Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2

We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC₂₀) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P₁ and S1P₂ receptors. S1P activated G_q, G₁₃, and all G_i isoforms and stimulated PLC-1, PLC-3, and Rho kinase activities. PLC- activity was partially inhibited by pertussis toxin (PTX), G or G_q antibody, PLC-1 or PLC-3 antibody, and by expression of G_q or G_i minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of G₁₃ or G_q minigene and abolished by expression of both. S1P stimulated Ca²⁺ release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC₅₀ 1 nM). Initial contraction and MLC₂₀ phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and G_q or G antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC₂₀ phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1) initial contraction mediated by S1P₂ and S1P₁ involving concurrent activation of PLC-1 and PLC-3 via G_q and G_i, respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca²⁺ release and MLCK-mediated MLC₂₀ phosphorylation, and 2) sustained contraction exclusively mediated by S1P₂ involving activation of RhoA via G_q and G₁₃, resulting in Rho kinase- and PKC-dependent MLC₂₀ phosphorylation. muscle contraction; signal transduction     

The Ca2+-sensing receptor couples to Galpha12/13 to activate phospholipase D in Madin-Darby canine kidney cells

The Ca²⁺-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: G_i to inhibit the activity of adenylyl cyclase and activate ERK, G_q to stimulate phospholipase C and phospholipase A₂, and G to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to G_12/13, we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known G_12/13 target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (CaR^WT) or the nonfunctional mutant CaR^R796W as a negative control, prelabeled these cells with [³H]palmitic acid, and measured CaR-stimulated PLD activity as the formation of [³H]phosphatidylethanol (PEt). The formation of [³H]PEt increased in a time-dependent manner in the cells that overexpress the CaR^WT but not the CaR^R796W. Treatment of the cells with C₃ exoenzyme inhibited PLD activity, which indicates that the CaR activates the Rho family of small G proteins, targets of G_12/13. To determine which G protein(s) the CaR couples to in order to activate Rho and PLD, we pretreated the cells with pertussis toxin to inactivate G_i or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for G_i and G_q, and a p115RhoGEF construct containing the RGS domain for G_12/13). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaR^WT inhibited extracellular Ca²⁺-stimulated PLD activity, but pretreatment of cells with pertussis toxin and overexpression of RGS4 were without effect. The involvement of other signaling components such as protein kinase C, ADP-ribosylation factor, and phosphatidylinositol biphosphate was excluded. These findings demonstrate that the CaR couples to G_12/13 to regulate PLD via a Rho-dependent mechanism and does so independently of G_i and G_q. This suggests that the CaR may regulate cytoskeleton via G_12/13, Rho, and PLD. calcium-sensing receptor; G proteins; RGS proteins     

Involvement of G protein betagamma-subunits in diverse signaling induced by G(i/o)-coupled receptors: study using the Xenopus oocyte expression system

We studied the functions of -subunits of G_i/o protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl^– currents in oocytes expressing ₂-adrenoceptor and the protein kinase A-dependent Cl^– channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [D-Ala², D-Leu⁵]-enkephalin (DADLE), and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing ₂-adrenoceptor-CFTR and 5-HT_1A receptor (5-HT_1AR), -opioid receptor, or GABA_B receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT_1AR. The 5-HT-induced enhancement of G_s-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin (G_t). The 5-HT-induced enhancement was further augmented by coexpression of the G-activated form of adenylate cyclase (AC) type II but not AC type III. Thus -subunits of G_i/o protein contribute to the enhancement of G_s-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT_1AR or -opioid receptor alone. They elicited Ca²⁺-activated Cl^– currents in oocytes coexpressing these receptors with the G-activated form of phospholipase C (PLC)-2 but not with PLC-1. These currents were inhibited by pretreatment with PTX and coexpression of G_t, suggesting that -subunits of G_i/o protein activate PLC-2 and then cause intracellular Ca²⁺ mobilization. Our results indicate that -subunits of G_i/o protein participate in diverse intracellular signals, enhancement of G_s-coupled receptor-mediated responses, and intracellular Ca²⁺ mobilization. G protein-coupled receptor; cystic fibrosis transmembrane conductance regulator gene; cross talk; electrophysiology     

