Regulation of TRPV2 channel surface expression in the retinal pigment epithelium

Abstract

Purpose: A large variety of retinal pigment (RPE) cell functions of importance for their close interaction with the photoreceptors are regulated by changes in intracellular free Ca²⁺. Transient-receptor-potential vanilloid-subtype-2 (TRPV2) channels were identified to contribute to angiotensin-2 or insulin-like growth-factor-1 (IGF-1) dependent signaling in the RPE. Thus, the regulation of surface expression of TRPV2 is the prerequisite of RPE function regulation.

Methods: Membrane currents of ARPE-19 cells were measured in the whole-cell configuration of the patch-clamp technique under K⁺-free conditions. TRPV2 protein surface expression was quantified by means of confocal microscopy. In situ expression of TRPV2 channels in the RPE was analyzed in sections of the mouse retina by means of immune histochemistry.

Results: Anti-TRPV2 staining of the mouse retina sections revealed the in situ expression of TRPV2 channels in the RPE, localized to both the apical and the basolateral membrane. A short increase of the bath temperature to +45° C led to activation of membrane conductance in ARPE-19 cells which could be blocked by the TPRV2 channel blockers La³⁺ (100 µM), ruthenium red (20 µM) and SKF96365 (20 µM). Application of IGF-1 (50ng/ml) also led activation of a comparable membrane conductance with ten times larger amplitudes which was blocked by ruthenium red. In the presence of IGF-1 a rise in the temperature showed no significant further current increase. Application of cannabidiol (15 µM) increased the membrane conductance which was completely blocked by SKF96365. However, the presence of cannabidiol the heat-evoked increase in the membrane conductance was significant larger than that under control conditions. Analysis of the surface expression of TRPV2 channel protein by means of confocal microscopy showed that both cannabidiol and IGF-1 increased its surface expression. Furthermore, both, the cannabidiol-dependent increase in membrane conductance or the cannadidiol-dependent increase in TRPV2 surface expression were sensitive to the PI3-kinase blocker LY294002 (30 µM).

Conclusions: TRPV2 channel is expressed in vivo and in vitro by RPE cells. The surface expression of TRPV2 channels regulated by a PI3-kinase-dependent mechanism which is an important modulator of angiotensin-2 or IGF-1 signaling. IGF-1 signaling requires both increased surface expression and concomitant direct activation of TRPV2 channels.

Keywords: 569 ion channels • 701 retinal pigment epithelium • 439 calcium