Functional characterization of the NCC27 nuclear protein in stable transfected CHO-K1 cells

NCC27 belongs to a family of small, highly conserved, organellar ion channe l proteins. It is constitutively expressed by native CHO-K1 and dominantly localized to the nucleus and nuclear membrane. When CHO-K1 cells are transf ected with NCC27-expressing constructs, synthesized proteins spill over int o the cytoplasm and ion channel activity can then be detected on the plasma as well as nuclear membrane. This provided a unique opportunity to directl y compare electrophysiological characteristics of the one cloned channel, b oth on the nuclear and cytoplasmic membranes. At the same time, as NCC27 is unusually small for an ion channel protein, we wished to directly determin e whether it is a membrane-resident channel in its own right. In CHO-K1 cel ls transfected with epitope-tagged NCC27 constructs, we have demonstrated t hat the NCC27 conductance is chloride dependent and that the electrophysiol ogical characteristics of the channels are essentially identical whether ex pressed on plasma or nuclear membranes. In addition, we show that a monoclo nal antibody directed at an epitope tag added to NCC27 rapidly inhibits the ability of the expressed protein to conduct chloride, but only when the an tibody has access to the tag epitope. By selectively tagging either the ami no or carboxyl terminus of NCC27 and varying the side of the membrane from which we record channel activity, we have demonstrated conclusively that NC C27 is a transmembrane protein that directly forms part of the ion channel and, further, that the amino terminus projects outward and the carboxyl ter minus inward. We conclude that despite its relatively small size, NCC27 mus t form an integral part of an ion channel complex.-Tonini, R., Ferroni, A., Valenzuela, S. M., Warton, K., Campbell, T. J., Breit, S. N., Mazzanti, M. Functional characterization of the NCC27 nuclear protein in stable transfe cted CHO-K1 cells.