Expression and Immunolocalization of Plasma Membrane Calcium – ATPase Isoforms in Corneal Epithelium

Abstract

Abstract: : Purpose: The plasma membrane Ca²⁺–ATPase (PMCA) is an integral membrane protein essential to the control of [Ca²⁺]_i. Four genes encode PMCA proteins termed PMCA1 – PMCA4. Little is known about the expression of these isoforms in corneal epithelium (CE) or about the functional roles of PMCAs in corneal biology. The purpose of this investigation is to characterize the expression and distribution of PMCAs in CE, leading to the development of a model for the role(s) of PMCAs in corneal biology. Methods: Native human (h) and rabbit (rb) CE was examined. Western blot analyses were used to identify and quantitate PMCA expression using isoform–specific antibodies (Abs) and a panPMCA antibody that recognizes all PMCAs. Distribution of PMCAs in cryostat and paraffin sections of hCE was determined by immunohistochemistry with the same Abs. In vivo wound healing studies using rabbits were done using the n–heptanol technique and immunohistochemistry to determine the cellular location of PMCAs during the healing process of CE. Results: In Western blot experiments, bands of approximately 140 kD were recognized for PMCA1 and PMCA4. The PMCA2 Ab labeled a band of approximately 126 kD. Immunohistochemistry in hCE showed that basal cells expressed all four PMCAs. PMCA1 was mainly found on basal cell membranes. PMCA2 was concentrated on cytoplasmic membranes, and exhibited a punctate distribution on the cell and cytoplasmic membranes of wing cells. PMCA3 had a perinuclear location in hCE. PMCA4 was the major PMCA isoform expressed in hCE, and was predominantly expressed on plasma membranes of all cells in all layers of hCE. The in vivo studies revealed that during wound healing, PMCA4 redistributed from the cell membrane to cytoplasmic membranes in epithelial cells near the wound margin. Conclusions: PMCA isoforms are selectively expressed and distributed in CE reflecting possible underlying functional differences in the Ca²⁺ handling requirements in the various layers of CE.

Keywords: calcium • cornea: epithelium • ion transporters