Dawson, David C.

Landstrom, Allison

Liu, Xuehong

O'Donnell, Nicolette

Elsevier - Publisher Connector 

322a Monday, February 22, 2010channel activity is mainly due to an increased open-time. The results shown
here suggest that NBD1 and NBD2 may employee different chemical mecha-
nisms in binding ATP and that NBD1 can be a potential molecular target for
developing CFTR potentiators for CF-related mutants. The effects of different
nucleotides (for example, GTP and UTP) on NBD1 and NBD2 will be studied
to gain a better understanding of the chemical mechanism underlying nucleo-
tide-NBD interaction.
1672-Pos
The Inhibition Mechanisms of the Regulatory Domain of Cyctic Fibrosis
Transmembrane Conductance Regulator
Guangyu Wang.
Gregory Fleming James Cystic Fibrosis Research Center, and Department of
Physiology and Biophysics, University of Alabama at Birmingham,
Birmingham, AL, USA.
The cyctic fibrosis transmembrane conductance regulator (CFTR) is a member
of the human C subfamily of ATP-binding cassette (ABC) transporters but
functions as a chloride channel. Activity of CFTR is tightly controlled not
only by ATP binding-induced NBD1-NBD2 dimerization but also by phos-
phorylation of the unique regulatory (R) domain by protein kinase A
(PKA). The R domain has multiple phosphorylation sites for which only
Ser737 and Ser768 are inhibitory. The underlying mechanisms are unclear be-
cause neither the structure of the R domain nor its interactions with other
parts of CFTR have been fully illuminated. Here I applied the crystal structure
of bacterial transporter Sav1866 and sulfhydryl-specific crosslinking strategy
to determine which part of CFTR interacts with the R domain regulating
channel activity. The results show that diamide-induced disulfide bond cross-
linking of S768C to H950C, K951C, H954C or S955C from cytoplasmic loop
3 (CL3) inhibited the channel activity and inhibition was reversed by DTT.
Similarly, disulfide crosslinking of S737C to H954C, S955C or Q958C also
suppressed the channel activity. Furthermore, mutation of these residues to al-
anines weakened the curcumin-induced relative PKA-dependence which was
completely removed by deletion of the R domain. Finally, activation of a dou-
ble mutant H950R/S768R CFTR did not need any PKA while either H950R
or S768R construct needed it. These results suggest that S768 and S737 may
form putative H-bonds with hydrophilic residues of CL3 and thus inhibit the
channel activity in the unphosphorylated state. In the phosphorylated state,
a putative ferrous bridge involving H950, H954, C832, D836, H775 and
H667 at the CL3-R interface may inhibit the channel activity. All these obser-
vations are consistent with the recent electron cryomicroscopy-based struc-
tural model on which the R domain is closed to cytoplasmic loops regulating
channel gating.
1673-Pos
CFTR Cytosolic Loop Mutations Allosterically Promote ATP Independent
Channel Gating
Wei Wang, Jianping Wu, Karen Bernard, Ge Li, Guangyu Wang,
Mark Bevensee, Kevin L. Kirk.
UAB, Birmingham, AL, USA.
CFTR channel gating normally depends on ATP binding and NBD dimeriza-
tion. Optimal CFTR channel activation further requires phosphorylation of the
R domain. How ATP binding at the NBDs and phosphorylation of the R do-
main regulate CFTR channel gating are not fully understood. In the present
study, we demonstrate that mutations in the CFTR Cytosolic Loops (CL)
markedly promote channel opening in the absence of ATP and NBD2, pre-
sumably by an allosteric mechanism. In excised inside-out patches, we ob-
served that single or double mutations of K978 and K190 in CL 3 and 1 in-
duced large ATP independent currents (5-70% of current before removing
ATP). These mutant channels deactivated with a slow time constant
(49.1154.58 sec) when ATP was removed by Hexokinase/glucose and subse-
quent bath perfusion. A K978 point mutation greatly increased the ATP sen-
sitivity of channel activation by decreasing the EC50 (by 8-fold) for ATP ac-
tivation, which is consistent with the slow deactivation following ATP
removal. K978 mutations markedly enhanced G551D channel activity, a dis-
ease mutant that fails to respond to ATP, and D1198-CFTR, a mutant that
lacks NBD2, indicating that the K978 mutations affect channel gating down-
stream of NBD dimerization. Interestedly, R domain phosphorylation further
stimulated K978/G551D and K978/D1198 combined mutants, indicating that
the R domain regulates channel activity independently of NBD dimerization.
