Regulation of Inwardly Rectifying K+ (Kir) Channels in the Retinal Pigment Epithelium (RPE) by Phosphatidylinositol 4, 5–Bisphosphate (PIP2)

Abstract

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. K_ir current was isolated as the Cs⁺–sensitive component of the whole–cell current.

Results: : As shown previously, K_ir current was sustained in cells dialyzed with 4 mM Mg–ATP but ran down rapidly (t_1/2 = 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 ( t_1/2 = 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 K_ir 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