the patches were excised into medium containing IP3. In about 50% of experiments excision caused the loss of activation of I minby IP3. In the remaining patches activation ofI min by IP3 was lost upon extensive washes of the patch surface. The ability of IP3 to activateI min was restored by treating the patches with rat cerebellar microsomes reach in IP3 receptors but not by control forebrain microsomes. The re-activatedI min had the same kinetic properties asI min when it is activated by Ca2+-mobilizing agonists in intact cells and by IP3 in excised plasma membrane patches and it was inhibited by the I crac inhibitor SKF95365. We propose that I min is a form ofI crac and is gated by IP3receptors.

Ca2+ signaling in polarized exocrine cells

Exocrine cells respond to a battery of neurotransmitters and hormones that act on G protein-coupled and G protein-independent receptors. Receptors transmit their signals by activation of biochemical pathways that change the concentration of second messengers, the most common of which are cAMP and intracellular Ca2+ ions ([Ca2+] i ). Many G protein-coupled receptors (GPCR) expressed in a given cell, activate the same biochemical pathway changing the concentration of the same second messengers. With the development of techniques that measure the concentration of second messengers in single cells and subcellular compartments, it became clear that different receptors using the same biochemical pathway could generate receptor-specific signals. In this short review, we summarize the current knowledge regarding a central question in cell signaling (Hunter, 2000: Weng et al., 1999) of how signaling specificity is achieved by GPCR expressed in a given cell using the same signaling pathway.