Expression and immunolocalization of plasma membrane calcium ATPase isoforms in human corneal epithelium.
OBJECTIVE
Plasma membrane Ca2+-ATPases (PMCAs) are integral membrane proteins essential to the control of intracellular Ca2+ ([Ca2+]i) concentration. Four genes encode PMCA proteins termed PMCA1-PMCA4. Little is known about the expression of these isoforms in corneal epithelium (CE). The purpose of this investigation is to characterize the expression and distribution of PMCAs in human CE (hCE).


METHODS
PMCA mRNA expression was examined by RT-PCR analysis of total RNA from native hCE using PMCA gene specific primers. PMCA isoform expression at the protein level in native hCE was examined by immunoblotting using isoform specific antibodies (Abs) and a panPMCA Ab that recognizes all PMCAs. Distribution of PMCAs in postmortem and surgical sections of hCE was determined by immunohistochemistry with the same Abs.


RESULTS
Immunoblot analysis with the panPMCA Ab yielded an intense band of approximately 135 kDa and several faintly staining bands above and below this major band. The isoform specific Abs labeled one or more bands that corresponded to bands detected with the panPMCA Ab. RT-PCR analysis of total RNA from hCE yielded PCR DNAs that were identified by sequencing as products of PMCA1, PMCA2, PMCA3, and PMCA4, thus confirming the immunoblot data. Immunohistochemistry demonstrated localization of PMCAs in all layers of hCE. PMCA4 was the predominant isoform, and was expressed along the plasma membrane of cells in all layers of CE, except with a notable absence along the basal cell membranes adjacent to the stroma. PMCA1 and PMCA2 were found mainly on basal and wing cells. In contrast to PMCA4, PMCA1 immunoreactivity (IR) was located on portions of basal cell plasma membranes adjacent to the stroma. PMCA2 IR was detected cytoplasmically within basal and wing cells in both central cornea and limbus. PMCA3 IR was located in basal cell nuclei in central cornea, but in a perinuclear location in the limbal, basal, and wing cells.


CONCLUSIONS
Human CE expresses multiple PMCA isoforms that are differentially expressed and localized among the layers and cells that comprise the CE. We propose that the differential expression of multiple PMCA isoforms affords CE the requisite flexibility to respond to the demands for Ca2+ regulation required during renewal and regeneration of its multiple cell types.
Talarico
Ernest F
EF
Department of Anatomy & Cell Biology, Indiana University School of Medicine-Northwest, Gary, IN 46408-1197, USA.
Kennedy
Brian G
BG
Marfurt
Carl F
CF
Loeffler
Karin U
KU
Mangini
Nancy J
NJ
D000368:Aged; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D027682:Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D019573:Epithelium, Corneal / Q000201:enzymology*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes; D008297:Male; D008875:Middle Aged; D053499:Plasma Membrane Calcium-Transporting ATPases; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
C506952:ATP2B1 protein, human; D000911:Antibodies, Monoclonal; D027682:Cation Transport Proteins; D007527:Isoenzymes; D012333:RNA, Messenger; D053499:Plasma Membrane Calcium-Transporting ATPases; C506958:ATP2B2 protein, human; C506962:ATP2B3 protein, human; D000252:Calcium-Transporting ATPases; D002118:Calcium