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Transplantation studies in mice and rats have shown that human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts (1-3), but two critical issues related to their electrophysiological behaviour [...]

L-type voltage-gated [Ca.sup.2+] channels (LVGCs) are functionally downregulated in arterial smooth muscle (SM) cells (ASMCs) of mice with SM-specific knockout of [Na.sup.+]/[Ca2.sup.+] exchanger type-1 ([NCX1.sup.SM-/-]) (32). Here, using activators and inhibitors of protein kinase C (PKC), we explore the regulation of these channels by a PKC-dependent mechanism. In both wild-type (WT) and [NCX1.sup.SM-/-] myocytes, the PKC activator phorbol 12,13-dibutyrate (PDBu) increases LVGC conductance, decreases channel closing rate, and shifts the voltage dependence of channel opening to more negative potentials. Three different PKC inhibitors, bisindolylmaleimide, Ro-31-8220, and PKC 19-31, all decrease LVGC currents in WT myocytes and prevent the PDBu-induced increase in LVGC current. Dialysis of WT ASMCs with activated PKC increases LVGC current and decreases channel closing rate. These results demonstrate that PKC activates LVGCs in ASMCs. The phosphatase inhibitor calyculin A increases LVGC conductance by over 50%, indicating that the level of LVGC activation is a balance between phosphatase and PKC activities. PDBu causes a larger increase in LVGC conductance and a larger shift in voltage dependence in [NCX1.sup.SM-/-] myocytes than in WT myocytes. The inhibition of PKC with PKC 19-31 decreased LVGC conductance by 65% in WT myocytes but by only 37% in [NCX1.sup.SM-/-] myocytes. These results suggest that LVGCs are in a state of low PKC-induced phosphorylation in [NCX1.sup.SM-/-] myocytes. We conclude that in [NCX1.sup.SM-/-] myocytes, reduced [Ca2.sup.+] entry via NCX1 lowers cytosolic [Ca2.sup.+], thereby reducing PKC activation that lowers LVGC activation. sodium/calcium exchanger type-1 knockdown; calcium channels doi: 10.1152/ajpheart.00965.2009.

Mice with smooth muscle (SM)-specific knockout of [Na.sup.+]/[Ca2.sup.+] exchanger type-1 ([NCX1.sup.SM-/-]) and the NCX inhibitor, SEA0400, were used to study the physiological role of NCX1 in mouse mesenteric arteries. NCX1 protein expression was greatly reduced in arteries from [NCX1.sup.SM-/-] mice generated with Cre recombinase. Mean blood pressure (BP) was 6-10 mmHg lower in [NCX1.sup.SM-/-] mice than in wild-type (WT) controls. Vasoconstriction was studied in isolated, pressurized mesenteric small arteries from WT and [NCX1.sup.SM-/-] mice and in heterozygotes with a global null mutation ([NCX1.sup.Fx-/-]). Reduced NCX1 activity was manifested by a marked attenuation of responses to low extracellular [Na.sup.+] concentration, nanomolar ouabain, and SEA0400. Myogenic tone (MT, 70 mmHg) was reduced by ~15% in [NCX1.sup.SM-/-] arteries and, to a similar extent, by SEA0400 in WT arteries. MT was normal in arteries from [NCX1.sup.Fx/-] mice, which had normal BP. Vasoconstrictions to phenylephrine and elevated extracellular [K.sup.+] concentration were significantly reduced in [NCX1.sup.SM-/-] arteries. Because a high extracellular [K.sup.+] concentration-induced vasoconstriction involves the activation of L-type voltage-gated [Ca2.sup.+] channels (LVGCs), we measured LVGC-mediated currents and [Ca2.sup.+] sparklets in isolated mesenteric artery myocytes. Both the currents and the sparklets were significantly reduced in [NCX1.sup.sM-/-] (vs. WT or [NCX1.sup.Fx/-]) myocytes, but the voltage-dependent inactivation of LVGCs was not augmented. An acute application of SEA0400 in WT myocytes had no effect on LVGC current. The LVGC agonist, Bay K 8644, eliminated the differences in LVGC currents and [Ca2.sup.+] sparklets between [NCXI.sup.SM-/-] and control myocytes, suggesting that LVGC expression was normal in [NCX1.sup.SM-/-] myocytes. Bay K 8644 did not, however, eliminate the difference in myogenic constriction between WT and [NCX1.sup.SM-/-] arteries. We conclude that, under physiological conditions, NCX1-mediated [Ca2.sup.+] entry contributes significantly to the maintenance of MT. In [NCX1.sup.SM-/-] mouse artery myocytes, the reduced [Ca.sup.2+] entry via NCX1 may lower cytosolic [Ca2.sup.+] concentration and thereby reduce MT and BP. The reduced LVGC activity may be the consequence of a low cytosolic [Ca2.sup.+] concentration. mesenteric arteries; myogenic tone; patch clamp; calcium sparklers; Bay K 8644 doi: 10.1152/ajpheart.00964.2009.

