Abstract
The innexins ShakingB Lethal (SBL) and ShakingB Neural+16 (SBN16) form heterotypic gap junctions in the Giant Fiber System of Drosophilia melanogaster. The junctions favor arthodromic transmission of action potentials and exhibit properties of rectification when expressed in vivo. Sequence analysis reveals that SBL and SBN16 are similar in size but differ significantly in sequence within the amino terminus (NT), first transmembrane domain (TM1) and first extracellular loop (EL1). It was previously shown that the NT carries properties required for rectification in the sense that homotypic ShakBL junctions can be induced to rectify by replacing the NT of SBL with that of SBN16 in one of the cells. While this result provided a strong first impression about the importance of the NT in rectification, further experiments suggest complex interactions and structural features are required. This is highlighted by the behavior of the reciprocal chimera (SBN16 with the NT of SBL) which does not induce rectification when paired heterotypically with SBN16 but displays voltage-dependent gating similar to SBL when paired homotypically. We previously suggested that the NT but must be paired with an appropriate innexin body in order to induce voltage-dependent rectification and are further investigating features of the innexin channel essential for rectification. We are also investigating the role of various innexin domains in transjunctional (Vj)-dependent gating. Using chimeric constructs that focus on domains with significant sequence variation between SBL and SBN16 (namely NT, TM1 and E1) in combination with Xenopus oocyte expression and electrophysiology we hope to provide insight into the structural determinants of electrical rectification and Vj-dependent gating in innexin-based junctions. The attributes of using splice variants, which are unique to innexin-based gap junctions, will be discussed.
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