Localization & Physiology of L-type Ca2+ Channels in Human RPE

Abstract

Purpose: : Best’s vitelliform macular dystrophy is characterized by a reduced light peak (LP) in the EOG. Recent evidence suggests that L-type voltage-dependent Ca²⁺ channels (VDCCs) are involved in the generation of the LP. The aim of this study is to characterize the localization and function of L-type Ca²⁺ channels in human fetal RPE cells (hfRPE).

Methods: : Primary cultures of hfRPE and native hfRPE tissues were used in all experiments. L-type VDCC subunit mRNA and protein expression were determined by RT-PCR and Western blot respectively. Polarized membrane distribution was determined by immunofluorescence (IF) microscopy. Intracellular microelectrode recordings were used to measure apical and basal membrane potential (V_Ba), transepithelial potential (TEP) and resistance. Fura-2 Ca²⁺ imaging was used to monitor intracellular free Ca²⁺.

Results: : mRNA expression of Cav1.3 subunits (α1D, β2, 3, 4) were detected in native and cultured hfRPE cells. Immunocytochemistry showed that the α1D subunit was mainly localized at the apical membrane. Intracellular recordings demonstrated that addition of the L-type VDCC blocker, nimodipine, to the apical bath decreased TEP and hyperpolarized V_Ba. In contrast, basal bath addition of nimodipine had little effect. The apical nimodipine-induced hyperpolarization of V_Ba was blocked by the removal of apical [Ca²⁺]_o or by the addition of basal DIDS (500μM), consistent with decreased activity of basolateral membrane Ca²⁺-activated Cl^- channels. Apical, but not basal bath addition of the L-type VDCC activator BayK8644 evoked an increase in intracellular Ca²⁺ and hyperpolarized both the apical and basolateral membranes. This response was abolished in apical Ca²⁺-free Ringer or in the presence of apical La³⁺ (200 μM). The BayK8644 evoked-hyperpolarization was blocked by Ba²⁺ in both the apical and basal baths, suggesting that the elevation of [Ca²⁺]_i activates Ca²⁺-dependent K⁺ channels.

Conclusions: : Our results indicate that L-type VDCCs are present predominantly at the apical, but not at the basolateral membrane of hfRPE cells. L-type Ca²⁺ channels mediate extracellular Ca²⁺ influx from the apical membrane and this influx is required for maintaining the activity of basolateral Ca²⁺-activated Cl^- channels. Based on this data, we discuss possible mechanisms for LP generation.

Keywords: calcium • ion channels • retinal pigment epithelium