[Ca.sup.+]-activated [Cl.sup.-] channel (CLCA) proteins are encoded by a family of highly related and clustered genes in mammals that are markedly upregulated in inflammation and have been shown to affect chloride transport. Here we describe the cellular processing and regulatory sequences underlying murine (m) CLCA4 proteins. The 125-kDa mCLCA4 gene product is cleaved to 90- and 40-kDa fragments, and the N[H.sub.2]- and COOH-terminal fragments are secreted, where they are found in cell media and associated with the plasma membrane. The 125-kDa full-length protein is only found in the endoplasmic reticulum (ER), and specific luminal diarginine retention and dileucine forward trafficking signals contained within the CLCA4 sequence regulate export from the ER and proteolytic processing. Mutation of the dileucine luminal sequences resulted in ER trapping of the immaturely glycosylated 125-kDa peptide, indicating that proteolytic cleavage occurs following recognition of the trafficking motifs. Moreover, the mutated dileucine and diarginine signal sequences directed processing of a secreted form of enhanced green fluorescent protein in a manner consistent with the effects on mCLCA4. chloride channel; asthma; protein trafficking; epithelium; endoplasmic reticulum retention/trafficking signal

Author(s): Wilhelm Roell [1, 2, 9]; Thorsten Lewalter [3, 9]; Philipp Sasse [1, 9]; Yvonne N. Tallini [6]; Bum-Rak Choi [7]; Martin Breitbach [1]; Robert Doran [6]; Ulrich M. Becher [...]

Genetically encoded sensor proteins provide unique opportunities to advance the understanding of complex cellular interactions in physiologically relevant contexts; however, previously described sensors have proved to be of limited use to report cell signaling in vivo in mammals. Here, we describe an improved [Ca.sup.2+] sensor, GCaMP2, its inducible expression in the mouse heart, and its use to examine signaling in heart cells in vivo. The high brightness and stability of GCaMP2 enable the measurement of myocyte [Ca.sup.2+] transients in all regions of the beating mouse heart and prolonged pacing and mapping studies in isolated, perfused hearts. Transgene expression is efficiently temporally regulated in cardiomyocyte GCaMP2 mice, allowing recording of in vivo signals 4 weeks after transgene induction. High-resolution imaging of [Ca.sup.2+] waves in GCaMP2-expressing embryos revealed key aspects of electrical conduction in the preseptated heart. At embryonic day (e.d.) 10.5, atrial and ventricular conduction occur rapidly, consistent with the early formation of specialized conduction pathways. However, conduction is markedly slowed through the atrioventricular canal in the e.d. 10.5 heart, forming the basis for an effective atrioventricular delay before development of the AV node, as rapid ventricular activation occurs after activation of the distal AV canal tissue. Consistent with the elimination of the inner AV canal muscle layer at e.d. 13.5, atrioventricular conduction through the canal was abolished at this stage. These studies demonstrate that GCaMP2 will have broad utility in the dissection of numerous complex cellular interactions in mammals, in vivo. atrioventricular node | [Ca.sup.2+] imaging | genetic sensor | heart development | fluorescent [Ca.sup.2+] sensor

