May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
Regulation of Inwardly Rectifying K+ (Kir) Channels in the Retinal Pigment Epithelium (RPE) by Phosphatidylinositol 4, 5–Bisphosphate (PIP2)
Author Affiliations & Notes
  • B.R. Pattnaik
    University of Michigan, Ann Arbor, MI
    Ophthalmology and Visual Sciences,
  • B.A. Hughes
    University of Michigan, Ann Arbor, MI
    Ophthalmology and Visual Sciences and Molecular and Integrative Physiology,
  • Footnotes
    Commercial Relationships  B.R. Pattnaik, None; B.A. Hughes, None.
  • Footnotes
    Support  NIH Grants EY08850 and EY07003
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 4722. doi:
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      B.R. Pattnaik, B.A. Hughes; Regulation of Inwardly Rectifying K+ (Kir) Channels in the Retinal Pigment Epithelium (RPE) by Phosphatidylinositol 4, 5–Bisphosphate (PIP2) . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4722.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Kir7.1 channels in the RPE apical membrane participate in the homeostasis of the outer retina by mediating K+ secretion into the subretinal space. Although it has been established that Kir channel activity in the RPE is highly dependent on the hydrolysis of cellular ATP, the underlying mechanism remains unknown. Given that Kir channels are gated by PIP2, we hypothesize that the dependence of RPE Kir channel activity on hydrolysable ATP stems the requirement for this nucleotide in the synthesis of PIP2 by phosphatidyl–inositol (PI) kinases. The purpose of this study was to investigate the regulation of Kir channels in the RPE by PIP2.

Methods: : Whole–cell currents were recorded from freshly dissociated bovine RPE cells at room temperature. The standard pipette solution contained (in mM): 30 KCl, 83 K–gluconate, 10 HEPES, 5.5 EGTA, 0.5 CaCl2, and 4 Mg–ATP, pH 7.2. The control bath solution contained 135 NaCl, 5 KCl, 10 HEPES, 10 glucose, 1.8 CaCl2, and 1.0 MgCl2, pH 7.4. Kir current was isolated as the Cs+–sensitive component of the whole–cell current.

Results: : As shown previously, Kir current was sustained in cells dialyzed with 4 mM Mg–ATP but ran down rapidly (t1/2 = 2.7 ± 0.2 min; n = 5) and completely when the pipette solution lacked ATP but contained 4 mM Mg2+. Kir current in cells dialyzed with a ATP– and Mg2+–free solution containing a cocktail of lipid phosphatase inhibitors (FVPP) ran down partially, with 79 ± 5 % of the initial current remaining after 6 minutes of dialysis (n = 7). The addition of exogenous PIP2 (50 µM) to ATP–free pipette solution containing Mg2+ slowed and reduced rundown, such that 55 ± 4 % (n = 6) the initial Kir current remained after 8 minutes of dialysis. On the other hand, inclusion of 50 µM neomycin, a PIP2 scavenger, in Mg–ATP containing pipette solution resulted in rapid rundown of Kir current ( t1/2 = 3.96 ± 0.4 min; n = 5) to 22 ± 2.3 % (n = 5) of its initial value. Finally, bath application of the PI–kinase inhibitors quercetin (100 µM) or phenylarsine oxide (100 µM) inhibited 66 ± 2.4% (n=6) and 42 ± 2.7% (n =9 ) of the Kir current, respectively, in cells dialyzed with standard Mg–ATP pipette solution.

Conclusions: : The results demonstrate that reduction in membrane PIP2 content by targeting the enzymes responsible for its degradation or regeneration causes a decrease in Kir channel activity in the RPE, and that this can be mitigated by exogenous PIP2. We conclude that PIP2 metabolism may provide an important mechanism for the regulation of the RPE apical membrane K+ conductance.

Keywords: ion channels • electrophysiology: non-clinical • signal transduction: pharmacology/physiology 

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