Expression and Localisation of Voltage-gated L-type Ca2+ ion Channels in Adult Human Retinal Pigment Epithelium

Abstract

Abstract: : Purpose: L-type channel currents have been described in freshly isolated and cultured retinal pigment epithelial (RPE) cells from various species. Recent data point out a role of L-type channels in growth factor secretion in RPE cells. To gain further information about the role of these channels for RPE function, we determined expression and localisation of L-type Ca²⁺-channel subunits α_1C and α_1D in adult human RPE. Methods: Human RPE cells in situ (frozen sections) and in vitro (cultured serum-supplemented or serum-free for up to 3 passages) were immunostained with polyclonal antibodies against α_1C and α_1D subunits. Specific staining was confirmed using corresponding blocking peptides. Antibody-labelling in retina sections was performed by LSAB-2, in cultured cells by immunofluorescence. Results: In vivo, human RPE cells mainly express α_1D subunits, which are localised in basolateral membranes. Proliferating RPE cells (cultured under serum supplementation) expressed both α_1D and α_1C subunits during subculture at least until passage 3. α_1D subunits were mainly localised in the cell membrane, whereas α_1C was mainly found in the endoplasmic reticulum. α_1D subunit expression decreased during subculture, while α_1C subunit expression increased. Quiescent, repigmented RPE cells (cultured serum-free) preferentially expressed Ca²⁺-channel subunit α_1D. In these cells α_1D subunits were localised in the basolateral membrane. Conclusions: In vivo and in vitro, human RPE cells express mainly the neuroendocrine subtype of L-type Ca²⁺-channels (corresponding with the α_1D subunit). In both, in vivo and in vitro, the Ca²⁺-channels are localised in the basolateral membrane. The expression and localisation of the correct L-type channel subtype is dependent on the cell cycle. The expression of the neuroendocrine subtype, which is localized in the basolateral membrane, implies that L-type channels in the RPE regulate secretion of growth factors to the basolateral side of the RPE.

Keywords: retinal pigment epithelium • ion channels • calcium