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Dual role of protein kinase C on BK channel regulation.

ABSTRACT: Large conductance voltage- and Ca(2+)-activated potassium channels (BK channels) are important feedback regulators in excitable cells and are potently regulated by protein kinases. The present study reveals a dual role of protein kinase C (PKC) on BK channel regulation. Phosphorylation of S(695) by PKC, located between the two regulators of K(+) conductance (RCK1/2) domains, inhibits BK channel open-state probability. This PKC-dependent inhibition depends on a preceding phosphorylation of S(1151) in the C terminus of the channel alpha-subunit. Phosphorylation of only one alpha-subunit at S(1151) and S(695) within the tetrameric pore is sufficient to inhibit BK channel activity. We further detected that protein phosphatase 1 is associated with the channel, constantly counteracting phosphorylation of S(695). PKC phosphorylation at S(1151) also influences stimulation of BK channel activity by protein kinase G (PKG) and protein kinase A (PKA). Though the S(1151)A mutant channel is activated by PKA only, the phosphorylation of S(1151) by PKC renders the channel responsive to activation by PKG but prevents activation by PKA. Phosphorylation of S(695) by PKC or introducing a phosphomimetic aspartate at this position (S(695)D) renders BK channels insensitive to the stimulatory effect of PKG or PKA. Therefore, our findings suggest a very dynamic regulation of the channel by the local PKC activity. It is shown that this complex regulation is not only effective in recombinant channels but also in native BK channels from tracheal smooth muscle.

SUBMITTER: Zhou XB

PROVIDER: S-EPMC2867903 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Dual role of protein kinase C on BK channel regulation.

Zhou Xiao-Bo XB Wulfsen Iris I Utku Emine E Sausbier Ulrike U Sausbier Matthias M Wieland Thomas T Ruth Peter P Korth Michael M

Proceedings of the National Academy of Sciences of the United States of America 20100412 17

Large conductance voltage- and Ca(2+)-activated potassium channels (BK channels) are important feedback regulators in excitable cells and are potently regulated by protein kinases. The present study reveals a dual role of protein kinase C (PKC) on BK channel regulation. Phosphorylation of S(695) by PKC, located between the two regulators of K(+) conductance (RCK1/2) domains, inhibits BK channel open-state probability. This PKC-dependent inhibition depends on a preceding phosphorylation of S(1151 ...[more]

PMID: 20385812

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Project description:The internal vestibule of large-conductance Ca(2+) voltage-activated K(+) (BK) channels contains a ring of eight negative charges not present in K(+) channels of lower conductance (Glu386 and Glu389 in hSlo) that modulates channel conductance through an electrostatic mechanism (Brelidze, T.I., X. Niu, and K.L. Magleby. 2003. Proc. Natl. Acad. Sci. USA. 100:9017-9022). In BK channels there are also two acidic amino acid residues in an extracellular loop (Asp326 and Glu329 in hSlo). To determine the electrostatic influence of these charges on channel conductance, we expressed wild-type BK channels and mutants E386N/E389N, D326N, E329Q, and D326N/E329Q channels on Xenopus laevis oocytes, and measured the expressed currents under patch clamp. Contribution of E329 to the conductance is negligible and single channel conductance of D326N/E329Q channels measured at 0 mV in symmetrical 110 mM K(+) was 18% lower than the control. Current-voltage curves displayed weak outward rectification for D326N and the double mutant. The conductance differences between the mutants and wild-type BK were caused by an electrostatic effect since they were enhanced at low K(+) (30 mM) and vanished at high K(+) (1 M K(+)). We determine the electrostatic potential change, Deltaphi, caused by the charge neutralization using TEA(+) block for the extracellular charges and Ba(2+) for intracellular charges. We measured 13 +/- 2 mV for Deltaphi at the TEA(+) site when turning off the extracellular charges, and 17 +/- 2 mV for the Deltaphi at the Ba(2+) site when the intracellular charges were turned off. To understand the electrostatic effect of charge neutralizations, we determined Deltaphi using a BK channel molecular model embedded in a lipid bilayer and solving the Poisson-Boltzmann equation. The model explains the experimental results adequately and, in particular, gives an economical explanation to the differential effect on the conductance of the neutralization of charges D326 and E329.

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Dual role of protein kinase C on BK channel regulation.

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Dual role of protein kinase C on BK channel regulation.

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