Research ArticlePhysiology

A molecular signature in the pannexin1 intracellular loop confers channel activation by the α1 adrenoreceptor in smooth muscle cells

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Science Signaling  17 Feb 2015:
Vol. 8, Issue 364, pp. ra17
DOI: 10.1126/scisignal.2005824
  • Fig. 1 Pharmacological inhibition of Panx1 reduces vasoconstriction and ATP release selectively upon activation of α1AR.

    (A to D) Effect of 10Panx1 (300 μM) and probenecid (2 mM) on contractile response of pressurized TDAs stimulated with the indicated concentrations of agonists. n = 5 to 7. *P < 0.05 compared to untreated response (black curves) using two-way analysis of variance (ANOVA). (E) Relative ATP released from intact TDAs in response to phenylephrine (PE) in the presence or absence of 10Panx1 (300 μM), serotonin (5-HT), or endothelin-1 (ET-1). Data are presented as a percent increase in ATP concentration from unstimulated conditions. The insert shows an image of a TDA in a well of a 96-well dish. n = 5 to 11. *P < 0.05 compared to phenylephrine using a Kruskal-Wallis test.

  • Fig. 2 Inducible SMC deletion of Panx1 selectively inhibits vasoconstriction and ATP release upon α1AR stimulation.

    (A) Representative immunofluorescence micrographs showing Panx1 labeling (red) on cross sections of TDAs isolated from mice of the indicated genotypes. All mice had been injected with tamoxifen for 10 days. The far right panel shows a negative control (secondary antibody only) on a cross section of a TDA isolated from Cre/Panx1WT mice. The autofluorescence of the internal elastic lamina (IEL) appears in green, and the nuclei were labeled with DAPI (4′,6-diamidino-2-phenylindole) (blue). * indicates the lumen. Scale bar, 10 μm. (B) Basal tone exhibited by TDAs from each genotype (exposed to tamoxifen for 10 days). (C to F) Contraction of pressurized TDAs isolated from Cre/Panx1WT mice (black curves), Cre/Panx1Fl mice (dark gray curves), Cre+/Panx1WT mice (light gray curves), and Cre+/Panx1Fl (green curves) all injected with tamoxifen for 10 days and stimulated with cumulative concentrations of phenylephrine (n = 6 to 16), noradrenaline (n = 4 to 8), serotonin (n = 5 to 10), or endothelin-1 (n = 4 to 8). *P < 0.05 compared to Cre/Panx1WT using a two-way ANOVA. (G) Histogram showing the phenylephrine (PE)–induced ATP release from intact TDAs isolated from mice of the indicated genotypes, all injected with tamoxifen for 10 days. Data are presented as a percent increase in ATP concentration from unstimulated conditions. *P < 0.05 compared to Cre/Panx1WT using a Kruskal-Wallis test. n = 4 to 8.

  • Fig. 3 Inducible SMC deletion of Panx1 reduces blood pressure in freely moving mice.

    (A) Difference in the 24-hour mean arterial pressure (MAP) of mice of the indicated genotypes before and after tamoxifen injections. (B) Difference in the MAP during the day cycle (12-hour light: 6:00 a.m. to 6:00 p.m.) of mice of the indicated genotypes before and after tamoxifen injections. (C) Difference in the MAP during the night cycle (12-hour no light: 6:00 p.m. to 6:00 a.m.) of mice of the indicated genotypes before and after tamoxifen injections. *P < 0.05 comparing the MAP before and after tamoxifen injection using a nonparametric paired t test (Wilcoxon). n = 4 to 7.

  • Fig. 4 A peptide analog to a Panx1 intracellular loop sequence inhibits vasoconstriction and ATP release upon α1AR stimulation and reduces blood pressure.

