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TRANSMEMBRANE HELIX 6 (TM6)
GIILRKIFTTISFCIVLRMAV
(amino acids 330 thru 350)
This helix was the first one suggested (by Anderson, 1991) to be involved in forming pore. For information on pore, see helix1.
Mutations at several positions in TM6 affect biophysical parameters such as single-channel conductance, rectification and anion selectivity. TM6 and TM12 are the two most conserved helices, in terms of sequence, when comparing CFTRs from different species. TM6 is the most conserved helix and probably lines the pore. TM6 more than any other helix controls pore properties of CFTR.
If it turns out that CFTR has a discrete "ion selectivity filter", it will probably be located within TM1-TM6. Amino acid residues K335, F337, T338, and R347 in TM6, when mutated, significantly affect anion selectivity.
Point mutations in this helix, helix 1 and helix 5 have been shown to affect anion binding by CFTR. However, permeability ratios of ions was not changed. For this reason, it is believed that anion binding is the process most affected by structural changes. TM5 and 6 are likely to contribute to part of the pore.
Lysine (95?) and Lysine (335?) were mutated in this helix to aspartate and glutamate. Ion selectivity was changed such that iodine was favored over chloride. The arginine at position 334 is mutated in some CF alleles to glutamine or tryptophan, while the arginine found at 347 is mutated to cysteine, histidine, or proline in other naturally occurring mutations. This suggests a probable role important in pore function. The conductance of the pore was reduced to less than 30% of wild-type. It's interesting that when the mutation resulting in a histidine at position 347 was restored to normal wild type when the pH was dropped to 5.5 when histidine is positively charged. It probably binds the anion here.
Residues R334/K335 and R347 are believed based on mutagenesis data to be important in forming the pore, either directly or indirectly. R347D mutation does not bind DNDS or DIDS as well as wild-type. When there is a double mutation in two residues of helix 6 (T338A and T339C), larger anions are able to permeate thru the channel. There is also an increase in conductance of chloride. Interestingly, when mutated to cysteines, there was no reaction with MTS reagents, which suggests these amino acids do not directly line the pore. This means it's possible the backbone of helix 6 is the most important factor for determining anion selectivity.
11 residues were found by cysteine-accessibility method to be lining the pore. They are K329, I331, L333, R334, K335, F337, S341, I344, R347 , T351, R352, and Q353. The residue R352 was determined to be a site near the intracellular end of the pore and was involved in selectivity for anions over cations. It is known that the intracellular end of the predicted pore of CFTR presents a current barrier. Cheung and Akabas have suggested that R352 is involved in forming a pore loop which has the responsibility of helping with anion selectivity, while the rest of the M6 is in the alpha-helical form.
Channel-lining water accessible residues have been found in transmembrane helices 1, 3, and 6 using the substituted cysteine accessibility method.
Arg-352 is at cytoplasmic end of helix 6 and is believed to be important in charge discrimination within the pore. The pore probably narrows near this residue to form an electrostatic barrier to further transport of ions.
Arg-347 in M6 forms a salt bridge with Asp-924. Mutants R347D show different multiple anion effects. It's possible disruption of the salt bridge causes change in structure of an anion binding site elsewhere. Arg-347 block by SCN- is predicted based on electric field studies to be ~20% of the way thru pore from intracellular side, which is in good agreement with predicted structure based on hydropathy analysis.
The amino acids from Threonine 351 to Glutamine 353, which form the part of the helix near the cytoplasm, are believed to be part of the pore where it narrows and therefore may be involved in the selectivity filter which selects for anions over cations.
S341 is the major determinant of the channel inhibitor DPC binding to CFTR. Electric field studies indicate block occurs at this amino acid ~40% of way thru pore from intracellular side.
Mutation of Lys95 (in TM1) and Lys335 to negatively charged amino acids changes CFTR anion selectivity from Br>Cl>I>F to I>Br>Cl>F. Arg347 in this helix when mutated this way does not change the anion selectivity very significantly. (also true for Arg1030 in TM10).
But Tabacharania in 1992 found that Arg347 mutations alter both conductance and changes CFTR to a single anion pore (it is usually a multi-ion pore). CF patients with less severe cases sometimes have a mutation at this arginine.
Cytoplasmic end of this helix probably is a major determinant of anion selectivity.
Simultaneous mutation of two residues near middle of this helix (T338A and T339A) increases single-channel conductance. This means they may be near narrowest part of pore. T338 mutations has extensive effects on anion selectivity and may be contributing to the region with highest selectivity for anions in the pore. S341 may also lie in the selectivity region. Mutations in it affect conductance, selectivity, and sensitivity to block by DPC, NPPB and glibenclamide (McDonough et.al. 1994).
There are 6 positively charged amino acid residues in the transmembrane helices of CFTR (K95 in M1, R134 in M2, R334 and K335 as well as R347 in M6, R1030 in M10) and they seem to be highly conserved between species.
There appears to be a shift in secondary structure of TM6 from helix to beta-sheet in different organic solvents.
Recent Evidence: More Pieces to the Puzzle?
In October, 2001, McCarty and Zhang of Emory University reported on a study
of the possible 3D structure of the pore of CFTR. They
compared the relative importance of various sites previously studied and
identified new sites that contribute strongly to anion selectivity, using
chloride and substitute anions in oocytes expressing wild-type cystic fibrosis
transmembrane conductance regulator or 12-pore-domain mutants, and determined
relative permeability and relative conductance for 9 monovalent anions and 1
divalent anion. Their data indicate that ".. a region of
strong discrimination resides between T338 and S341 in transmembrane 6, where
mutations affected selectivity between chloride and both large and small anions.
Mutations further toward the extracellular end of the pore only strongly
affected selectivity between chloride and larger anions. Only mutations at S341
affected selectivity between monovalent and divalent anions. The data are
consistent with a narrowing of the pore between the extracellular end and a
constriction near the middle of the pore." Am J Physiol Lung Cell
Mol Physiol 2001 Oct;281
In December, 2000 Kogan et al (from the Hospital for Sick Children, Toronto, Ontario) reported that there may be cross-talk between the pore of CFTR and the ATPase activity at the NBDs. They showed that the R347D mutation and diphenylamine-2-carboxylate (DPC is an open pore inhibitor) also inhibit CFTR ATPase activity. They also found that glutathione (GSH) inhibits CFTR ATPase activity and this inhibition is different than in the CFTR-R347D variant.
At the Biophysical Society Meeting, 2/2001, Liu et al presented evidence that T338 side chain lies within the pore. They used mutagenesis to produce R334C and T338C and used the charge-inducing reagents MTSET+ and MTSES- to modify CFTR while adjusting the pH. They also provided "important verification of the notion that bath pH changes modify the time-average charge on the cysteine at 334." At the same meeting, Zang and McCarty (Emory University) presented a model-independent approach they used, called alanine-scanning mutagenesis, in an attempt to identify residues in TM6 involved in anion selectivity. They concluded "[our] results suggest that a region of high discrimination resides between T338 and S341...Mutations there affected selectivity to large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions."