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Individual PKC-phosphorylation sites in organic cation transporter 1 determine substrate selectivity and transport

Giuliano Ciarimboli1, Hermann Koepsell, Mariya Iordanova

  • 1Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, Universitätsklinikum Münster, Domagkstrasse 3a, Münster, D-48149 Germany. gciari@uni-muenster.de

Journal of the American Society of Nephrology : JASN
|April 15, 2005
PubMed

Insights

Protein kinase C (PKC) activation stimulates rat organic cation transporter type 1 (rOCT1) via five specific phosphorylation sites. Mutating these sites, especially all five, blocks stimulation, indicating their necessity for rOCT1 regulation.

Area of Science:

  • Molecular biology
  • Biochemistry
  • Cell biology

Background:

  • Rat organic cation transporter type 1 (rOCT1) plays a crucial role in transporting various organic cations.
  • Protein kinase C (PKC) is known to regulate transporter function, but the specific mechanisms for rOCT1 are not fully understood.
  • Identifying phosphorylation sites is key to understanding PKC-mediated regulation of rOCT1.

Purpose of the Study:

  • To investigate the molecular mechanisms by which PKC activation stimulates rOCT1.
  • To identify the specific PKC phosphorylation sites involved in rOCT1 regulation.
  • To determine the impact of these phosphorylation sites on rOCT1 substrate affinity and function.

Main Methods:

  • Site-directed mutagenesis was used to create rOCT1 mutants with single or all five putative PKC phosphorylation sites substituted with alanine.
  • Functional characterization of wild-type (WT) and mutant rOCT1 was performed in HEK293 cells using microfluorometric measurements with the fluorescent substrate ASP(+).
  • Apparent affinities for various substrates (TEA(+), TPA(+), quinine) and effects of PKC activation were assessed.

Main Results:

  • Substitution of single or all five PKC phosphorylation sites suppressed PKC-induced stimulation of ASP(+) uptake.
  • PKC regulation by p56(lck) tyrosine kinase remained conserved in all mutants.
  • Mutations differentially affected the apparent affinities for TEA(+), TPA(+), and quinine, and PKC activation no longer altered substrate affinity in mutants.
  • PKC activation did not significantly affect rOCT1 trafficking, but substitution of all sites abolished PKC-induced phosphorylation.

Conclusions:

  • The presence of all five potential PKC phosphorylation sites is essential for PKC-induced stimulation of rOCT1.
  • These phosphorylation sites, located in the large intracellular loop, are critical for modulating rOCT1 substrate binding or structure.
  • The findings provide crucial insights into the regulation of rOCT1 by PKC signaling pathways.

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