A study of the pore of gap junction hemichannels
Date of Award
Doctor of Philosophy (Ph.D.)
Physiology and Biophysics
First Committee Member
Gerhard Dahl - Committee Chair
Gap junctions are cell to cell channels that mediate intercellular communication. Each gap junction channel is made by docking of two hemichannels. Hemichannels are composed of subunits named connexins. More than 20 connexins have been identified in vertebrates. Connexins proteins have four transmembrane segments. The topology of connexins has been determined through extensive studies, but little is known about functional domain assignments.In the present study, pore structures of hemichannels are studied by swapping the putative pore lining domains between two connexins, Cx46, a rat lens connexin, and Cx32E143, a chimera made by replacing E1 of Cx32 with E1 of Cx43. Both Cx46 and Cx32E143 make open hemichannels and their single channel properties are distinct. There is a five amino acid difference between M1 (the first transmembrane domain) of Cx46 and Cx32E143. We find that both the unit conductance and channel gating properties are transferable by swapping the M1 domain. Swapping the second half of M1 from Cx32E 143 to Cx46 results in a channel (Cx46M132II) that displays also the unit conductance and gating properties of the M1 donor, Cx32E 143. However, swapping the first half of M1 yields a mutant, Cx46M 132I, which retains the channel properties of the M1 recipient, Cx46. It is known from the previous study that replacing leucine35 (located in the second half of M1 segment) in Cx46 with cysteine results in a mutant with reduced conductance when reacted with thiol reagent. I thus replaced leucine with glycine. This mutation results in a channel with larger single channel conductance, higher open probability and larger cut off limit for tracer molecule. We conclude that M1 domain, particularly the second half of M1, contains a conductance module and a gating module.
Hu, Xinge, "A study of the pore of gap junction hemichannels" (2001). Dissertations from ProQuest. 1717.