    (A) Diagram showing the position of each of the four peptides on mPanx1. The scissors indicate a caspase cleavage site. (B to H) Effects of the indicated peptide inhibitor on phenylephrine-induced constriction of pressurized TDAs (B to E), and effect of IL2 peptide on constriction of pressurized TDAs induced by the indicated concentrations of agonists (E to H). The black curves represent constriction in the absence of peptide. n = 4 to 7. *P < 0.05 compared to constriction in the absence of peptide using a two-way ANOVA. (I) Effect of the indicated peptide on phenylephrine (PE)–induced ATP release. Data are presented as a percent increase in ATP concentration from unstimulated conditions. n = 3 to 8. *P < 0.05 compared to no peptide using a Kruskal-Wallis test. (J) Difference between the MAPs (ΔMAP) measured before and after injection of saline, IL2 peptide, or its scrambled IL2 peptide in C57BL/6 mice. *P < 0.05 comparing before and after vehicle and peptide injections using a nonparametric paired t test (Wilcoxon). n = 7 to 9.

  • Fig. 5 Characterization of α1DAR-mediated ATP release by Panx1 using a heterologous system.

    (A) Left panel: Representative current-voltage (I-V) curve obtained from whole-cell patch clamp recording of HEK293 cells cotransfected with Panx1 and α1DAR before (control, black curve) and after stimulation with phenylephrine (green curve), and upon application of CBX (red curve). Right panel: Representative time course of whole-cell current recorded from cotransfected HEK293 cell showing the effect of phenylephrine and CBX. (B) Phenylephrine-induced Panx1 current (top panel) and phenylephrine-induced ATP release (bottom panel) in untransfected HEK293 cells or HEK293 cells transfected with the indicated constructs. (C) Effect of the IL2 peptide and scrambled IL2 peptide on phenylephrine-induced Panx1 current (top panel) and phenylephrine-induced ATP release (bottom panel). (D) Phenylephrine-induced Panx1 current (top panel) and phenylephrine-induced ATP release (bottom panel) in HEK293 cells cotransfected with α1DAR and the indicated Panx1 construct. Panx1 current data [(B) to (D), top panels] are presented as a percent increase of CBX-sensitive current at +80 mV, or as a percent increase of ATP concentration from unstimulated conditions [(B) to (D), bottom panels]. (B to D) *P < 0.05 compared to cotransfected conditions (B), no peptide (C), or wild-type Panx1 (Panx1WT) (D) using a Kruskal-Wallis test. (E) Phenylephrine-induced contraction of pressurized TDAs isolated from Cre+/Panx1Fl mice not injected with tamoxifen and electroporated without plasmid (black curve), Cre+/Panx1Fl mice after injection with tamoxifen for 10 days and then electroporated without plasmid (gray curve), or Cre+/Panx1Fl mice after injection with tamoxifen for 10 days and then electroporated with Panx1WT (pink curve). (F) Phenylephrine-induced contraction of pressurized TDAs from control mice as indicated in (E) (black and gray curves) and Cre+/Panx1Fl mice after injection with tamoxifen for 10 days and then electroporated with Panx1YLK>AAA (blue curve). (E and F) *P < 0.05 compared to control Cre+/Panx1Fl (black curve) using a two-way ANOVA. n = 6 mice.

  • Table 1 Effect of probenecid and 10Panx1 on TDA constriction in response to phenylephrine, noradrenaline, serotonin, or endothelin-1.

    EC50 and EMAX were calculated using the cumulative concentration response curves shown in Fig. 1 (A to D). EC50 represents the concentration needed to produce 50% of the maximum effect (EMAX). EMAX is expressed as the percentage of maximal diameter. Data are presented as means ± SEM. *P < 0.05 compared to control using a Kruskal-Wallis test (n = 5 to 7).

    PhenylephrineNoradrenaline
    ControlProbenecid10Panx1ControlProbenecid10Panx1
    EC50 (μM)1.21 ± 0.272.71 ± 1.362.38 ± 0.530.56 ± 0.251.30 ± 0.651.61 ± 0.49
    EMAX41.1 ± 2.2172.6 ± 3.51*60.6 ± 5.80*33.3 ± 1.0474.9 ± 3.04*73.4 ± 10.6*
    SerotoninEndothelin-1
    ControlProbenecid10Panx1ControlProbenecid10Panx1
    EC50 (nM)50.8 ± 18.983.3 ± 8.4247.9 ± 23.14.57 ± 0.8812.8 ± 4.056.53 ± 1.36
    EMAX34.8 ± 2.6639.5 ± 3.2930.9 ± 4.9241.9 ± 4.8234.6 ± 4.5041.5 ± 3.91
  • Table 2 Contractile properties of TDAs isolated from Cre/Panx1WT, Cre/Panx1Fl, Cre+/Panx1WT, or Cre+/Panx1F1 mice.

