C2 region-derived peptides of β-protein kinase C regulate cardiac Ca2+ channels

Zhi Hao Zhang, John A Johnson, Long Chen, Nabil El-Sherif, Daria Mochly-Rosen, Mohamed Boutjdir

Research output: Contribution to journalArticle

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Abstract

We have previously shown that α1-adrenergic activation inhibited β- adrenergic stimulated L-type Ca2+ current (1(Ca)). To determine the role of protein kinase C (PKC) in this regulation, the inositol trisphosphate pathway was bypassed by direct activation of PKC with 4β-phorbol 12-myristate 13- acetate (PMA). To minimize Ca2+-induced Ca2+ inactivation, Ba2+ current (I(Ba)) was recorded through Ca2+ channels in adult rat ventricular myocytes. We found that PMA (0.1 μmol/L) consistently inhibited basal I(Ba) by 40.5 ± 7.4% and isoproterenol (ISO, 0.1 μmol/L)-stimulated I(Ba), by 48.9±7.8%. These inhibitory effects were not observed with the inactive phorbol ester analogue α-phorbol 12,13-didecanoate (0.1 μmol/L). To identify the PKC isozymes that mediate these PMA effects, we intracellularly applied peptide inhibitors of a subclass of PKC isozymes, the C2-containing cPKCs. These peptides (βC2-2 and βC2-4) specifically inhibit the translocation and function of C2-containing isozymes (α-PKC, β-PKC, and β11-PKC), but not the C2-less isozymes (δ-PKC and ε-PKC). We first used the pseudosubstrate peptide (0.1 μmol/L in the pipette), which inhibits the catalytic activity of all the PKC isozymes, and found that PMA-induced inhibition of ISO-stimulated I(Ha) was reduced to 16.8±7.4% but was not affected by the scrambled pseudosubstrate peptide. The effects of PMA on basal and ISO-stimulated I(Ba) were then determined in the presence of C2- derived peptides or control peptides. When the pipette contained 0.1 μmol/L of βC2-2 or BC2-4, PMA-induced inhibition of basal I(Ba) was 26.1 ±4.5% and 23.6±2.2%, respectively. Similarly, ISO-stimulated I(Ba) was inhibited by 29.9±6.6% and 29.3±7.8% in the presence of βC2-2 and βC2-4, respectively. In contrast, there was no significant change in the effect of PMA in the presence of control peptides, scrambled βC2-4, or pentalysine. Finally, PMA- induced inhibition of basal and ISO-stimulated I(Ba) was almost completely abolished in cells dialyzed with both βC2-2 and βC2-4. Together, these data suggest a role for C2-containing isozymes in mediating PMA-induced inhibition of L-type Ca2+ channel activity.

Original languageEnglish (US)
Pages (from-to)720-729
Number of pages10
JournalCirculation Research
Volume80
Issue number5
DOIs
StatePublished - Jan 1 1997
Externally publishedYes

Fingerprint

Protein Kinase C
Acetates
Peptides
Isoenzymes
pentalysine
Adrenergic Agents
phorbol-12-myristate
Phorbol Esters
Inositol
Isoproterenol
Muscle Cells

Keywords

  • Ca current
  • cardiac myocyte
  • phorbol ester
  • protein kinase C isozyme
  • receptor

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

C2 region-derived peptides of β-protein kinase C regulate cardiac Ca2+ channels. / Zhang, Zhi Hao; Johnson, John A; Chen, Long; El-Sherif, Nabil; Mochly-Rosen, Daria; Boutjdir, Mohamed.

In: Circulation Research, Vol. 80, No. 5, 01.01.1997, p. 720-729.

