Expression of Orai Genes and Icrac Activation in the Human RPE

Abstract

Purpose: : The retinal pigment epithelium (RPE) fulfills a large variety of tasks which are important for visual function. Many of these tasks, such as phagocytosis, growth factor secretion or transepithelial ion transport, are regulated by increases in intracellular Ca²⁺ as second-messenger. Despite the multitude of Ca²⁺-dependently regulated functions only few Ca²⁺ channels have been described so far in the RPE to couple Ca²⁺ conductance and Ca²⁺ signaling.

Methods: : RT-PCR with mRNA from freshly isolated human RPE cells and from the human RPE cell line ARPE-19 was used to identify expression of Ca²⁺-conducting ion channels. Membrane currents of APRE-19 cells were measured in the whole-cell configuration under K⁺-free conditions. Fura-2 Ca²⁺-imaging in ARPE-19 cells was used to record changes in intracellular free Ca²⁺.

Results: : RT-PCR from mRNA of freshly isolated RPE cells as well from the RPE cell line ARPE-19 revealed the expression of the Icrac channel proteins Orai 1, 2 and 3 as well as their stimulators stim-1 and stim-2. Using the classic maneuver to stimulate capacitative Ca²⁺ entry (depletion of Ca²⁺ stores by 1 µM thapsigargin under extracellular Ca²⁺-free conditions and then re-adding extracellular Ca²⁺) led to an increase in intracellular free Ca²⁺ which could be blocked by application of a high concentration of 2-APB (75 µM) either before or during induction of capacitative Ca²⁺ entry. On the other hand application of a low concentration of 2-APB (2 µM) led to an enhancement of the Ca²⁺ increase induced by capacitative Ca²⁺ entry. Depletion of cytosolic Ca²⁺ stores by administering an extracellular divalent cation-free solution led to an increase in the whole-cell conductance monitored by voltage-ramps between -130 mV and +50 mV.

Conclusions: : Human RPE cells express all known three members of the Orai gene family and the two members of the stim family. Depletion of cytosolic Ca²⁺ stores increased the membrane conductance in RPE cells leading to increases in intracellular free Ca²⁺ with characteristics of Icrac channels. With these data we show a new Ca²⁺ entry mechanism linked to the Ca²⁺/inositolphosphate second-messenger system in RPE cells which help to further understand regulatory pathways of agonists such acetylcholine, ATP or IGF-1.

Keywords: ion channels • signal transduction • retinal pigment epithelium