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Two PKC consensus sites on human acid-sensing ion channel 1b differentially regulate its function.

Edlira Bashari1, Yawar J Qadri, Zhen-Hong Zhou

  • 1Dept. of Physiology and Biophysics, Univ. of Alabama at Birmingham, Birmingham, AL 35294, USA.

American Journal of Physiology. Cell Physiology
|December 19, 2008
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Protein kinase C (PKC) differentially regulates human acid-sensing ion channel 1b (hASIC1b) function at serine 40 and serine 499 sites. Understanding these specific PKC phosphorylation sites in hASIC1b may aid glioma treatment therapies.

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Area of Science:

  • Neuroscience
  • Molecular Biology
  • Cell Biology

Background:

  • Human acid-sensing ion channel 1b (hASIC1b) is a proton-gated cation channel implicated in glioma cell behavior.
  • Previous studies indicated amiloride-sensitive currents in glioma cells and that PKC modulates hASIC1b function.
  • hASIC1b interacts with PICK1, which recruits PKC to the plasma membrane.

Purpose of the Study:

  • To investigate the hypothesis that PKC regulates hASIC1b activity through specific phosphorylation sites.
  • To identify the critical PKC consensus sites on hASIC1b responsible for PKC-mediated modulation.

Main Methods:

  • Mutagenesis of hASIC1b at putative PKC phosphorylation sites (T26, S40, S499) to alanine (non-phosphorylatable) or glutamic acid/aspartic acid (phosphomimetic).
  • Expression of wild-type and mutant hASIC1b in Xenopus oocytes.
  • Two-electrode voltage clamp to measure acid-activated currents.
  • Treatment with PKC activators (PMA, phorbol 12,13-dibutyrate) and inhibitors (chelerythrine).
  • Assessment of surface expression of hASIC1b.

Main Results:

  • Mutations at T26 abolished channel activity, while mutations at S40 and S499 affected acid-activated currents.
  • S40E, S499A, and S499D mutants showed decreased currents compared to wild-type.
  • PKC activators inhibited wild-type and S499A hASIC1b, but not S40A mutants.
  • PKC inhibitor chelerythrine inhibited wild-type and S40A, but not S499A or S40A/S499A mutants.
  • PKC modulation did not alter hASIC1b surface expression.

Conclusions:

  • Serine 40 and serine 499 are critical sites for PKC-mediated regulation of hASIC1b.
  • These sites differentially mediate the effects of PKC activation and inhibition on hASIC1b function.
  • Findings provide insight into PKC regulation of hASIC1b in glioma cells, potentially informing therapeutic strategies.