Altered gating and regulation of a carboxy-terminal ClC channel mutant expressed in the Caenorhabditis elegans oocyte

CLH-3a and CLH-3b are swelling-activated, alternatively spliced Caenorhabditis elegans ClC anion channels that have identical membrane domains but exhibit marked differences in their cytoplasmic NH₂ and COOH termini. The major differences include a 71-amino acid CLH-3a NH₂-terminal extension and a 270-amino acid extension of the CLH-3b COOH terminus. Splice variation gives rise to channels with striking differences in voltage, pH, and Cl^– sensitivity. On the basis of structural and functional insights gained from crystal structures of bacterial ClCs, we suggested previously that these functional differences are due to alternative splicing of the COOH terminus that may change the accessibility and/or function of pore-associated ion-binding sites. We recently identified a mutant worm strain harboring a COOH-terminal deletion mutation in the clh-3 gene. This mutation removes 101 COOH-terminal amino acids unique to CLH-3b and an additional 64 upstream amino acids shared by both channels. CLH-3b is expressed in the worm oocyte, which allowed us to characterize the mutant channel, CLH-3bC, in its native cellular environment. CLH-3bC exhibits altered voltage-dependent gating as well as pH and Cl^– sensitivity that resemble those of CLH-3a. This mutation also alters channel inhibition by Zn²⁺, prevents ATP depletion-induced activation, and dramatically reduces volume sensitivity. These results suggest that the deleted COOH-terminal region of CLH-3bC functions to modulate channel sensitivity to voltage and extracellular ions. This region also likely plays a role in channel regulation and cell volume sensitivity. Our findings contribute to a growing body of evidence indicating that cytoplasmic domains play key roles in the gating and regulation of eukaryotic ClCs. chloride; cell volume; voltage-gated anion channel     

Inhibition of Galphaq-dependent PLC-beta1 activity by PKG and PKA is mediated by phosphorylation of RGS4 and GRK2

In smooth muscle of the gut, G_q-coupled receptor agonists activate preferentially PLC-1 to stimulate phosphoinositide (PI) hydrolysis and inositol 1,4,5-trisphosphate (IP₃) generation and induce IP₃-dependent Ca²⁺ release. Inhibition of Ca²⁺ mobilization by cAMP- (PKA) and cGMP-dependent (PKG) protein kinases reflects inhibition of PI hydrolysis by both kinases and PKG-specific inhibitory phosphorylation of IP₃ receptor type I. The mechanism of inhibition of PLC-1-dependent PI hydrolysis has not been established. Neither G_q nor PLC-1 was directly phosphorylated by PKA or PKG in gastric smooth muscle cells. However, both kinases 1) phosphorylated regulator of G protein signaling 4 (RGS4) and induced its translocation from cytosol to plasma membrane, 2) enhanced ACh-stimulated association of RGS4 and G_q·GTP and intrinsic G_q·GTPase activity, and 3) inhibited ACh-stimulated PI hydrolysis. RGS4 phosphorylation and inhibition of PI hydrolysis were blocked by selective PKA and PKG inhibitors. Expression of RGS4(S52A), which lacks a PKA/PKG phosphorylation site, blocked the increase in GTPase activity and the decrease in PI hydrolysis induced by PKA and PKG. Blockade of PKA-dependent effects was only partial. Selective phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which contains a RGS domain, by PKA augmented ACh-stimulated GRK2:G_q·GTP association; both effects were blocked in cells expressing GRK2(S685A), which lacks a PKA phosphorylation site. Inhibition of PI hydrolysis induced by PKA was partly blocked in cells expressing GRK2(S685A) and completely blocked in cells coexpressing GRK2(S685A) and RGS4(S52A) or G_q(G188S), a G_q mutant that binds GRK2 but not RGS4. The results demonstrate that inhibition of PLC-1-dependent PI hydrolysis by PKA is mediated via stimulatory phosphorylation of RGS4 and GRK2, leading to rapid inactivation of G_q·GTP. PKG acts only via phosphorylation of RGS4. regulators of G protein signaling; G protein-coupled receptor kinase 2; phospholipase C; cAMP-dependent protein kinase; cGMP-dependent protein kinase     

