Intracellular domains of mouse connexin26 and-30 affect diffusional and electrical properties of gap junction channels

To evaluate the influence of intracellular domains of connexin (Cx) on chan nel transfer properties, we analyzed mouse connexin (Cx) Cx26 and Cx30, whi ch show the most similar amino acid sequence identities within the family o f gap junction proteins. These connexin genes are tightly linked on mouse c hromosome 14. Functional studies were performed on transfected HeLa cells s tably expressing both mouse connexins. When we examined homotypic intercell ular transfer of microinjected neurobiotin and Lucifer yellow, we Found tha t gap junctions in Cx30-transfected cells, in contrast to Cx26 cells, were impermeable to Lucifer yellow. Furthermore, we observed heterotypic transfe r of neurobiotin between Cx30-transfectants and HeLa cells expressing mouse Cx30.3, Cx40, Cx-43 or Cx45, but not between Cx26 transfectants and HeLa c ells of the latter group. The main differences in amino acid sequence betwe en Cx26 and Cx30 are located in the presumptive cytoplasmic loop and C-term inal region of these integral membrane proteins. By exchanging one or both of these domains, using PCR-based mutagenesis, we constructed Cx26/30 chime ric cDNAs, which were also expressed in HeLa cells after transfection. Homo typic intercellular transfer of injected Lucifer yellow was observed exclus ively with those chimeric constructs that coded for both cytoplasmic domain s of Cx26 in the Cx30 backbone polypeptide chain. In contrast, cells transf ected with a construct that coded for the Cx26 backbone with the Cx30 cytop lasmic loop and C-terminal region did not show transfer of Lucifer yellow. Thus, Lucifer yellow transfer can be conferred onto chimeric Cx30 channels by exchanging the cytoplasmic loop and the C-terminal region of these conne xins. In turn, the cytoplasmic loop and C-terminal domain of Cx30 prevent L ucifer yellow transfer when swapped with the corresponding domains of Cx26, In chimeric Cx30/Cx26 channels where the cytoplasmic loop and C-terminal d omains had been exchanged, the unitary channel conductance was intermediate between those of the parental channels. Moreover, the voltage sensitivity was slightly reduced. This suggests that these cytoplasmic domains interfer e directly or indirectly with the diffusivity, the conductance and voltage gating of the channels.