Regulation of CFTR gating (normal and CF)
The Cystic fibrosis transmembrane conductance regulator
(CFTR) is a chloride channel that belongs to the
ATP-binding cassette (ABC) superfamily .
Mutations in CFTR-encoding gene cause cystic fibrosis (CF),
a genetic disease characterized by defective transport of chloride ions across several
epithelial tissues , . CFTR
has two nucleotide binding domains (NBD1 and NBD2) that control channel gating by binding
and hydrolysis of ATP. Upon dimerization of the two NBDs of
CFTR in a head-to-tail configuration, two
ATP-binding pockets (ABP1 and ABP2) are formed with the
ATP molecules sandwiched at the interface .
Each ABP plays a different role in CFTR gating; ABP2 is the
site critical for the ATP-dependent opening of the
CFTR channel, whereas ATP
binding to ABP1 is believed to contribute to the stability of the open channel
conformation , . CFTR also
has a large centrally localized regulatory domain (R domain) that is a special feature of
this ABC protein , , , .
Pyrophosphate is the product of the reaction of
cAMP synthesis from ATP.
Pyrophosphate is well-known for its ability to modulate
CFTR gating. Together with ATP,
Pyrophosphate can lock CFTR in
a stable open state. It has been reported that high concentrations of
Pyrophosphate lead to inhibition of CTFR gating. However, it
is unclear if Pyrophosphate inhibits the process directly or
indirectly through its ability to chelate Mg2+ , , . It is shown that
Pyrophosphate potentiates CFTR
only in human, but not mouse .
CFTR channel activity is modulated by phosphorylation by
cyclic AMP-dependent Protein
kinase A (PKA) . Two gating modes have been
reported: CFTR channel open bursts are long in the presence
of PKA, but shorten upon PKA
removal, presumably reflecting rapid partial CFTR
dephosphorylation . The mechanisms by which
ATP and PKA together regulate
CFTR channel gating are complex and controversial. Thus,
phosphorylated CFTR behaves as a conventional ligand-gated
channel employing cytoplasmic ATP as a readily available
cytoplasmic ligand . ATP binding leads to
channel opening whereupon its hydrolysis prompts channel closing, and phosphorylation
acts like a switch to drive gating of the transmembrane ion pore .
CFTR phosphorylation affects
ATP binding and not the subsequent steps of hydrolysis and
channel opening , .
AMP-activated protein kinase (AMPK) can also
phosphorylate CFTR and thus lead to reduced secretion of
chloride ions by inhibition of the channel activity without affecting the number of
CFTR channels in the plasma membrane. The exact molecular
mechanism of this event is unknown , , .
G551D is the third overall most common CF mutation with a worldwide frequency of ~3%.
This mutation is associated with a severe phenotype characterized by pulmonary
dysfunction and pancreatic insufficiency , . G551 is
located in the signature sequence of NBD1that, together with the Walker A and B motifs of
NBD2, forms ABP2, a critical site for channel opening by ATP
. G551D mutation more likely hampers conformational changes at ABP2 that
facilitate NBD dimerization, i.e., channel opening by ATP
. G551D-CFTR exhibited a markedly reduced
ATPase activity , .
Furthermore, G551D-CFTR does not respond to
ADP or changes in Mg('2+)
concentration. The residual low activity of G551D-CFTR
represents ATP-independent gating events , .
Micromolar Cd('2+) and
Zn('2+) can dramatically increase the activity of
G551D-CFTR in the absence of
ATP. This effect of Cd('2+) and
Zn('2+) is not seen in wild-type channels .
Some CFTR potentiators are examined as correctors of gating. These potentiators are
Anthracene-9-carboxylic acid (9-Anthroic acid) , Phloxine B , ,
benzimidazolone analogs NS004  and
NS1619, Genistein , , ,
and VX-770 (Vertex Pharmaceuticals Inc.)
Many of these potentiators (e.g., Phloxine B , , benzimidazolone analogs NS004,
Genistein , ,
9-Anthroic acid ) can both stimulate, and
inhibit wild-type and mutant CFTR channel activity in a
dose-dependent manner. Phosphorylation status of CFTR is a
very important for action of potentiators on this channel,
Different CFTR domains may be important for action of different potentiators. It is
suggested, that Phloxine B, benzimidazolone analogs
NS004 and NS1619, and
Genistein stimulate CFTR via
interaction with NBD2 domain, and inhibit CFTR via binding
to NBD1 domain or by occluding the pore , .
9-Anthroic acid binding sites for potentiating and
inhibitory effects on CFTR channels are located outside of
the R-domain .
Curcumin strongly activates
G551D-CFTR channel. Stimulatory effect of
Curcumin does not require dimerization of the two NBDs.
However, stimulation by curcumin is nonetheless strongly
dependent on prior phosphorylation of the channel by PKA,
even though neither ATP nor NBD2 are required for this
activation. Thus, induction of CFTR opening can be carried
out via an alternative mechanism that bypasses the normal requirement for
ATP binding and NBD dimerization .
VX-770 (Vertex Pharmaceuticals Inc.), an investigational
oral potentiator, is designed to act directly on the malfunctioning
CFTR protein to help restore the balance of salt and water.
Clinical development of VX-770 is currently focused on a
subset of CF patients who have a G551D CFTR mutation
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