[Ca.sup.2+]-induced [Ca.sup.2+] release (CICR) from the sarcoplasmic reticulum (SR) occurs in smooth muscle as spontaneous SR [Ca.sup.2+] release or [Ca.sup.2+] sparks and, in some spiking tissues, as [Ca.sup.2+] release that is triggered by the activation of sarcolemmal [Ca.sup.2+] channels. Both processes display spatial localization in that release occurs at a higher frequency at specific subcellular regions. We have used two-photon flash photolysis (TPFP) of caged [Ca.sup.2+] (DMNP-EDTA) in Fluo-4-loaded urinary bladder smooth muscle cells to determine the extent to which spatially localized increases in [Ca.sup.2+] activate SR release and to further understand the molecular and biophysical processes underlying CICR. TPFP resulted in localized [Ca.sup.2+] release in the form of [Ca.sup.2+] sparks and [Ca.sup.2+] waves that were distinguishable from increases in [Ca.sup.2+] associated with [Ca.sup.2+] uncaging, unequivocally demonstrating that [Ca.sup.2+] release occurs subsequent to a localized rise in [[[Ca.sup.2+]].sub.i]. TPFP-triggered [Ca.sup.2+] release was not constrained to a few discharge regions but could be activated at all areas of the cell, with release usually occurring at or within several microns of the site of photolysis. As expected, the process of CICR was dominated by ryanodine receptor (RYR) activity, as ryanodine abolished individual [Ca.sup.2+] sparks and evoked release with different threshold and kinetics in FKBP12.6-null cells. However, TPFP CICR was not completely inhibited by ryanodine; [Ca.sup.2+] release with distinct kinetic features occurred with a higher TPFP threshold in the presence of ryanodine. This high threshold release was blocked by xestospongin C, and the pharmacological sensitivity and kinetics were consistent with CICR release at high local [[[Ca.sup.2+]].sub.i] through inositol trisphosphate ([InsP.sub.3]) receptors ([InsP.sub.3]Rs). We conclude that CICR activated by localized [Ca.sup.2+] release bears essential similarities to those observed by the activation of [I.sub.Ca] (i.e., major dependence on the type 2 RYR), that the release is not spatially constrained to a few specific subcellular regions, and that [Ca.sup.2+] release through [InsP.sub.3]R can occur at high local [[[Ca.sup.2+]].sub.i].

In this study we examined the expression of RyR subtypes and the role of RyRs in neurotransmitterand hypoxia-induced [Ca.sup.2+] release and contraction in pulmonary artery smooth muscle cells (PASMCs). Under perforated patch clamp conditions, maximal activation of RyRs with caffeine or inositol triphosphate receptors ([IP.sub.3]Rs) with noradrenaline induced eqnivalent increases in [[Ca.sup.2+]]i and [Ca.sup.2+-] activated [C1.sup.-] currents in freshly isolated rat PASMCs. Following maximal [IP.sub.3]-induced Ca9+ release, neither caffeine nor chloro-m-cresol induced a response, whereas prior application of caffeine or chloro-m-cresol blocked [IP.sub.3]-induced [Ca.sup.2+] release. In cultured human PASMCs, which lack functional expression of RyRs, caffeine failed to affect ATP-induced increases in [[Ca.sup.2+]]i in the presence and absence of extracellular [Cd.sup.2+]. The RyR antagonists ruthenium red, ryanodine, tetracaine, and dantrolene greatly inhibited submaximal noradrenaline- and hypoxia-induced [Ca.sup.2+] release and contraction in freshly isolated rat PASMCs, but did not affect ATP-induced [Ca.sup.2-] release in cultured human PASMCs. Real-time quantitative RT-PCR and immunofluoreseence staining indicated similar expression of all three RyR subtypes (RyR1, RyR2, and RyR3) in freshly isolated rat PASMCs. In freshly isolated PASMCs from RyR3 knockout ([RyR3.sup.-/-]) mice, hypoxia-induced, but not submaximal noradrenaline-induced, [Ca.sup.2+] release and contraction were significantly reduced. Ruthenium red and tetracaine can further inhibit hypoxic increase in [[[Ca.sup.2+]].sub.i] in [RyR3.sup. -/-] mouse PASMCs. Collectively, our data suggest that (a) RyRs play an important role in submaximal noradrenaline- and hypoxia-induced [Ca.sup.2+] release and contraction; (b) all three subtype RyRs are expressed; and (c) RyR3 gene knockout significantly inhibits hypoxia-, but not submaximal noradrenaline-induced [Ca.sup.2+] and contractile responses in PASMCs. KEY WORDS: ryanodine receptor * inositol triphosphate receptor * hypoxia * neurotransmitter * pulmonary artery smooth muscle cell

