The role of Ca²⁺ in PIP₂ and palmitoyl CoA regulation of TRPV1 currents.

<p>(A) Representative whole-cell recordings (n = 7) and (B) inside-out recording (n = 5) of currents elicited by 1 µM capsaicin in the absence of Ca²⁺. (C) Grouped data of the effect of Ca²⁺ on the desensitization of TRPV1 currents. (D) Representative inside-out recordings and histograms of the effect of 1 µM palmitoyl CoA (n = 7), 25 µM PIP₂ (n = 4) or 1 µM palmitoyl CoA combined with 25 µM PIP₂ (n = 9) on currents elicited by 1 µM capsaicin (n = 9) in the absence of Ca²⁺. (E) Representative inside-out recordings and grouped data of the application of 1 µM capsaicin (n = 6), 1 µM palmitoyl CoA (n = 8) or 25 µM PIP₂ (n = 8) in the presence of 2 mM Ca²⁺. (F) Representative inside-out recordings and grouped data of the effect of 1 µM capsaicin (n = 12), 1 µM palmitoyl CoA (n = 11) or 25 µM PIP₂ (n = 10) in the presence of 2 mMCa²⁺ and 2 µM U73122. *P<0.05. Dashed line denotes zero current level.</p

The effect of palmitoyl CoA on TRPV1 currents.

<p>(A) Representative whole-cell recordings elicited by 1 µM capsaicin (n = 5) in the presence of (B) 25 µM PIP₂ (n = 12) or (C) 1 µM palmitoyl CoA (n = 9). (D) Representative whole-cell recordings elicited by acidic pH (pH = 5.5, n = 5) in the presence of (E) 1 µM palmitoyl CoA (n = 5). (F) Grouped data of the effects of PIP₂ and palmitoyl CoA on TRPV1 currents. *P<0.05. Dashed line denotes zero current level.</p

Chain-length, saturation and voltage-dependent effects of LC-CoA modulation of TRPV1 currents.

<p>(A) Representative inside-out recordings of the application various LC-CoAs (0.1 µM) in the presence, or (B) absence of 1 µM capsaicin. (C) Grouped data of the effect of 0.1 µM LC-CoAs in the presence or absence of 1 µM capsaicin. (D) I–V plot of currents elicited by 1 µM capsaicin in the presence of increasing concentration of palmitoyl CoA. (E) Concentration-effect curve of palmitoyl CoA modulation of currents elicited by 1 µM capsaicin. *P<0.05.</p

Kinetic parameters of WT TRPV1 and R702A mutant currents in the presence and absence of palmitoyl CoA.

<p>Currents were elicited by pH5.5. * Significantly different from values in the WT control (first pulse) (P<0.05). ** Significantly different from values in the WT control (second pulse) (P<0.05).</p

The role of K711 in LC-CoA modulation of TRPV1 channel function.

<p>(A) Amino acid sequence alignment of TRPV1. (B) Representative current trace of WT TRPV1 during repeated exposure to 1 µM capsaicin. (C) Representative current traces of the K711A TRPV1 mutant in the presence or absence of 1 µM palmitoyl CoA following repeated exposure to 1 µM capsaicin. (B,C) inset: Overlay of WT and K711A current traces normalized to maximum current illustrate the similar kinetics. (D) Grouped data of the effect of the K711A mutation on TRPV1 channel kinetics in response to 1 µM capsaicin. (E) Grouped data of the effect of 1 µM palmitoyl CoA on WT and K711A TRPV1 channel kinetics in response 1 µM capsaicin. *P<0.05, **P<0.01. Dashed line denotes zero current level.</p

The role of R702 in LC-CoA modulation of TRPV1 channel function.

<p>(A) Representative current trace of WT TRPV1 during repeated exposure to 1 µM capsaicin. (B) Representative current traces of the R702A TRPV1 mutant in the presence or absence of 1 µM palmitoyl CoA following repeated exposure to acidic activating solution of pH 5.5. (C) Grouped data of the effect of the R702A mutation on TRPV1 channel kinetics in response to acidic solution of pH 5.5. (D) Grouped data of the effect of 1 µM palmitoyl CoA on WT and R702A TRPV1 channel kinetics in response to the pH 5.5 solution. *P<0.05, **P<0.01. Dashed line denotes zero current level.</p

The effect of LC-CoAs on [Ca²⁺]_i.

<p>(A) Representative intracellular paired pulse Ca²⁺ recordings and grouped data of the effect of intracellular LC-CoAs on capsaicin-elicited (1 µM) Ca²⁺ levels in tsA201 cells expressing recombinant TRPV1 channel following repeated exposure to capsaicin, Ad-Scramble (n = 5) and Ad-ACSL-1 (n = 6) or (B) Jurkat 6.1 T-cells expressing endogenous channels, Ad-Scramble (n = 8) and Ad-ACSL-1 (n = 9). (C) Representative intracellular Ca²⁺ recordings and grouped data of the effect of intracellular LC-CoAs on Jurkat 6.1 T-cells, Ad-Scramble (n = 8) and Ad-ACSL-1 (n = 9) following application of 20 µg/ml PHA. Grouped data showing the effect of 1 µM capsazepine on intracellular calcium levels in Jurkat 6.1 T-cells, Ad-ACSL-1 (n = 9) and Ad-ACSL-1+CPZ (n = 10) following application of 20 µg/ml PHA. *P<0.05, **P<0.01.</p

Kinetic parameters of WT TRPV1 and K711A mutant currents in the presence and absence of palmitoyl CoA.

<p>Currents were induced by 1 mmol/l capsaicin.</p><p>* Significantly different from values in the WT control (first pulse) (P<0.05).</p><p>** Significantly different from values in the WT control (second pulse) (P<0.05).</p

Calreticulin Induces Dilated Cardiomyopathy

<div><p>Background</p><p>Calreticulin, a Ca²⁺-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart.</p><p>Methodology/Principal Findings</p><p>We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca²⁺ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca²⁺-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca²⁺-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin.</p><p>Conclusions/Significance</p><p>We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy <i>in vivo</i> leading to heart failure. This is due to an alternation in changes in a subset of Ca²⁺ handling genes, gap junction components and left ventricle remodeling.</p></div

Suppression of Cx43 promoter by calreticulin.

<p>(A) A schematic representation of calreticulin and calreticulin domains. CRT, calreticulin full length tagged with HA epitope; CRT-PC, P+C domain of calreticulin tagged with HA epitope. HA, hemagglutinin; KDEL, ER retrieval amino acid sequence. (B) Western blot analysis with anti-HA antibodies (reporting recombinant calreticulin and calreticulin PC domain) and anti-Cx43 antibodies of cell lysates from H9C2 cells (<i>H9C2</i>), H9C2 cells expression full length calreticulin (<i>H9C2+CRT</i>), and H9C2 cells expression Ca²⁺ buffering PC domain of calreticulin (<i>H9C2+CRT-PC</i>). HA, hemagglutinin; CRT, calreticulin; CRT-PC, calreticulin P+C domain. GAPDH was used as a loading control. (C) Control H9C2 cells (<i>H9C2</i>), H9C2 cell lines expression full length calreticulin (<i>H9C2+CRT</i>) or H9C2 cells expression Ca²⁺ buffering PC domain of calreticulin (<i>H9C2+CRT-PC</i>) were transfected with luciferase reporter vector under control of the Cx43 promoter and β-galactosidase expression vector. Cell lysates were harvested and assayed for luciferase activity. **<i>p</i><0.01 (n = 9).</p