Cloning and Heterologous Expression of Anoctamin Isoforms

Abstract

Anoctamin1 (Ano1) has Ca2+-activated Cl- channel (CaCC) character which has an important role in salivation. There are nine other members in the anoctamin family, and not much is known about them except Ano1. In this study, mouse Anoctamin9 (mAno9) and human Anoctamin6 (hAno6) gene were cloned, epitope tagged with enhanced green fluorescent protein (EGFP) or hemagglutinin (HA) tag to the C-terminus, and heterologously expressed in human embryonic kidney (HEK) 293 cell. Consequently, location of the expressed EGFP-linked anoctamin channel proteins in the cell was examined and proteins were extracted in order to detect anoctamin protein with EGFP-specific antibody. As a result, both mAno9 and hAno6 cDNA expression plasmids were heterologously expressed in HEK293 cell. mAno9 appeared to be intracellularly localized in an uniformly diffuse pattern, while hAno6 was expressed in both plasma membrane and intracellular compartments, suggesting distinct roles within the cellular compartments.