Research output: Contribution to journalArticle

Zhang, ZH, Johnson, JA, Chen, L, El-Sherif, N, Mochly-Rosen, D & Boutjdir, M 1997, 'C2 region-derived peptides of β-protein kinase C regulate cardiac Ca2+ channels', Circulation Research, vol. 80, no. 5, pp. 720-729. https://doi.org/10.1161/01.RES.80.5.720
Zhang, Zhi Hao ; Johnson, John A ; Chen, Long ; El-Sherif, Nabil ; Mochly-Rosen, Daria ; Boutjdir, Mohamed. / C2 region-derived peptides of β-protein kinase C regulate cardiac Ca2+ channels. In: Circulation Research. 1997 ; Vol. 80, No. 5. pp. 720-729.
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abstract = "We have previously shown that α1-adrenergic activation inhibited β- adrenergic stimulated L-type Ca2+ current (1(Ca)). To determine the role of protein kinase C (PKC) in this regulation, the inositol trisphosphate pathway was bypassed by direct activation of PKC with 4β-phorbol 12-myristate 13- acetate (PMA). To minimize Ca2+-induced Ca2+ inactivation, Ba2+ current (I(Ba)) was recorded through Ca2+ channels in adult rat ventricular myocytes. We found that PMA (0.1 μmol/L) consistently inhibited basal I(Ba) by 40.5 ± 7.4{\%} and isoproterenol (ISO, 0.1 μmol/L)-stimulated I(Ba), by 48.9±7.8{\%}. These inhibitory effects were not observed with the inactive phorbol ester analogue α-phorbol 12,13-didecanoate (0.1 μmol/L). To identify the PKC isozymes that mediate these PMA effects, we intracellularly applied peptide inhibitors of a subclass of PKC isozymes, the C2-containing cPKCs. These peptides (βC2-2 and βC2-4) specifically inhibit the translocation and function of C2-containing isozymes (α-PKC, β-PKC, and β11-PKC), but not the C2-less isozymes (δ-PKC and ε-PKC). We first used the pseudosubstrate peptide (0.1 μmol/L in the pipette), which inhibits the catalytic activity of all the PKC isozymes, and found that PMA-induced inhibition of ISO-stimulated I(Ha) was reduced to 16.8±7.4{\%} but was not affected by the scrambled pseudosubstrate peptide. The effects of PMA on basal and ISO-stimulated I(Ba) were then determined in the presence of C2- derived peptides or control peptides. When the pipette contained 0.1 μmol/L of βC2-2 or BC2-4, PMA-induced inhibition of basal I(Ba) was 26.1 ±4.5{\%} and 23.6±2.2{\%}, respectively. Similarly, ISO-stimulated I(Ba) was inhibited by 29.9±6.6{\%} and 29.3±7.8{\%} in the presence of βC2-2 and βC2-4, respectively. In contrast, there was no significant change in the effect of PMA in the presence of control peptides, scrambled βC2-4, or pentalysine. Finally, PMA- induced inhibition of basal and ISO-stimulated I(Ba) was almost completely abolished in cells dialyzed with both βC2-2 and βC2-4. Together, these data suggest a role for C2-containing isozymes in mediating PMA-induced inhibition of L-type Ca2+ channel activity.",
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TY - JOUR

T1 - C2 region-derived peptides of β-protein kinase C regulate cardiac Ca2+ channels