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 [Ca.sup.2+] release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary [Ca.sup.2+] release events ([Ca.sup.2+] sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in [Ca.sup.2+] release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), [Ca.sup.2+] sparks, [Ca.sup.2+]- induced [Ca.sup.2+] release, and [Ca.sup.2+] waves in smooth muscle cells freshly isolated from wild-type, [FKBP12.6.sup.-/-], and [RYR3.sup.-/-] mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous [Ca.sup.2+] sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous [Ca.sup.2+] sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. [Ca.sup.2+]-induced [Ca.sup.2+] release was similarly augmented in [FKBP12.6.sup.-/-], but not in RYR3 null cells relative to wild-type. Finally, [Ca.sup.2+] wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal [Ca.sup.2+] wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked [Ca.sup.2+] sparks in smooth muscle, and the finding of normal [Ca.sup.2+] sparks and CICR in RYR3 null mice, indicate that [Ca.sup.2+] release through RYR2 molecules contributes to the formation of spontaneous and evoked [Ca.sup.2+] sparks, and associated STOCs, in smooth muscle. KEY WORDS: [Ca.sup.2+]-induced [Ca.sup.2+] release * ryanodine receptor * FKBP12.6 * RYR3 * knockout mouse

Intracellular [Ca.sup.2+] release through ryanodine receptors (RyRs) plays important roles in smooth muscle excitation-contraction coupling, but the underlying regulatory mechanisms are poorly understood. Here we show that FK506 binding protein of 12.6 kDa (FKBP12.6) associates with and regulates type 2 RyRs (RyR2) in tracheal smooth muscle. FKBP12.6 binds to RyR2 but not other RyR or inositol 1,4,5-trisphosphate receptors, and FKBP12, known to bind to and modulate skeletal RyRs, does not associate with RyR2. When dialyzed into tracheal myocytes, cyclic ADP-ribose (cADPR) alters spontaneous [Ca.sup.2+] release at lower concentrations and produces macroscopic [Ca.sup.2+] release at higher concentrations; neurotransmitter-evoked [Ca.sup.2+] release is also augmented by cADPR. These actions are mediated through FKBP12.6 because they are inhibited by molar excess of recombinant FKBP12.6 and are not observed in myocytes from FKBP12.6-knockout mice. We also report that force development in FKBP12.6-null mice, observed as a decrease in the concentration/tension relationship of isolated trachealis segments, is impaired. Taken together, these findings point to an important role of the FKBP12.6/RyR2 complex in stochastic (spontaneous) and receptor-mediated [Ca.sup.2+] release in smooth muscle. FK506 binding protein 12.6; ryanodine receptor type 2; calcium sparks; calcium-activated chloride currents

Ion channels subserve diverse cellular functions. Reactive oxygen and nitrogen species modulate ion channel function by a number of mechanisms including 1) transcriptional regulation of gene expression, 2) posttranslational modifications of channel proteins, i.e. nitrosylation, nitration, and oxidation of key amino acid residues, 3) by altering the gain in other signaling pathways that may in turn lead to changes in channel activity or channel gene expression, and 4) by modulating trafficking or turnover of channel proteins, as typified by oxygen radical activation of NF-kB, with subsequent changes in proteasomal degradation of channel degradation. Regardless of the mechanism, as was discussed in a symposium at the 2003 Experimental Biology Meeting in San Diego, CA, changes in the cellular level of reactive oxygen and nitrogen species can have profound effects on the activity of ion channels and cellular function. calcium channels; potassium channels; sodium channels; chloride channels; nitric oxide; cystic fibrosis transmembrane conductance regulator

Author(s): Hong-Bo Xin [1, 3]; Takaaki Senbonmatsu [2]; Dong-Sheng Cheng [1]; Yong-Xiao Wang [3]; Julio A. Copello [1]; Guang-Ju Ji [3]; Mei Lin Collier [4]; Ke-Yu Deng [3]; Loice H. [...]