Similarly, K978 mutations also increased the activation rate at low dose (3 U/
ml) of PKA, indicating that K978 mutations also enhance the PKA sensitivity
of channel activation. Our results support an allosteric gating mechanism in
which loops 1 and 3 functionally link ATP binding and NBD dimerization
to CFTR channel opening.1674-Pos
Accessibility of Cysteines Within the NBD Interface in a CFTR Channel
Luiz A.P. Chaves.
Rockefeller University, New York, NY, USA.
Opening and closing of a CFTR channel is accompanied by ATP-driven forma-
tion and hydrolysis-triggered disruption of a head-to-tail NBD1-NBD2 hetero-
dimer where composite interfacial sites, between the Walker motifs of one
NBD and the LSGGQ-like (ABC signature) sequence of the other, each enclose
an ATP molecule. Only the ‘‘NBD2’’ composite site (containing NBD2Walker
motifs) is catalytically competent. The ATP-bound tight NBD1-NBD2 hetero-
dimer is linked to the open-channel state, but the disposition of the NBDs in the
closed-channel state of CFTR, in the absence of ATP or after its hydrolysis, re-
mains unknown. To address this, we assess accessibility to various size MTS
reagents of single interfacial target cysteines introduced into the ABC signature
sequence of the competent site (at NDB1 position S549), or of the dead site (at
NBD2 position S1347), or at mid-interface positions S605 of NBD1 or A1374
of NBD2, in full-length cys-depleted CFTR-C(832-1458)S,expressed in Xeno-
pus oocytes and examined in excised patches. Cysteines at all four positions
were readily accessible to MTSET in closed channels in the absence of ATP.
For channels opening and closing in 3 mM ATP, the reaction rate depended
on MTSET concentration and was ~105 M1s1 at 5 mM at position 549,
and was similarly rapid at corresponding position 1347. In closed channels
without ATP, cysteines at 549, 605, 1347, and 1374 were all also readily acces-
sible to MTS reagents of increasing size, e.g., MTS-biotin, MTS-THAE (trihex-
ylammonium-ethyl) and MTS-verapamil, up to ~11A˚x16A˚x10A˚, suggesting
substantial separation between NBD1 and NBD2, throughout the NBD inter-
face, in closed CFTR channels. (Supported by NIH DK51767).
1675-Pos
Strict Coupling Between CFTR’s Catalytic Cycle and Gating of its
Channel Pore Revealed by Distributions of Open Burst Durations
La´szlo´ Csana´dy1, Paola Vergani2, David C. Gadsby3.
1Semmelweis University, Budapest, Hungary, 2University College London,
London, United Kingdom, 3The Rockefeller University, New York,
NY, USA.
CFTR, the ABC protein defective in cystic fibrosis, functions as an anion chan-
nel. Once phosphorylated by protein kinase A, a CFTR channel is opened and
closed by events at its two cytosolic nucleotide binding domains (NBDs). For-
mation of a head-to-tail NBD1/NBD2 heterodimer, by ATP binding in two in-
terfacial composite sites between conserved Walker A and B motifs of one
NBD and the ABC-specific signature sequence of the other, has been proposed
to trigger channel opening. ATP hydrolysis at the only catalytically competent
interfacial site is suggested to then destabilize the NBD dimer and prompt chan-
nel closure. But this gating mechanism, and how tightly CFTR channel opening
and closing are coupled to its catalytic cycle, remain controversial. Here we de-
termine the distributions of open burst durations of individual CFTR channels,
and use maximum likelihood to evaluate fits to equilibrium and non-equilib-
rium mechanisms and estimate the rate constants that govern channel closure.
