INTRODUCTION Plasma membrane proteins are generally trafficked from the ER to the cell surface via the secretory pathway. Connexins (Cx), the subunit proteins of gap junction communication channels are thought to progress along this canonical pathway during which oligomerisation into hexameric connexon hemichannels occurs (Evans et al., 1999; Musil and Goodenough, 1991; Yeager et al., 1998), a process occurring concurrently or soon after co-translational folding during protein insertion into the endoplasmic reticulum (Diez et al., 1999; Falk et al., 1997). Gap junctions are ubiquitous intercellular channels underpinning direct cell to cell communication and tissue integration (Kumar and Gilula, 1996). About 20 different connexins have been identified in mammalian genomic maps. All connexins are believed to maintain a common topography in the membrane with four transmembrane domains, cytoplasmic N- and C-termini, and one intracellular and two extracellular loops. Connexins vary mainly in the amino acid sequences in the cytoplasmic intracellular loop and the carboxyl tail with the length of the latter varying from 16 amino acids in the nonphosphorylated Cx26 to 156 amino acids in hyperphosphorylated Cx43. The docking of hexameric connexons generates a channel that transfers cytoplasmic ions and molecules <1200 Da across the intercellular gap at cell- cell contact regions. (Nicholson et al., 2000; Bukauskas et al., 2000). Multiple determinants govern connexin oligomerisation, which may result in the formation of homo or heteromeric connexons. This is an area of intense interest, especially since mutations detected in connexin communicationopathies such as Charcot-Marie Tooth X-linked disease, sensori-neural deafness and skin abnormalities appear to influence connexin trafficking and their assembly into gap junction channels (Deschenes et al., 1997; Martin et al., 1999; Martin et al., 2000a; Martin and Evans, 2000; VanSlyke et al., 2000; Castro et al., 1999). Connexins are characterised by rapid turnover rates (half-lives of 1-4 hours) (Laing and Beyer, 2000; Laird et al., 1995). The synthesis and breakdown of gap junctions are complex processes influenced by hormones that can induce a rapid increase in gap junction expression, especially those constructed predominantly if not exclusively of Cx26 (e.g. in breast during lactation) (Monaghan et al., 1994; Locke et al., 2000), and in livers subject to hormonal activation (Kojima et al., 1994; Kojima et al., 1996). In Cx32-knockout mice, the circadian expression of Cx26 and acute phase responses are not influenced by abolishing Cx32 expression (Temme et al., 2000). These results in tissues co-expressing Cx32 and Cx26 suggest that the presence of independent gap junction assembly pathways can provide a mechanism that accounts for such disparate expression profiles. Cx26 has previously been shown 3845 The assembly of gap junctions was investigated in mammalian cells expressing connexin (Cx) 26, 32 and 43 fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Targeting of Cx32-CFP and 43-GFP to gap junctions and gap junctional communication was inhibited in cells treated with Brefeldin A, a drug that disassembles the Golgi. However gap junctions constructed of Cx26-GFP were only minimally affected by Brefeldin A. Nocodazole, a microtubule disruptor, had little effect on the assembly of Cx43-GFP gap junctions, but perturbed assembly of Cx26- GFP gap junctions. Co-expression of Cx26-YFP and Cx32- CFP in cells treated with Brefeldin A resulted in assembly of gap junctions constructed of Cx26-YFP. Two amino acids that distinguish Cx26 from Cx32 in transmembrane domains were mutated in Cx32 to investigate underlying mechanisms determining trafficking routes to gap junctions. One mutation, Cx32I28L, conferred on it partial Cx26-like trafficking properties as well the post- translational membrane insertion characteristics of Cx26, suggesting that a key determinant regulating trafficking was present in the first transmembrane domain. The results provide a protein trafficking basis for specifying and regulating connexin composition of gap junctions and thus selectivity of intercellular signaling, with Cx32 and 43 trafficking through the secretory pathway and Cx26 also following an alternative pathway. Key words: Connexin-fluorescent protein chimera, Gap junction, Trafficking pathways. SUMMARY Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap junction intercellular communication channels Patricia E. M. Martin* ,‡ , Geraldine Blundell, Shoeb Ahmad, Rachel J. Errington and W. Howard Evans ‡ Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK *Present address: Department of Radiology, Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK ‡ Authors for correspondence (e-mail: martinpe@cf.ac.uk) Accepted 24 July 2001 Journal of Cell Science 114, 3845-3855 (2001) © The Company of Biologists Ltd RESEARCH ARTICLE