Mitochondrial Ca2+-induced K+ influx increases respiration and enhances ROS production while maintaining membrane potential

We recently demonstrated a role for altered mitochondrial bioenergetics and reactive oxygen species (ROS) production in mitochondrial Ca²⁺-sensitive K⁺ (mtK_Ca) channel opening-induced preconditioning in isolated hearts. However, the underlying mitochondrial mechanism by which mtK_Ca channel opening causes ROS production to trigger preconditioning is unknown. We hypothesized that submaximal mitochondrial K⁺ influx causes ROS production as a result of enhanced electron flow at a fully charged membrane potential (_m). To test this hypothesis, we measured effects of NS-1619, a putative mtK_Ca channel opener, and valinomycin, a K⁺ ionophore, on mitochondrial respiration, _m, and ROS generation in guinea pig heart mitochondria. NS-1619 (30 µM) increased state 2 and 4 respiration by 5.2 ± 0.9 and 7.3 ± 0.9 nmol O₂·min^–1·mg protein^–1, respectively, with the NADH-linked substrate pyruvate and by 7.5 ± 1.4 and 11.6 ± 2.9 nmol O₂·min^–1·mg protein^–1, respectively, with the FADH₂-linked substrate succinate (+ rotenone); these effects were abolished by the mtK_Ca channel blocker paxilline. _m was not decreased by 10–30 µM NS-1619 with either substrate, but H₂O₂ release was increased by 44.8% (65.9 ± 2.7% by 30 µM NS-1619 vs. 21.1 ± 3.8% for time controls) with succinate + rotenone. In contrast, NS-1619 did not increase H₂O₂ release with pyruvate. Similar results were found for lower concentrations of valinomycin. The increase in ROS production in succinate + rotenone-supported mitochondria resulted from a fully maintained _m, despite increased respiration, a condition that is capable of allowing increased electron leak. We propose that mild matrix K⁺ influx during states 2 and 4 increases mitochondrial respiration while maintaining _m; this allows singlet electron uptake by O₂ and ROS generation. mitochondrial bioenergetics; heart mitochondria     

Characterization of G proteins involved in activation of nonselective cation channels and arachidonic acid release by norepinephrine/alpha1A-adrenergic receptors

We demonstrated recently that norepinephrine activates Ca²⁺-permeable nonselective cation channels (NSCCs) in Chinese hamster ovary cells stably expressing _1A-adrenergic receptors (CHO-_1A). Moreover, extracellular Ca²⁺ through NSCCs plays essential roles in norepinephrine-induced arachidonic acid release. The purpose of the present study was to identify the G proteins involved in the activation of NSCCs and arachidonic acid release by norepinephrine. For these purposes, we used U73122, an inhibitor of phospholipase C (PLC), and dominant negative mutants of G₁₂ and G₁₃ (G₁₂G228A and G₁₃G225A, respectively). U73122 failed to inhibit NSCCs activation by norepinephrine. The magnitudes of norepinephrine-induced extracellular Ca²⁺ influx in CHO-_1A microinjected with G₁₃G225A were smaller than those in CHO-_1A. In contrast, the magnitudes of norepinephrine-induced extracellular Ca²⁺ influx in CHO-_1A microinjected with G₁₂G228A were similar to those in CHO-_1A. In addition, neither a Rho-associated kinase (ROCK) inhibitor nor a phosphoinositide 3-kinase inhibitor affected norepinephrine-induced extracellular Ca²⁺ influx. G₁₃G225A, but not G₁₂G228A, also inhibited arachidonic acid release partially. These results demonstrate that 1) the G_q/PLC-pathway is not involved in NSCCs activation by norepinephrine, 2) G₁₃ couples with CHO-_1A and plays important roles for norepinephrine-induced NSCCs activation, 3) neither ROCK- nor PI3K-dependent cascade is involved in NSCCs activation, and 4) G₁₃ is involved in norepinephrine-induced arachidonic acid release in CHO-_1A. norepinephrine; _1A-adrenergic receptor; nonselective cation channel; G₁₃ protein; arachidonic acid release     

