Tetracycline-regulated expression enables purification and functional analysis of recombinant connexin channels from mammalian cells

Abstract

Intercellular coupling mediated by gap junction channels composed of connexin protein underlies numerous physiological processes, such as cellular differentiation, tissue synchronization and metabolic homoeostasis. The distinct molecular permeability of junctional channels composed of different connexin isoforms allows cellular control of coupling via regulation of isoform expression. However, the permeability properties of most connexin isoforms have not been well characterized due to the difficulty of manipulating and measuring the diffusible concentrations of cytoplasmic messenger molecules and metabolites, and to a lack of control over channel isoform composition, in vivo. Here we present a method to express and purify active connexin hemichannels of a single isoform or a consistent ratio of two isoforms from cultured cells using the Tet-On inducible expression system and one-step anti-haemagglutinin immunoaffinity purification. The procedure yields 10-20 microg of pure connexin protein from 2.5x10(8) HeLa cells. The purified channels are shown to be useful for in vitro permeability analysis using well established techniques. This method has substantial advantages over existing methods for heterologous connexin expression, such as the ease of co-expression of two isoforms at a constant ratio, consistently high expression levels over many passages, and the ability to study channel properties in situ as well as in purified form. Furthermore, the generic cloning site of the new pBI-GT vector and the commercial availability of anti-haemagglutinin (clone HA-7)-agarose make this affinity tagging and purification procedure easily applicable to other proteins.

Connexin 32 and its derived homotypic gap junctional intercellular communication inhibit the migration and invasion of transfected HeLa cells via enhancement of intercellular adhesion

“…HeLa cells that express Cx32 under the control of a bidirectional tetracycline-inducible promoter were described and characterized previously (13). In this stably transfected cell line, the Cx32 coding sequence is followed by a sequence coding for a thrombin-cleavable COOH-terminal epitope tag consisting of an HA epitope followed by a 6 x (His-Asn) sequence.…”

“…The so-called 'parachute' assay for GJIC was performed as described by Koreen et al (13). Donor and receiver cells were grown to confluence.…”

Inhibition of gap junctions relieves the hepatotoxicity of TNF-α

“…After the formation of stable GJs, the small molecule calcine-AM (which exhibits green fluorescence) from the "donor cells" will enter the adjacent cell (receiver cells) through the GJ; this can be observed using a fluorescent inverted microscope. Recording the number of receiver cells around a donor cell serves as an index of GJ function (Koreen et al, 2004;Wentlandt et al, 2005). To observe the effects of GJ inhibition on calcine-AM transfer, oleamide was dissolved with DMSO to a final concentration of 25 μM and added to the culture.…”

The Effects of 2-Aminoethoxydiphenyl Borate and Diphenylboronic Anhydride on Gap Junctions Composed of Connexin43 in TM4 Sertoli Cells

“…27,28) Donor and receiver cells were grown to confluence. The donor cells were double-labeled with 5 mM CM-DiI, a membrane dye that does not spread to coupling cells, and 5 mM calcein-acetoxymethyl ester, which is converted into calcein in intracellular plasma to be GJ-permeable, for 30 min at 37°C.…”