Altered formation of hemichannels and gap junction channels caused by C-terminal connexin-32 mutations.
Hexamers of connexins (Cxs) form hemichannels that dock tightly in series via their extracellular domains to give rise to gap junction channels. Here we examined the ability of a variety of C-terminal Cx32 mutations, most of which have been identified in X-linked Charcot-Marie-Tooth disease, to form hemichannels and to complete gap junction channels using the Xenopus oocyte system. First, we show that undocked wild-type Cx32 hemichannels at the plasma membrane can be detected as opening channels activated by depolarization. We have been able to estimate the efficiency of assembly of complete channels by measuring the time-dependent incorporation of preformed hemichannels into gap junction channels after cell-to-cell contact. These data offer strong evidence that hemichannels are the direct precursors of gap junction channels. Of 11 Cx32 mutants tested, a group of 5 mutations prevented the formation of functional hemichannels at the cell surface, whereas 4 mutations were fully able to form precursors but reduced the ability of hemichannels to assemble into complete channels, and 2 mutants formed channels normally. The data revealed that a minimum length of human Cx32 including the residue Arg-215 is required for the expression of hemichannels at the cell surface and that the efficiency of hemichannel incorporation into complete channels decreased gradually with the progressive shortening of the cytoplasmic C-terminal domain.
Castro
C
C
Unidad de Neurología Experimental-Consejo Superior de Investigaciones Científicas, Departamento de Investigación, Hospital "Ramón y Cajal," 28034 Madrid, Spain.
Gómez-Hernandez
J M
JM
Silander
K
K
Barrio
L C
LC
D000818:Animals; D002607:Charcot-Marie-Tooth Disease / Q000235:genetics / Q000503:physiopathology; D017630:Connexins / Q000235:genetics*; D005260:Female; D017629:Gap Junctions / Q000502:physiology*; D006801:Humans; D015640:Ion Channel Gating; D008564:Membrane Potentials / Q000502:physiology; D009154:Mutation; D018408:Patch-Clamp Techniques; D014981:Xenopus; D000097002:Gap Junction beta-1 Protein
D017630:Connexins; D000097002:Gap Junction beta-1 Protein