Calsequestrin targeting to sarcoplasmic reticulum of skeletal muscle fibers

Calsequestrin (CS) is the low-affinity, high-capacity calcium binding protein segregated to the lumen of terminal cisternae (TC) of the sarcoplasmic reticulum (SR). The physiological role of CS in controlling calcium release from the SR depends on both its intrinsic properties and its localization. The mechanisms of CS targeting were investigated in skeletal muscle fibers and C₂C₁₂ myotubes, a model of SR differentiation, with four deletion mutants of epitope (hemagglutinin, HA)-tagged CS: CS-HA24_NH2, CS-HA2D, CS-HA3D, and CS-HAHT, a double mutant of the NH₂ terminus and domain III. As judged by immunofluorescence of transfected skeletal muscle fibers, only the double CS-HA mutant showed a homogeneous distribution at the sarcomeric I band, i.e., it did not segregate to TC. As shown by subfractionation of microsomes derived from transfected skeletal muscles, CS-HAHT was largely associated to longitudinal SR whereas CS-HA was concentrated in TC. In C₂C₁₂ myotubes, as judged by immunofluorescence, not only CS-HAHT but also CS-HA3D and CS-HA2D were not sorted to developing SR. Condensation competence, a property referable to CS oligomerization, was monitored for the several CS-HA mutants in C₂C₁₂ myoblasts, and only CS-HA3D was found able to condense. Together, the results indicate that 1) there are at least two targeting sequences at the NH₂ terminus and domain III of CS, 2) SR-specific target and structural information is contained in these sequences, 3) heterologous interactions with junctional SR proteins are relevant for segregation, 4) homologous CS-CS interactions are involved in the overall targeting process, and 5) different targeting mechanisms prevail depending on the stage of SR differentiation. protein-protein interactions; oligomerization; intracellular sorting     

Laminin-alpha1 globular domains 3 and 4 induce heterotrimeric G protein binding to alpha-syntrophin's PDZ domain and alter intracellular Ca2+ in muscle

-Syntrophin is a component of the dystrophin glycoprotein complex (DGC). It is firmly attached to the dystrophin cytoskeleton via a unique COOH-terminal domain and is associated indirectly with -dystroglycan, which binds to extracellular matrix laminin. Syntrophin contains two pleckstrin homology (PH) domains and one PDZ domain. Because PH domains of other proteins are known to bind the -subunits of the heterotrimeric G proteins, whether this is also a property of syntrophin was investigated. Isolated syntrophin from rabbit skeletal muscle binds bovine brain G-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, - and -dystroglycan, or dystrophin precipitate a complex with G from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of G binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of G_s and G occurs and active G_s, measured as GTP-³⁵S bound, decreases. Because intracellular Ca²⁺ is elevated in Duchenne muscular dystrophy and G_s is known to activate the dihydropyridine receptor Ca²⁺ channel, whether laminin also altered intracellular Ca²⁺ was investigated. Laminin-1 decreases active (GTP-S-bound) G_s, and the Ca²⁺ channel is inhibited by laminin-1. The laminin ₁-chain globular domains 4 and 5 region, the region bound by DGC -dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to -dystroglycan inhibits G binding by syntrophin in C₂C₁₂ myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor. Duchenne muscular dystrophy; protein G -subunit; pleckstrin homology domain     

