Parallel Detection of Na,K-ATPase Alpha Subunit Isoforms by Pan-Specific Monoclonal mAb 9A7

Abstract

While emphasis has been placed upon those proteins which either mediate or respond to the rapid influx of calcium following depolarization, there has been little emphasis upon those proteins which aid in the reequilibration of the membrane potential. In an effort to identify presynaptic membrane proteins implicated in neurosecretion, monoclonal antibodies were screened against proteins which cosegregated with neuronal voltage-dependent calcium channels (VDCC) following immunoprecipitation. One monoclonal antibody (mAb 9A7) identified a 110-kDa protein. Micropeptide sequencing of (i) the mAb 9A7 immunoaffinity purified antigen and (ii) the 110-kDa protein present in the neuronal (N-type) VDCC preparation (McEneryet al.,1991,Proc. Natl. Acad. Sci.88, 11095–11099) indicated identity with the alpha subunit(s) of the Na,K-ATPase. Further characterization by Western blotting, immunochemical localization, and immunoaffinity purification indicated that mAb 9A7 not only recognized the alpha3 isoform which is predominant in neuronal tissues but also identified the alpha1 and alpha2 isoforms. mAb 9A7 exhibited a wide cross-species reactivity and recognized human, rat, and mouse alpha subunit isoforms at an internal epitope. The pan-specificity of mAb 9A7 and the differential mobility of the alpha1 isoform relative to the alpha2 and alpha3 permitted parallel detection of multiple alpha isoforms. Western blot analysis of undifferentiated rat pheochromocytoma cell line (PC12) and human neuroblastoma (IMR32) cells indicated coexpression of the alpha1 and alpha3 isozymes. Upon differentiation of IMR32 cells by dibutrylyl-cAMP, a substantial increase in the alpha3 relative to the alpha1 isoform was observed. While the enrichment of total Na,K-ATPase may reflect the increased demand for ATP-dependent ion transport as IMR32 cells become more excitable, the specific increase in the alpha3 isoform suggests a unique role of this isoform during IMR32 cell differentiation.