We examine partially- and fully-phosphorylated, wild-type CFTR channels,
and two mutant CFTR channels each bearing a deleterious mutation in one
or other composite ATP binding site. We show that the wild-type CFTR chan-
nel gating cycle is essentially irreversible and tightly coupled to the ATPase cy-
cle, and that this coupling is completely destroyed by the NBD2Walker-B mu-
tation D1370N but only partially disrupted by the NBD1 Walker-A mutation
K464A. [NIH R01-DK051767, NIH FIC R03-TW007829, Wellcome Trust
081298/Z/06/Z]
1676-Pos
DF508 CFTR Expressed In Xenopus Oocytes Exhibits Unique Thermal
Sensitivity
Xuehong Liu, Allison Landstrom, Nicolette O’Donnell, David C. Dawson.
Oregon Health & Science University, Portland, OR, USA.
The deletion of a phenylalanine at position 508 is the most common, disease-
related mutation in the CFTR protein. DF508 CFTR channels are assembled in
mammalian cells, but exhibit two deficiencies thought to underlie the disease
phenotype: impaired trafficking and defective gating. The CFTR trafficking de-
fect is partially mitigated at low temperature so that Xenopus oocytes are ideal
for studying the physical properties of DF508 channels under physiological
conditions. In order to investigate possible effects of the Phe deletion on the in-
trinsic stability of CFTR channel function in the plasma membrane, we moni-
tored channel activity at room temperature (~23C) and during a brief (10-12
minute) period of elevated temperature (28C to 37C). In oocytes expressing
wild type CFTR, a temperature challenge resulted in a reversible increase in the
conductance; a result of a simultaneous increase in single-channel conductance
and open probability. Both parameters, however, returned to baseline values
Monday, February 22, 2010 323awhen the temperature was returned to 23C. In marked contrast, conductance
due to DF508 CFTR channels exhibited a transient increase within a minute af-
ter the temperature challenge, followed by a quasi-exponential decline of about
80-90% of the initial conductance (t1/2 ¼ 4 minutes at 37C). The temperature
induced decrease in DF508 CFTR conductance was not reversed by returning
the temperature to 23C. The second-site reverent construct, R553M/ DF508
CFTR, previously shown to rescue CFTR function in mammalian cells
(Teem et al. 1993. Cell. 73:335-346), exhibited a thermal response that was in-
distinguishable from wild type. Preliminary data suggests that this ‘‘thermal in-
stability’’ that is readily detectable when DF508 CFTR is expressed in Xenopus
oocytes, reflects an intrinsic structural defect in the channel protein that results
in a temperature-sensitive alteration in gating and could potentially trigger the
retrieval of surface protein documented in mammalian cells.
1677-Pos
Riding the Conformational Wave to the Open Channel State in the CFTR
Chloride Channel
Guiying Cui, Cody Freeman, Nael A. McCarty.
Emory University School of Medicine, Atlanta, GA, USA.