Activation of PLC-delta1 by Gi/o-coupled receptor agonists

The mechanism of phospholipase (PLC)- activation by G protein-coupled receptor agonists was examined in rabbit gastric smooth muscle. Ca²⁺ stimulated an eightfold increase in PLC-1 activity in permeabilized muscle cells. Treatment of dispersed or cultured muscle cells with three G_i/o-coupled receptor agonists (somatostatin, -opioid agonist [D-Pen²,D-Pen⁵]enkephalin, and A₁ agonist cyclopentyl adenosine) caused delayed increase in phosphoinositide (PI) hydrolysis (8- to 10-fold) that was strongly inhibited by overexpression of dominant-negative PLC-1(E341R/D343R; 65–76%) or constitutively active RhoA(G14V). The response coincided with capacitative Ca²⁺ influx and was not observed in the absence of extracellular Ca²⁺, but was partly inhibited by nifedipine (16–30%) and strongly inhibited by SKF-96365, a blocker of store-operated Ca²⁺ channels. Treatment of the cells with a G_q/13-coupled receptor agonist, CCK-8, caused only transient, PLC-1-mediated PI hydrolysis. Unlike G_i/o-coupled receptor agonists, CCK-8 activated RhoA and stimulated RhoA:PLC-1 association. Inhibition of RhoA activity with C3 exoenzyme or by overexpression of dominant-negative RhoA(T19N) or G₁₃ minigene unmasked a delayed increase in PI hydrolysis that was strongly inhibited by coexpression of PLC-1(E341R/D343R) or by SKF-96365. Agonist-independent capacitative Ca²⁺ influx induced by thapsigargin stimulated PI hydrolysis (8-fold), which was partly inhibited by nifedipine (25%) and strongly inhibited by SKF-96365 (75%) and in cells expressing PLC-1(E341R/D343R). Agonist-independent Ca²⁺ release or Ca²⁺ influx via voltage-gated Ca²⁺ channels stimulated only moderate PI hydrolysis (2- to 3-fold), which was abolished by PLC-1 antibody or nifedipine. We conclude that PLC-1 is activated by G_i/o-coupled receptor agonists that do not activate RhoA. The activation is preferentially mediated by Ca²⁺ influx via store-operated Ca²⁺ channels. phospholipase C; G protein     

ATP stimulates Na+-glucose cotransporter activity via cAMP and p38 MAPK in renal proximal tubule cells

Extracellular ATP plays an important role in the regulation of renal function. However, the effect of ATP on the Na⁺-glucose cotransporters (SGLTs) has not been elucidated in proximal tubule cells (PTCs). Therefore, this study was performed to examine the action of ATP on SGLTs and their related signal pathways in primary cultured rabbit renal PTCs. ATP increased [¹⁴C]--methyl-D-glucopyranoside (-MG) uptake in a time-dependent (>1 h) and dose-dependent (>10^–6 M) manner. ATP stimulated -MG uptake by increasing in V_max without affecting K_m. ATP-induced increase of -MG uptake was correlated with the increase in both SGLT1 and SGLT2 protein expression levels. ATP-induced stimulation of -MG uptake was blocked by suramin (nonspecific P2 receptor antagonist), RB-2 (P2Y receptor antagonist), and MRS-2179 (P2Y₁ receptor antagonist), suggesting a role for the P2Y receptor. ATP-induced stimulation of -MG uptake was blocked by pertussis toxin (PTX, a G_i protein inhibitor), SQ-22536 (an adenylate cyclase inhibitor), and PKA inhibitor amide 14-22 (PKI). ATP also increased cAMP formation, which was blocked by PTX and RB-2. However, pretreatment of adenosine deaminase did not block ATP-induced cAMP formation. In addition, ATP-induced stimulation of -MG uptake was blocked by SB-203580 (p38 MAPK inhibitor), but not by PD-98059 (p44/42 MAPK inhibitor) or SP-600125 (JNK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK. In conclusion, ATP increases -MG uptake via cAMP and p38 MAPK in renal PTCs. adenosine 5'-triphosphate; mitogen-activated protein kinase     

iNOS initiates and sustains metabolic arrest in hypoxic lung adenocarcinoma cells: mechanism of cell survival in solid tumor core

