Abstract
Abstract: :
Purpose: The plasma membrane Ca2+-ATPase (PMCA) is an integral membrane protein essential to the control of [Ca2+]i. Four genes known as ATP2B1 - ATP2B4 encode PMCA proteins termed PMCA1 - PMCA4, respectively, with tissue-specific isoform expression allowing tissues to adapt to specific Ca2+ handling requirements. The purpose of this investigation is to characterize, for the first time, PMCA isoform expression and distribution in corneal epithelium. Methods: Native and cultured rabbit corneal epithelial cells were examined. Western blot analysis was used to identify and quantitate PMCA expression using an antibody (mAb 5F10) that recognizes all four PMCA isoforms, and isoform specific antibodies (Abs). Distribution of PMCA in cryostat sections of rabbit cornea was determined by immunohistochemistry with mAb 5F10. To identify PMCA splice variants expressed in corneal epithelium, RT-PCR using gene specific primers was applied to total RNA extracted from epithelial samples. Resultant PCR DNAs were sequenced. Results: In Western blot experiments, a labeled band of approximately 150 kD was recognized for PMCA1 and PMCA4 in native and cultured rabbit corneal epithelium. The PMCA3 Ab labeled a band of approximately 233 kD and numerous smaller bands (<142 kD) in both native and cultured tissue. No bands were identified for PMCA2. Cryostat sections of whole rabbit cornea showed strong immunoreactivity to mAb 5F10. Immunoreactivity was localized to the epithelial cell plasma membrane with the heaviest labeling in the basal cell and wing cell layers, and lesser staining in the squamous cell layer. PCR at site A detected PMCA1 and PMCA4 transcripts. Direct sequencing of the 450 bp product identified that PCR cDNA fragments corresponded to exons 6, 7, and 8 of the A-site x-variant for PMCA1. These data represented significant homology with human PMCA1 and with PMCA1 from rabbit smooth muscle. Direct sequencing of the 311 bp and 602 bp site A products corresponded to the x-variant for PMCA4, again with significant homology to human PMCA4. Conclusions: PMCA1, 3 and 4 are expressed in rabbit corneal epithelium. Of the bands demonstrated for PMCA3, the higher molecular weight bands may represent oligomerization of PMCA3 protein controlled by the CaM-binding domain (Carofoli 1991). PMCA1 and PMCA4 are characterized as the A-site x-variants. Possible roles for PMCA activity in corneal epithelial biology, including wound healing, will be discussed.
Keywords: 334 calcium • 370 cornea: basic science • 372 cornea: epithelium