BACK TO DIAGRAM CFTR REVIEW PAGE
N TERMINAL END (amino acids 1 thru 80)
MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSV
DSADNLSEKLEREWDRELASKKNPKLINALRRC
The only high-resolution x-ray crystal structure of an ABC transporter family member is for that of MsbA (4.5 Å). It shows part of the N-terminal portion of MsbA as forming a helix (residues 10-21) parallel with the lipid bilayer. MsbA has residues 10-Trp, 13-Phe, and 17-Trp intercalating into the inner leaflet side of the cell membrane. Chang and Roth, Science 293, 9/7/01 pgs 1793-1800.
The N-terminal end of CFTR has been found to interact with a protein called syntaxin 1A. This effectively couples CFTR regulation to membrane traffic machinery. Syntaxin 1A also inhibits channel (at least in part) directly. Syntaxin 1A was first found in the brain neurons and is an integral protein important for fusion of synaptic vesicles to the presynaptic cell membrane. It has since been found at lower levels in airway and gut epithelia. It is therefore part of the protein complexes involved in membrane trafficking. The family of proteins it is part of is called T-SNARES (or Q-SNARES). The CFTR binding site on syntaxin 1A maps to the third predicted helical domain (H3) of the syntaxin 1A membrane protein. Naren, et al. Proc Natl Acad Sci U S A 1998 Sep 1;95(18):10972-7.
When syntaxin 1A is coexpressed in xenopus oocytes with CFTR, it is found that CFTR current activity is inhibited. It was also found that by adding Munc-18a (known to bind syntaxin well), CFTR activity was restored. Inhibitors of syntaxin 1A also worked this way in colonic epithelial cells.
Syntaxin 1A is the only isoform tested which interacts with CFTR. It does so in a 1 to 1 ratio. The N-terminal of CFTR normally interacts directly with the R-domain, and syntaxin 1A seems to block this interaction.
This interaction may serve to coordinate the regulation of vesicle trafficking with chloride transport as is seen with N and L-type calcium channels. There is also a plant syntaxin which regulates potassium and chloride channels in guard cells of the leaf.
The negatively regulated interaction with syntaxin 1A makes this a possible target for future drug treatments. It may provide a very specific treatment which effects only CFTR and not other ion channels, and may therefore not have the unintended side effects often seen when new drugs are discovered.
Munc-18, another protein involved in vesicle fusion, reciprocally regulates CFTR channel function with syntaxin 1A. It relieves inhibition by syntaxin 1A. It probably also interacts at the N-terminal region.
There is a highly conserved and highly charged motif from D44 thru E60 in the N-terminal region of CFTR. Within this region, D47, E51, E54, and D58 were found to be involved in regulation of channel gating via the R-domain (residues 708-740, which contain three highly conserved positively charged motifs). It is possible that the N-terminal tail directly controls the access of the activated (phosphorylated) R-domain to inhibitory or stimulatory sites within the pore region.
The first methionine codon in CFTR is not the only one capable of functioning as a start codon. Even methionines past amino acid position 150 may initiate translation. The methionine at position 281 functioned as a start site and produced functional CFTR, but with a lower conductance and similar anion selectivity. When mutations past TM4 were made, the channel was nonfunctional. When TM1-4 were removed, the reduction was only ~30% in conduction with no effect on ion selectivity. This suggests parts of TM1-4 line the pore but do not function in anion selectivity. They do not appear to function significantly in channel gating, since activation by PKA and ATP gave only lower than normal open probabilities.
CFTR has no apparent N-terminal signal peptide.
A Flag epitope was added to the NH(2) termini of full length CFTR by Chan KW; Csanady L; Seto-Young D; Nairn AC; Gadsby DC in August 2000.
Recent Evidence: More Pieces of the Puzzle?
Writing in the October 2001 Journal of Physiology, Fu, Narin, and Kirk (from
the UAB), state that a cystic fibrosis-associated mutant CFTR lacking one of the
acidic residues (D58N) in its N-terminal tail exhibited "lower macroscopic
currents in Xenopus oocytes and faster deactivation following washout of a cAMP
-activating cocktail than wild-type CFTR."