Nitric oxide (NO) modulates cellular metabolism by competitively inhibiting the reduction of O₂ at respiratory complex IV. The aim of this study was to determine whether this effect could enhance cell survival in the hypoxic solid tumor core by inducing a state of metabolic arrest in cancer cells. Mitochondria from human alveolar type II-like adenocarcinoma (A549) cells showed a fourfold increase in NO-sensitive 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) fluorescence and sixfold increase in Ca²⁺-insensitive NO synthase (NOS) activity during equilibration from PO₂s of 10023 mmHg, which was abolished by N-nitro-L-arginine methyl ester-HCl (L-NAME) and the inducible NOS (iNOS) inhibitor, N⁶-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL). Similarly, cytosolic and compartmented DAF-FM fluorescence increased in intact cells during a transition between ambient PO₂ and 23 mmHg and was abolished by transfection with iNOS antisense oligonucleotides (AS-ODN). In parallel, mitochondrial membrane potential (_m), measured using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), decreased to a lower steady state in hypoxia without change in glycolytic rate, adenylate energy charge, or cell viability. However, L-NAME or iNOS AS-ODN treatment maintained _m at normoxic levels irrespective of hypoxia and caused a marked activation of glycolysis, destabilization energy charge, and cell death. Comparison with other cancer-derived (H441) or native tissue-derived (human bronchial epithelial; alveolar type II) lung epithelial cells revealed that the hypoxic suppression of _m was common to cells that expressed iNOS. The controlled dissipation of _m, absence of an overt glycolytic activation, and conservation of viability suggest that A549 cells enter a state of metabolic suppression in hypoxia, which inherently depends on the activation of iNOS as PO₂ falls. cancer; oxygen conformation; mitochondrial nitric oxide synthase; mitochondrial metabolism     

Cd2+-induced swelling-contraction dynamics in isolated kidney cortex mitochondria: role of Ca2+ uniporter, K+ cycling, and protonmotive force

The nephrotoxic metal Cd²⁺ causes mitochondrial damage and apoptosis of kidney proximal tubule cells. A K⁺ cycle involving a K⁺ uniporter and a K⁺/H⁺ exchanger in the inner mitochondrial membrane (IMM) is thought to contribute to the maintenance of the structural and functional integrity of mitochondria. In the present study, we have investigated the effect of Cd²⁺ on K⁺ cycling in rat kidney cortex mitochondria. Cd²⁺ (EC₅₀ 19 µM) induced swelling of nonenergized mitochondria suspended in isotonic salt solutions according to the sequence KCl = NaCl > LiCl >> choline chloride. Cd²⁺-induced swelling of energized mitochondria had a similar EC₅₀ value and showed the same cation dependence but was followed by a spontaneous contraction. Mitochondrial Ca²⁺ uniporter (MCU) blockers, but not permeability transition pore inhibitors, abolished swelling, suggesting the need for Cd²⁺ influx through the MCU for swelling to occur. Complete loss of mitochondrial membrane potential (_m) induced by K⁺ influx did not prevent contraction, but addition of the K⁺/H⁺ exchanger blocker, quinine (1 mM), or the electroneutral protonophore nigericin (0.4 µM), abolished contraction, suggesting the mitochondrial pH gradient (pH_m) driving contraction. Accordingly, a quinine-sensitive partial dissipation of pH_m was coincident with the swelling-contraction phase. The data indicate that Cd²⁺ enters the matrix through the MCU to activate a K⁺ cycle. Initial K⁺ load via a Cd²⁺-activated K⁺ uniporter in the IMM causes osmotic swelling and breakdown of _m and triggers quinine-sensitive K⁺/H⁺ exchange and contraction. Thus Cd²⁺-induced activation of a K⁺ cycle contributes to the dissipation of the mitochondrial protonmotive force. bongkrekic acid; cyclosporin A; lanthanum; Ru360; ruthenium red     

CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to increase plasma membrane localization and 86Rb+ uptake

The carboxy terminus (CT) of the colonic H⁺-K⁺-ATPase is required for stable assembly with the -subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HK₂, we selected 84 amino acids in the CT of the -subunit of mouse colonic H⁺-K⁺-ATPase (CT-HK₂) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HK₂ with CD63, recombinant CT-HK₂ and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HK₂ protein (but not CT-HK₁) coprecipitated with CD63, confirming stable assembly of HK₂ with CD63. In HEK-293 transfected with HK₂ plus ₁-Na⁺-K⁺-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HK₂/NK₁ and ⁸⁶Rb⁺ uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HK₂-NK₁ complex in the cell membrane. protein assembly; cell surface localization     