The pore structure of the CFTR chloride channel is unknown. We showed pre-
viously that R352 in TM6 forms a salt bridge with D993 in TM9; charge-de-
stroying mutations at either site destabilized the open state, affecting conduc-
tance, selectivity, and pore blockade. Other pairs of interacting residues also
contribute to stabilizing the open state. We continued these experiments to de-
termine how steps leading to the dimerization of the NBDs upon binding of nu-
cleotide relate to the steps leading to pore opening, using single-channel record-
ings of WT-CFTR and channels bearing a cysteine or alanine at 352, 993, or
both. In R352C-CFTR, but not R352A-CFTR, modification of the cysteine
by positively-charged MTSETþ and MTSEAþ recovered the stability of the
open state. In D993C-CFTR, but not D993A-CFTR, negatively-charged
MTSES- recovered the stability of the open state. In contrast, D993C-CFTR
modified by MTSETþ retained the instability of the open state. The R352C/
D993C-CFTR double mutant exhibited instability of the open state in both
the absence and presence of DTT, suggesting that R352C did not form a disul-
fide with D993C. In WT-CFTR, exposure to AMP-PNP led to greatly pro-
longed channel openings, as expected. However, this response was not found
for R352A-CFTR. Surprisingly, R352C/D993C-CFTR could be latched open
by the bifunctional crosslinker, MTS-2-MTS, such that channels could not
close upon washout of ATP. MD simulations based on CFTR homology models
(see Dawson Lab abstract) predict conformational states in which R352 and
D993 approach each other to within van der Waals distances. These results sug-
gest that the binding of ATP at CFTR’s NBDs initiates a conformational wave,
which leads to a change in pore structure from the closed to the open state, the
latter being stabilized by inter-TM interactions including the R352-D993 salt
bridge. (Support: NIH-2R56DK056481-07)
1678-Pos
Homology Modeling and Molecular Dynamics Simulation Predict Side-
Chain Orientations and Conformational Changes in the Pore of the
CFTR Chloride Channel
Christopher Alexander1, Anthony Ivetac2, Yohei Norimatsu1,
Mark Sansom3, David C. Dawson1.
1Oregon Health and Science University, Portland, OR, USA, 2University of
California San Diego, La Jolla, CA, USA, 3University of Oxford,
Oxford, United Kingdom.
We recently presented two homology models of the CFTR chloride channel,
one based on homology to the prokaryotic ABC transporter, Sav1866, and
a second based on a 5 ns molecular dynamics (MD) simulation of the first
(Alexander et al, Biochemistry in press, 2009). Predictions for side-chain ori-
entations were in excellent agreement with the results of a cysteine scan of
transmembrane segment six (TM6) using both channel-permeant and chan-
nel-impermeant, thiol directed probes. Here we present the results of an ex-
tended MD simulation, along with the results of a cysteine scan of TM12.
Scanning results confirm the model predictions for ‘‘pore-lining’’ and ‘‘not
pore-lining’’ residues in TM12 and support the notion that pore narrowing
prevents the reaction of deeper-lying cysteines in TM12 toward larger,
thiol-directed probes like MTSETþ and MTSES- when these compounds enter
the channel from the outside. The extended MD simulation predicts move-
ments of pore elements that are in agreement with previously reported results
of state-dependent reactivity of a cysteine at position 337 (Norimatsu et al,
Biophysical Journal 96(3):468a-469a, 2009), and the postulated formation
of a salt-bridge between R352 (TM6) and D993 (TM9) (Cui et al, Biophysical
Journal 91(5):1737-48, 2008, and poster from the McCarty Lab). Supported
by NIH, the Cystic Fibrosis Foundation, the American Lung Association,
the Wellcome Trust, and the BBSRC.1679-Pos
Identification of Possible Binding Sites for the CFTR Pore Blocker,
GlyH-101
Yohei Norimatsu1, Anthony Ivetac2, John Kirkham1, Leah Frye3,
Mark Brewer3, Mark Sansom4, David C. Dawson1.
1Oregon Health & Science University, Portland, OR, USA, 2University of
California San Diego, La Jolla, CA, USA, 3Schrodinger Inc., Portland,
OR, USA, 4University of Oxford, Oxford, United Kingdom.
The last decade has seen the discovery by means of high throughput screening of
a wide range of small-molecule modulators of the CFTR chloride channel. These
compounds act by altering anion conduction, channel gating and/or trafficking of
the CFTR protein. However, binding sites for these molecules on CFTR or other
cellular constituents have yet to be identified. GlyH-101 is a CFTR modulator
that blocks the channel by entering from the extracellular side and binding to
a site within the pore. In an effort to identify possible GlyH-101 binding sites
within the pore of the CFTR channel, we applied the small-molecule docking
program, ‘‘Glide’’ (Schrodinger, Inc.), to a series of molecular models of
CFTR, derived by means of molecular dynamics simulation from a homology
model based on the prokaryotic ABC transporter, Sav1866 (Dawson and Locher,
Nature 443: 180-185, 2006; Alexander et al., Biochemistry in press, 2009). One
of the potential GlyH-101 binding sites identified by Glide lies in close proxim-
ity to two residues in the sixth transmembrane segment (TM6), F337 and T338,
where substituted cysteines are ‘‘protected’’ by GlyH-101 from reaction with
thiol-directed probes (Norimatsu et al., Biophys. Journal 96: 468a-469a,
2009). These results suggest an approach to identifying the binding site(s) for
GlyH-101 and other small molecules within the CFTR protein. Supported by
NIH, Cystic Fibrosis Foundation, American Lung Association, the Wellcome
Trust, and the BBSRC.