    The EC50 and EMAX are calculated from the data presented in Fig. 2 (C to F). EC50 represents the concentration needed to produce 50% of the maximum effect (EMAX). EMAX is expressed as the percentage of maximal diameter. Data are presented as means ± SEM. *P < 0.05, #P < 0.07 compared to Cre/Panx1WT using a Kruskal-Wallis test (n = 4 to 16).

    Phenylephrine
    Cre/Panx1WTCre/Panx1FLCre+/Panx1WTCre+/Panx1FL
    EC50 (μM)0.66 ± 0.130.64 ± 0.371.79 ± 1.137.84 ± 5.60
    EMAX51.8 ± 2.6646.3 ± 5.9453.5 ± 4.9169.1 ± 3.06*
    Noradrenaline
    Cre/Panx1WTCre/Panx1FLCre+/Panx1WTCre+/Panx1FL
    EC50 (μM)6.28 ± 1.4510.3 ± 1.36.78 ± 3.2050.5 ± 24.8
    EMAX41.4 ± 5.1045.6 ± 5.4048.2 ± 1.8256.4 ± 5.61#
    Serotonin
    Cre/Panx1WTCre/Panx1FLCre+/Panx1WTCre+/Panx1FL
    EC50 (nM)23.8 ± 5.0765.2 ± 40.940.7 ± 12.214.2 ± 5.53
    EMAX36.1 ± 2.6830.4 ± 1.7840.1 ± 4.5938.2 ± 5.60
    Endothelin-1
    Cre/Panx1WTCre/Panx1FLCre+/Panx1WTCre+/Panx1FL
    EC50 (nM)18.4 ± 10.93.77 ± 0.565.64 ± 2.9430.1 ± 14.6
    EMAX41.8 ± 4.9542.2 ± 6.5042.5 ± 4.8238.9 ± 6.90
  • Table 3 Amino acid sequences of intracellular loop and C-terminal region peptides.

    All listed peptides were linked with a TAT sequence (YGRKKQRRR). The numbers indicate the peptide location in the mPanx1 amino acid sequence. The CT1 peptide has previously been published to inhibit NMDA-mediated Panx1 opening (21).

    Amino acid sequence (mPanx1)
    IL1178-VGQSLWEISE-187
    IL2191-KYPIVEQYLK-200
    CT1305-RRLKVYEILPTFDVLH-318
    CT2381-IPTSLQTKGE-390
    Scrambled IL2IYLYVEQKPY
  • Table 4 Effect of four Panx1 mimetic peptides on constriction of TDAs in response to phenylephrine, noradrenaline, serotonin, or endothelin-1.

    The EC50 and EMAX are calculated from the data presented in Fig. 4 (B to H). EC50 represents the concentration needed to produce 50% of the maximum effect (EMAX). EMAX is expressed as the percentage of maximal diameter. Data are presented as means ± SEM. *P < 0.05 compared to no peptide using a Mann-Whitney test (n = 4 to 7).

    Phenylephrine
    No peptideCT1No peptideCT2
    EC50 (μM)1.67 ± 1.131.43 ± 0.591.06 ± 0.324.78 ± 3.93
    EMAX52.5 ± 3.3050.7 ± 9.0849.0 ± 2.4952.3 ± 4.86
    No peptideIL1No peptideIL2
    EC50 (μM)3.26 ± 1.531.14 ± 0.431.15 ± 0.350.57 ± 0.22
    EMAX40.1 ± 5.1032.3 ± 3.8147.70 ± 1.668.6 ± 5.11*
    NoradrenalineSerotoninEndothelin-1
    No peptideIL2No peptideIL2No peptideIL2
    EC50 (μM)0.49 ± 0.422.00 ± 1.9011.7 ± 1.655.03 ± 0.82*6.01 ± 3.740.88 ± 0.39
    EMAX27.1 ± 4.6953.4 ± 7.00*38.6 ± 5.7036.7 ± 3.5238.2 ± 6.6741.9 ± 3.73
  • Table 5 Effect of transfection of TDAs with Panx1WT or Panx1YLK>AAA on phenylephrine-induced constriction.