Functional analysis of the R1086H malignant hyperthermia mutation in the DHPR reveals an unexpected influence of the III-IV loop on skeletal muscle EC coupling

Malignant hyperthermia (MH) is an inherited pharmacogenetic disorder caused by mutations in the skeletal muscle ryanodine receptor (RyR1) and the dihydropyridine receptor (DHPR) _1S-subunit. We characterized the effects of an MH mutation in the DHPR cytoplasmic III-IV loop of _1S (R1086H) on DHPR-RyR1 coupling after reconstitution in dysgenic (_1S null) myotubes. Compared with wild-type _1S, caffeine-activated Ca²⁺ release occurred at approximately fivefold lower concentrations in nonexpressing and R1086H-expressing myotubes. Although maximal voltage-gated Ca²⁺ release was similar in _1S- and R1086H-expressing myotubes, the voltage dependence of Ca²⁺ release was shifted 5 mV to more negative potentials in R1086H-expressing myotubes. Our results demonstrate that _1S functions as a negative allosteric modulator of release channel activation by caffeine/voltage and that the R1086H MH mutation in the intracellular III-IV linker disrupts this negative regulatory influence. Moreover, a low caffeine concentration (2 mM) caused a similar shift in voltage dependence of Ca²⁺ release in _1S- and R1086H-expressing myotubes. Compared with _1S-expressing myotubes, maximal L channel conductance (G_max) was reduced in R1086H-expressing myotubes (_1S 130 ± 10.2, R1086H 88 ± 6.8 nS/nF; P < 0.05). The decrease in G_max did not result from a change in retrograde coupling with RyR1 as maximal conductance-charge movement ratio (G_max/Q_max) was similar in _1S- and R1086H-expressing myotubes and a similar decrease in G_max was observed for an analogous mutation engineered into the cardiac L channel (R1217H). In addition, both R1086H and R1217H DHPRs targeted normally and colocalized with RyR1 in sarcoplasmic reticulum (SR)-sarcolemmal junctions. These results indicate that the R1086H MH mutation in _1S enhances RyR1 sensitivity to activation by both endogenous (voltage sensor) and exogenous (caffeine) activators. excitation-contraction coupling; calcium channel; muscle disease     

Unique temperature-activated neurons from pit viper thermosensors

Rattlesnakes, copperheads, and other pit vipers have highly sensitive heat detectors known as pit organs, which are used to sense and strike at prey. However, it is not currently known how temperature change triggers cellular and molecular events that activate neurons supplying the pit organ. We dissociated and cultured neurons from the trigeminal ganglia (TG) innervating the pit organs of the Western Diamondback rattlesnake (Crotalus atrox) and the copperhead (Agkistrodon contortix) to investigate electrophysiological responses to thermal stimuli. Whole cell voltage-clamp recordings indicated that 75% of the TG neurons from C. atrox and 74% of the TG neurons from A. contortix showed a unique temperature-activated inward current (I_T). We also found an I_T-like current in 15% of TG neurons from the common garter snake, a species that does not have a specialized heat-sensing organ. A steep rise in the current-temperature relationship of I_T started just below 18°C, and cooling temperature-responsive TG neurons from 20°C resulted in an outward current, suggesting that I_T is on at relatively low temperatures. Ion substitution and Ca²⁺ imaging experiments indicated that I_T is primarily a monovalent cation current. I_T was not sensitive to capsaicin or amiloride, suggesting that the current did not show similar pharmacology to other mammalian heat-sensitive membrane proteins. Our findings indicate that a novel temperature-sensitive conductance with unique ion permeability and low-temperature threshold is expressed in TG neurons and may be involved in highly sensitive heat detection in snakes. snake; thermosensory; trigeminal; ion conductance     

Coupling of GABAB receptor GABAB2 subunit to G proteins: evidence from Xenopus oocyte and baby hamster kidney cell expression system

