Connexin: Difference between revisions
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=Differences between wild type and mutant connexin 26:= | =Differences between wild type and mutant connexin 26:= | ||
In general, single site mutations are spread fairly evenly across the whole protein with TM2 having the highest mutation density (number of amino acids with NHLS mutations divided by the total number of amino acids in the domain) at 67% to M1 and E1 having the lowest density of mutations with their respective domains at 33%. According to this criterion, TM4 has a mutation density of 40%. Of the four transmembrane helices, M1, M2 and M3 have attracted the most attention, because of the controversies involved in models with different helix assignments, based on lower resolution cryo-electron crystallographic structures and scanning cysteine accessibility mutagenesis. Far less is known about TM4 and how side chains interact with the other helices and with the lipid bilayer. <ref name='mutant | In general, single site mutations are spread fairly evenly across the whole protein with TM2 having the highest mutation density (number of amino acids with NHLS mutations divided by the total number of amino acids in the domain) at 67% to M1 and E1, having the lowest density of mutations with their respective domains at 33%. According to this criterion, TM4 has a mutation density of 40%. Of the four transmembrane helices, M1, M2 and M3 have attracted the most attention, because of the controversies involved in models with different helix assignments, based on lower resolution cryo-electron crystallographic structures and scanning cysteine accessibility mutagenesis. Far less is known about TM4 and how side chains interact with the other helices and with the lipid bilayer. <ref name='mutant int'/> | ||
Two structural crystallographic studies have been commenced on Cx26, the first one describing the WT protein in a resolution of 3.5 A⁰, and the second one deals with two types of mutations in the N terminus of the protein. | |||
Gap junction channels are unique in that they possess multiple mechanisms for channel closure, several of which involve the <scene name='70/701426/N_terminal/1'>N terminus</scene> (blue coloured) as a key component in gating, and possibly assembly. <ref name='pdb'>pmid 21094651</ref> | |||
The 3D structure of a mutant human connexin 26 <scene name='70/701426/Mutant_connexin26_-cx26m34a/1'>(Cx26M34A)</scene> channel shows an unexpected density within the vestibule of each hemichannel compared to the <scene name='70/701426/Wild_type_connexin/1'>wild type connexin 26</scene> , which is called a plug <ref name='pdb'/> , That plug was decreased in the the human mutant connexin 26 <scene name='70/701426/Deletion_of_cx26m34adel2-7/1'<(Cx26M34A4adel2-7)</scene> structure, indicating that the N terminus significantly contributes to form this plug feature. Experiments with this mutant show significantly reduced dye coupling between [http://en.wikipedia.org/wiki/HeLa HeLa cells] transiently expressing Cx26M34A gap junctions. <ref name='pdb'/> | |||
Functional analysis of the Cx26M34A channels revealed that these channels are predominantly closed, with the residual electrical conductance showing normal voltage gating. N-terminal deletion mutants with and without the M34A mutation showed no electrical activity in paired Xenopus oocytes and significantly decreased dye permeability in HeLa cells. Comparing this closed structure with the published X-ray structure of wild-type Cx26, which is proposed to be in an open state, revealed a radial outward shift in the transmembrane helices in the closed state, presumably to accommodate the N-terminal plug occluding the pore. Because both Cx26del2-7 and Cx26M34Adel2-7 channels are closed, the N terminus appears to have a prominent role in stabilizing the open configuration. <ref name='pdb'/> | |||