There was also a lower open probability attributed to shortened open channel
bursts. They then replaced the D58 and two nearby acidic
residues with alanines (D47A, E54A, D58A), and these changes also reduced
channel activity and had almost no effect on PKA phosphorylation or on the ATP
dependence of channel activation. However, the
N-tail triple mutant "exhibited a markedly inhibited response to AMP-PNP, a
poorly hydrolysable ATP analogue that can nearly lock open the wild-type
channel." They also stated that "the
N-tail mutant had both a slower response to AMP-PNP and a lower steady-state
open probability following AMP-PNP addition. When they
introduced the N-tail mutations into K1250A CFTR, an NBD2 hydrolysis mutant that
normally exhibits very long open channel bursts, it destabilized the activity of
this mutant as evidenced by decreased macroscopic currents and shortened open
channel bursts." They conclude by proposing that
"...this cluster of acidic residues modulates the stability of CFTR channel
openings at a step that is downstream of ATP binding and upstream of ATP
hydrolysis, probably at NBD2." J Physiol 2001 Oct 15;536(Pt
2):459-70
In Septermer, 2001, Fu and Kirk (of the University of
Alabama-Birmingham) determined that the inhibitory effect of the
N-terminal tail of CFTR is due to the loss of negative charge and that the
amino-terminal tail is also able to modulate other aspects of channel
gating. They introduced cysteines at two positions (E54C/D58C)
and then tested a series of methanethiosulfonate (MTS) reagents for their
effects on the gating properties of these cysteine mutants in intact Xenopus
oocytes and excised membrane patches. They reported that
"Covalent modification of these sites with either neutral (MMTS) or charged
(MTSET)) reagents markedly inhibited channel open probability primarily by
reducing the rate of channel opening. The MTS reagents had
negligible effects on the gating of the wild type channel or a corresponding
double alanine mutant (E54A/D58A) under the same conditions.
The inhibition of the opening rate of the E54C/D58C mutant channel by MMTS could
be reversed by the reducing agent dithiothreitol or by elevating the bath ATP
concentration above that required to activate maximally the wild type
channel. Interestingly, the three MTS reagents had
qualitatively different effects on the duration of channel openings (i.e.
channel closing rate), namely the duration of openings was negligibly changed by
the neutral MMTS, decreased by the positively charged MTSET, and increased by
the negatively charged MTSCE." They concluded by saying their
results indicated that "...the CFTR amino tail modulates both the rates of
channel opening and channel closing and that the negative charges at residues 54
and 58 are important for controlling the duration of channel
openings." J Biol Chem 2001 Sep 21;276(38):35660-8
"We report (April, 1999) the cloning of a cDNA encoding human syntaxin 8
(STX8), using the regulator (R) domain of the cystic fibrosis transmembrane
conductance regulator (CFTR) as a bait to screen a human fetal lung cDNA library
by the yeast two-hybrid system. This gene was found broadly transcribed and its
mRNA size is about 1.3 kb. The STX8 gene maps to chromosomal band 17p12 and it
encodes a 236-amino-acid protein. Syntaxin 8 contains in its C-terminal half a
coiled-coil domain found highly conserved in the t-SNARE (SNAP receptor on
target membrane) superfamily of proteins, which are involved in vesicular
trafficking and docking. In syntaxin 8, a C-terminal hydrophobic domain may
constitute a transmembrane anchor. It was recently shown that CFTR-mediated
chloride currents can be regulated by syntaxin 1A, a t-SNARE family member,
through direct protein-protein interaction. This raises the possibility that
syntaxin 8 may also be involved in such regulations." Thoreau et.al,
Biochem Biophys Res Commun 1999 Apr 13;257(2):577-83.
In October, 2000 Deken, Beckman, Boos, and Quick (of University of Alabama at Birmingham) found that the N-terminal cytoplasmic domain of the GABA transporter GAT1 was able to regulate substrate transport rates and that this domain was able to bind directly to syntaxin 1A and that this interaction caused a reduction in transporter transport rates.
Di et al., confirmed in February, 2001 using single-channel techniques in human airway epithelial cells that disrupting SNARE-CFTR interaction "results in increased CFTR chloride current via an increase in open-state probability." They also measured exocytosis using a dye as well as capacitive changes in the cell membrane and concluded that "this channel activity is not likely to result from increased fusion of vesicles containing preformed CFTR, but instead, may result from loss of current inhibition by Syntaxin 1A." Biophysical Society Meeting, 2/2001
Naren, et al in 1999 reported, using a mutant CFTR lacking the N-terminal tail, that the N-terminal tail is an important component of the CFTR gating mechanism. When adding the peptide corresponding to the tail, they got stimulation of the channel function. They mapped the activity to a cluster of negatively charged residues in the tail which turn out to be strictly conserved between species. They are D47, E51, E54, and D58. The write that "replacement of the negative charges eliminated the stimulatory effect of the N-tail peptide on the activity of the N-tail deletion mutant." They concluded: "the N-tail mutations influence CFTR gating in part by destabilizing the activated state of the channel....the amino terminal tail seems to exert its effects on CFTR chloride channel gating via an interaction with the R-domain" They found that a recombinant peptide (a.a. 595-855) containing the R-domain plus the distal portion of NBD1 binds the N-tail peptide in vitro with moderately high affinity." The N-tail needed an intact R-domain to control gating. "These findings support a model in which CFTR activity is stabilized by an interaction between the amino terminal tail and the R-domain and/or NBD1" They also pointed out that the N-terminal tails of several types of potassium channels (ex: Shaker) are able to take part in intramolecular interactions that modulate gating. "Perhaps the N-tail of CFTR stabilizes channel activity by controlling access of the phosphorylated R-domain to inhibitory or stimulatory sites within the channel, such as the NBDs". Science 286: 544-8