AU - Zhang, Zhi Hao

AU - Johnson, John A

AU - Chen, Long

AU - El-Sherif, Nabil

AU - Mochly-Rosen, Daria

AU - Boutjdir, Mohamed

PY - 1997/1/1

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N2 - We have previously shown that α1-adrenergic activation inhibited β- adrenergic stimulated L-type Ca2+ current (1(Ca)). To determine the role of protein kinase C (PKC) in this regulation, the inositol trisphosphate pathway was bypassed by direct activation of PKC with 4β-phorbol 12-myristate 13- acetate (PMA). To minimize Ca2+-induced Ca2+ inactivation, Ba2+ current (I(Ba)) was recorded through Ca2+ channels in adult rat ventricular myocytes. We found that PMA (0.1 μmol/L) consistently inhibited basal I(Ba) by 40.5 ± 7.4% and isoproterenol (ISO, 0.1 μmol/L)-stimulated I(Ba), by 48.9±7.8%. These inhibitory effects were not observed with the inactive phorbol ester analogue α-phorbol 12,13-didecanoate (0.1 μmol/L). To identify the PKC isozymes that mediate these PMA effects, we intracellularly applied peptide inhibitors of a subclass of PKC isozymes, the C2-containing cPKCs. These peptides (βC2-2 and βC2-4) specifically inhibit the translocation and function of C2-containing isozymes (α-PKC, β-PKC, and β11-PKC), but not the C2-less isozymes (δ-PKC and ε-PKC). We first used the pseudosubstrate peptide (0.1 μmol/L in the pipette), which inhibits the catalytic activity of all the PKC isozymes, and found that PMA-induced inhibition of ISO-stimulated I(Ha) was reduced to 16.8±7.4% but was not affected by the scrambled pseudosubstrate peptide. The effects of PMA on basal and ISO-stimulated I(Ba) were then determined in the presence of C2- derived peptides or control peptides. When the pipette contained 0.1 μmol/L of βC2-2 or BC2-4, PMA-induced inhibition of basal I(Ba) was 26.1 ±4.5% and 23.6±2.2%, respectively. Similarly, ISO-stimulated I(Ba) was inhibited by 29.9±6.6% and 29.3±7.8% in the presence of βC2-2 and βC2-4, respectively. In contrast, there was no significant change in the effect of PMA in the presence of control peptides, scrambled βC2-4, or pentalysine. Finally, PMA- induced inhibition of basal and ISO-stimulated I(Ba) was almost completely abolished in cells dialyzed with both βC2-2 and βC2-4. Together, these data suggest a role for C2-containing isozymes in mediating PMA-induced inhibition of L-type Ca2+ channel activity.

AB - We have previously shown that α1-adrenergic activation inhibited β- adrenergic stimulated L-type Ca2+ current (1(Ca)). To determine the role of protein kinase C (PKC) in this regulation, the inositol trisphosphate pathway was bypassed by direct activation of PKC with 4β-phorbol 12-myristate 13- acetate (PMA). To minimize Ca2+-induced Ca2+ inactivation, Ba2+ current (I(Ba)) was recorded through Ca2+ channels in adult rat ventricular myocytes. We found that PMA (0.1 μmol/L) consistently inhibited basal I(Ba) by 40.5 ± 7.4% and isoproterenol (ISO, 0.1 μmol/L)-stimulated I(Ba), by 48.9±7.8%. These inhibitory effects were not observed with the inactive phorbol ester analogue α-phorbol 12,13-didecanoate (0.1 μmol/L). To identify the PKC isozymes that mediate these PMA effects, we intracellularly applied peptide inhibitors of a subclass of PKC isozymes, the C2-containing cPKCs. These peptides (βC2-2 and βC2-4) specifically inhibit the translocation and function of C2-containing isozymes (α-PKC, β-PKC, and β11-PKC), but not the C2-less isozymes (δ-PKC and ε-PKC). We first used the pseudosubstrate peptide (0.1 μmol/L in the pipette), which inhibits the catalytic activity of all the PKC isozymes, and found that PMA-induced inhibition of ISO-stimulated I(Ha) was reduced to 16.8±7.4% but was not affected by the scrambled pseudosubstrate peptide. The effects of PMA on basal and ISO-stimulated I(Ba) were then determined in the presence of C2- derived peptides or control peptides. When the pipette contained 0.1 μmol/L of βC2-2 or BC2-4, PMA-induced inhibition of basal I(Ba) was 26.1 ±4.5% and 23.6±2.2%, respectively. Similarly, ISO-stimulated I(Ba) was inhibited by 29.9±6.6% and 29.3±7.8% in the presence of βC2-2 and βC2-4, respectively. In contrast, there was no significant change in the effect of PMA in the presence of control peptides, scrambled βC2-4, or pentalysine. Finally, PMA- induced inhibition of basal and ISO-stimulated I(Ba) was almost completely abolished in cells dialyzed with both βC2-2 and βC2-4. Together, these data suggest a role for C2-containing isozymes in mediating PMA-induced inhibition of L-type Ca2+ channel activity.

KW - Ca current

KW - cardiac myocyte

KW - phorbol ester

KW - protein kinase C isozyme

KW - receptor

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