Coupling of functional GABA_B receptors (GABA_BR) to G proteins was investigated with an expression system of baby hamster kidney (BHK) cells and Xenopus oocytes. Fluorescence resonance energy transfer (FRET) analysis of BHK cells coexpressing GABA_B1a receptor (GB_1aR) fused to Cerulean, a brighter variant of cyan fluorescent protein, and GABA_B2 receptor (GB₂R) fused to Venus, a brighter variant of yellow fluorescent protein, revealed that GB_1aR-Cerulean and GB₂R-Venus form a heterodimer. The GABA_BR agonists baclofen and 3-aminopropylphosphonic acid (3-APPA) elicited inward-rectifying K⁺ currents in a concentration-dependent manner in oocytes expressing GB_1aR and GB₂R, or GB_1aR-Cerulean and GB₂R-Venus, together with G protein-activated inward-rectifying K⁺ channels (GIRKs), but not in oocytes expressing GB_1aR alone or GB₂R alone together with GIRKs. Oocytes coexpressing GB_1aR + G_i2-fused GB₂R (GB₂R-G_i2) caused faster K⁺ currents in response to baclofen. Furthermore, oocytes coexpressing GB_1aR + GB₂R fused to G_qi5 (a chimeric G_q protein that activates PLC pathways) caused PLC-mediated Ca²⁺-activated Cl^– currents in response to baclofen. In contrast, these responses to baclofen were not observed in oocytes coexpressing GB_1aR-G_i2 or GB_1aR-G_qi5 together with GB₂R. BHK cells and Xenopus oocytes coexpressing GB_1aR-Cerulean + a triplet tandem of GB₂R-Venus-G_qi5 caused FRET and Ca²⁺-activated Cl^– currents, respectively, with a similar potency in BHK cells coexpressing GB_1aR-Cerulean + GB₂R-Venus and in oocytes coexpressing GB_1aR + GB₂R-G_qi5. Our results indicate that functional GABA_BR forms a heterodimer composed of GB₁R and GB₂R and that the signal transducing G proteins are directly coupled to GB₂R but not to GB₁R. fluorescence resonance energy transfer     

Dominant-negative effects of human P/Q-type Ca2+ channel mutations associated with episodic ataxia type 2

Episodic ataxia type 2 (EA2) is an inherited autosomal dominant disorder related to cerebellar dysfunction and is associated with mutations in the pore-forming _1A-subunits of human P/Q-type Ca²⁺ channels (Cav2.1 channels). The majority of EA2 mutations result in significant loss-of-function phenotypes. Whether EA2 mutants may display dominant-negative effects in human, however, remains controversial. To address this issue, five EA2 mutants in the long isoform of human _1A-subunits were expressed in Xenopus oocytes to explore their potential dominant-negative effects. Upon coexpressing the cRNA of _1A-WT with each _1A-mutant in molar ratios ranging from 1:1 to 1:10, the amplitude of Ba²⁺ currents through wild-type (WT)-Cav2.1 channels decreased significantly as the relative molar ratio of _1A-mutants increased, suggesting the presence of an _1A-mutant-specific suppression effect. When we coexpressed _1A-WT with proteins not known to interact with Cav2.1 channels, we observed no significant suppression effects. Furthermore, increasing the amount of auxiliary subunits resulted in partial reversal of the suppression effects in nonsense but not missense EA2 mutants. On the other hand, when we repeated the same coinjection experiments of _1A-WT and mutant using a splice variant of _1A-subunit that contained a considerably shorter COOH terminus (i.e., the short isoform), no significant dominant-negative effects were noted until we enhanced the relative molar ratio to 1:10. Altogether, these results indicate that for human WT-Cav2.1 channels comprising the long-_1A-subunit isoform, both missense and nonsense EA2 mutants indeed display prominent dominant-negative effects. channelopathy; voltage clamp; Xenopus oocytes; cerebellum; splice variants     

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/G_q-coupled P2Y₂ receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of - and -actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of - or -actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca²⁺-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis. lung; mucus; exocytosis     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A