    The EC50 and EMAX are calculated from the data presented in Fig. 4 (E and F). EC50 represents the concentration needed to produce 50% of the maximum effect (EMAX). EMAX is expressed as the percentage of maximal diameter. Data are presented as means ± SEM. *P < 0.05 in comparison to no tamoxifen, and #P < 0.05 in comparison to Cre+/Panx1FL + Panx1WT using a Kruskal-Wallis test.

    Phenylephrine
    Cre+/Panx1FL No tamoxifenCre+/Panx1FL With tamoxifenCre+/Panx1FL + Panx1WTCre+/Panx1FL + Panx1YLK>AAA
    EC50 (μM)0.64 ± 0.231.13 ± 0.770.45 ± 0.330.63 ± 0.53
    EMAX32.9 ± 4.8557.5 ± 9.20*#34.2 ± 6.1556.9 ± 5.84*#

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/364/ra17/DC1

    Fig. S1. Effect of SMC deletion of Panx1 on Panx2 and Panx3 abundance, ATP content, α1DAR abundance, and phenylephrine-induced contraction of aortic rings.

    Fig. S2. Effect of IL2 scrambled peptide and TAT peptide on phenylephrine-mediated constriction and ATP release.

    Fig. S3. Effect of mutations in the Panx1 IL2 region on the channel function.

    Table S1. Contractile properties of aortic rings isolated from control Cre+/Panx1Fl mice and Cre+/Panx1Fl injected with tamoxifen for 10 days.

    Table S2. Effect of scrambled IL2 peptide and TAT peptide on constriction of TDAs in response to phenylephrine.

    Table S3. Primers used for the generation of Panx1KYP>AAA, Panx1IVEQ>AAAA, and Panx1YLK>AAA plasmids.

  • Supplementary Materials for:

    A molecular signature in the pannexin1 intracellular loop confers channel activation by the α1 adrenoreceptor in smooth muscle cells

    Marie Billaud, Yu-Hsin Chiu, Alexander W. Lohman, Thibaud Parpaite, Joshua T. Butcher, Stephanie M. Mutchler, Leon J. DeLalio, Mykhaylo V. Artamonov, Joanna K. Sandilos, Angela K. Best, Avril V. Somlyo, Roger J. Thompson, Thu H. Le, Kodi S. Ravichandran, Douglas A. Bayliss, Brant E. Isakson*

    *Corresponding author. E-mail: brant{at}virginia.edu

    This PDF file includes:

    • Fig. S1. Effect of SMC deletion of Panx1 on Panx2 and Panx3 abundance, ATP content, α1DAR abundance, and phenylephrine-induced contraction of aortic rings.
    • Fig. S2. Effect of IL2 scrambled peptide and TAT peptide on phenylephrine-mediated constriction and ATP release.
    • Fig. S3. Effect of mutations in the Panx1 IL2 region on the channel function.
    • Table S1. Contractile properties of aortic rings isolated from control Cre+/Panx1Fl mice and Cre+/Panx1Fl injected with tamoxifen for 10 days.
    • Table S2. Effect of scrambled IL2 peptide and TAT peptide on constriction of TDAs in response to phenylephrine.
    • Table S3. Primers used for the generation of Panx1KYP>AAA, Panx1IVEQ>AAAA, and Panx1YLK>AAA plasmids.

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    Citation: M. Billaud, Y.-H. Chiu, A. W. Lohman, T. Parpaite, J. T. Butcher, S. M. Mutchler, L. J. DeLalio, M. V. Artamonov, J. K. Sandilos, A. K. Best, A. V. Somlyo, R. J. Thompson, T. H. Le, K. S. Ravichandran, D. A. Bayliss, B. E. Isakson, A molecular signature in the pannexin1 intracellular loop confers channel activation by the α1 adrenoreceptor in smooth muscle cells. Sci. Signal. 8, ra17 (2015).

    © 2015 American Association for the Advancement of Science

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