Mechanosensitive Channels
1680-Pos
Mechanosensitivity of a Voltage-Gated Ion Channel, Nav1.5
Arthur Beyder, James L. Rae, Cheryl Bernard, Gianrico Farrugia.
Mayo Clinic, Rochester, MN, USA.
Voltage-gated ion channels are often found in tissues where electrical and me-
chanical stimuli coexist. The mechanosensitive, voltage-gated sodium channel
Nav1.5 (encoded by SCN5A) is expressed in two such electromechanical organs,
the heart and the gastrointestinal tract. Mutations in SCN5A are frequently path-
ogenic and may affect mechanoelectrical coupling. The aim of this study was to
assess mechanical sensitivity of Nav1.5 at the molecular level. SCN5A was ex-
pressed inHEKcells and studiedusing a pipette pulled andfire polished to ensure
that a small number (2-50) of channels were reliably present in cell-attached mi-
cropatches. This allowed resolution of both single channel events and averaged
behavior. Both positive and negative pressures (up to 50mmHg) produced visi-
ble patch distention, an increase in patch current at all voltages and large hyper-
polarizing shifts in steady-state voltage-sensitivity of activation and inactiva-
tion. From voltage dependence of activation at rest (V1/2 ¼ 30mV at
0mmHg), pressure resulted in graded shifts ofV1/2 for activation and inactivation
of0.71mV/mmHg and0.72mV/mmHg, respectively. Channel kinetics were
predictably affected by the voltage shifts, but channel opening and fast inactiva-
tion were otherwise unaffected by pressure. Single channel traces showed that
unitary conductance was unaffected, rather peak currents appeared to increase
due to an increase in the number of active channels in the patch. These effects
were minimally reversible for as long as 30 minutes after a single stretch stimu-
lus. Patch excision resulted in an immediate shift of activation V1/2=-75mV and
loss of stretch sensitivity. Application of the inhibitor of actin polymerization,
cytochalasin D, diminished sensitivity to stretch (0.42mV/mmHg). Our
work demonstrates that mechanical stress at physiologically relevant levels af-
fects voltage sensing of Nav1.5 channels, without affecting the pore, channel
gate and fast inactivation. Supported by NIH DK52766.
1681-Pos
Integrin-Dependent and -Independent Potentiation of L-type Calcium
Current (Cav1.2) by Cell Stretch
Peichun Gui1, Gerald W. Zamponi2, George E. Davis1, Michael J. Davis1.
1Department of Medical Pharmacology & Physiology, University of
Missouri-Columbia School of Medicine, Columbia, MO, USA, 2Molecular
Neuroscience Research Group, University of Calgary, Calgary, AB, Canada.
Stretch-induced (myogenic) contraction of vascular smooth muscle (VSM)
requires calcium influx through L-type calcium channels (Cav1.2). Integrins
play a role in this process because alpha5 beta1 and alphaVbeta3 integrin block-
ing antibodies prevent myogenic constriction. Recent studies in our lab indicate
that Cav1.2 current is potentiated by alpha5beta1 integrin activation and requires
phosphorylation by PKA and c-Src of Cav1.2 C-terminal residues. To test


ΔF508 CFTR Expressed In Xenopus Oocytes Exhibits Unique Thermal Sensitivity 

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ΔF508 CFTR Expressed In Xenopus Oocytes Exhibits Unique Thermal Sensitivity

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