C. elegans AVA command interneurons play important roles in escape behavior, and contact A-type cholinergic motor neurons (A-MNs) through both electrical and chemical synapses. Our recent study shows that the gap junctions (GJs) between AVA and A-MNs only allow antidromic currents (from A-MNs into AVA), and that the function of these GJs depends on UNC-7 innexin in AVA and UNC-9 innexin in A-MNs (Liu et al., Nat Commun 2017). However, molecular basis of the antidromic rectification is unknown. To address this question, we began by expressing UNC-7 and UNC-9 in Xenopus oocytes, and analyzing biophysical properties of homotypic and heterotypic GJs formed by them. While UNC-9 has only one isoform, UNC-7 has at least three different isoforms (UNC-7a, UNC-7b and UNC-7c), which differ in the length of the amino terminal. UNC-7c has a short amino terminal (24 residues before the 1st membrane-spanning domain/TM1) like UNC-9 (26 residues before TM1) whereas UNC-7a and UNC-7c have additional 120 and 52 residues, respectively, before the first amino acid of UNC-7c. We recorded junctional currents (Ij) from paired oocytes by holding one oocyte at a constant voltage (-30 mV) while applying voltage steps of -150 mV to +50 mV (at 10-mV intervals) to the other oocyte. The voltage difference between the two oocytes is the junctional voltage (Vj). We measured the steady-state Ij at all the Vj steps (-120 to +120 mV), plotted the Gj - Vj relationship, and fitted the Gj - Vj relationship to a Boltzmann function. We found that homotypic GJs of UNC-9 and UNC-7c are similar in the Gj - Vj relationship but are very different from those of UNC-7a and UNC-7b. Although all the UNC-7 isoforms may form heterotypic GJs with UNC-9, only one of them can form heterotypic GJs that allow unidirectional current flow (from UNC-9 oocyte to UNC-7 oocyte). Expression of this specific UNC-7 isoform in AVA interneurons in an unc-7
mutant significantly restored the antidromic junctional currents between AVA and A-MNs whereas the other two UNC-7 isoforms had either no effect or a much weaker effect. Taken together, our results suggest that 1) the amino terminal domain of UNC-7 plays important roles in GJ gating; 2) GJs between AVA and A-MNs probably consist of UNC-9 in A-MNs and a specific UNC-7 isoform in AVA; and 3) interactions between UNC-7 and UNC-9 hemichannels can reciprocally influence their gating properties.