Site-Directed Spin Labeling Reveals Pentameric Ligand-
Gated Ion Channel Gating Motions
Cosma D. Dellisanti1, Borna Ghosh1, Susan M. Hanson1, James M. Raspanti1, Valerie A. Grant1,
Gaoussou M. Diarra1, Abby M. Schuh1, Kenneth Satyshur1, Candice S. Klug2, Cynthia Czajkowski1*
1Department of Neuroscience, University of Wisconsin, Madison, Wisconsin, United States of America, 2Department of Biophysics, Medical College of Wisconsin,
Milwaukee, Wisconsin, United States of America
Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic
transmission. In pLGICs, binding of agonist to the extracellular domain triggers a structural rearrangement that leads to the
opening of an ion-conducting pore in the transmembrane domain and, in the continued presence of neurotransmitter, the
channels desensitize (close). The flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7,
and 9) to the transmembrane channel domain (M2–M3 loop) are essential for coupling ligand binding to channel gating.
Comparing the crystal structures of two bacterial pLGIC homologues, ELIC and the proton-activated GLIC, suggests channel
gating is associated with rearrangements in these loops, but whether these motions accurately predict the motions in
functional lipid-embedded pLGICs is unknown. Here, using site-directed spin labeling (SDSL) electron paramagnetic
resonance (EPR) spectroscopy and functional GLIC channels reconstituted into liposomes, we examined if, and how far, the
loops at the ECD/TMD gating interface move during proton-dependent gating transitions from the resting to desensitized
state. Loop 9 moves ,9 A˚inward toward the channel lumen in response to proton-induced desensitization. Loop 9 motions
were not observed when GLIC was in detergent micelles, suggesting detergent solubilization traps the protein in a
nonactivatable state and lipids are required for functional gating transitions. Proton-induced desensitization immobilizes
loop 2 with little change in position. Proton-induced motion of the M2–M3 loop was not observed, suggesting its
conformation is nearly identical in closed and desensitized states. Our experimentally derived distance measurements of
spin-labeled GLIC suggest ELIC is not a good model for the functional resting state of GLIC, and that the crystal structure of
GLIC does not correspond to a desensitized state. These findings advance our understanding of the molecular mechanisms
underlying pLGIC gating.
Citation: Dellisanti CD, Ghosh B, Hanson SM, Raspanti JM, Grant VA, et al. (2013) Site-Directed Spin Labeling Reveals Pentameric Ligand-Gated Ion Channel
Gating Motions. PLoS Biol 11(11): e1001714. doi:10.1371/journal.pbio.1001714
Academic Editor: Richard W. Aldrich, University of Texas at Austin, United States of America
Received June 3, 2013; Accepted October 8, 2013; Published November 19, 2013
Copyright: ? 2013 Dellisanti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by an NIH grant (NS34727) to CC. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: ELIC, Erwinia chrysanthemi ligand-gated ion channel; GLIC, Gloeobacter violaceus ligand-gated ion channel; MTSL, 1-oxyl-2,2,5,5-tetramethyl-3-
pyrroline-3-methyl methanethiosulfonate spin label; pLGICs, pentameric ligand-gated ion channels; SDSL EPR, site-directed spin labeling electron paramagnetic
* E-mail: firstname.lastname@example.org
Chemical signaling in the brain and periphery relies on the
rapid opening and closing of pentameric ligand-gated ion channels
(pLGICs), which include nicotinic acetylcholine (nAChRs), sero-
tonin-type-3 (5-HT3Rs), c-aminobutyric acid-A (GABAARs), and
glycine (GlyRs) receptors . These receptors exist in at least
three distinct, interconvertible states: resting (unliganded, closed
channel), activated (liganded, open channel), and desensitized
(liganded, closed channel), and the binding of agonists, antago-
nists, and allosteric drugs alters the equilibria between these states.
Neurotransmitter binding in the extracellular ligand-binding
domain triggers rapid opening of an intrinsic ion channel more
than 60 A˚away in the transmembrane domain of the receptor,
and with prolonged neurotransmitter exposure, the channel moves
into a nonconducting desensitized state. Although we know a fair
amount about the structure of these receptors, the mechanisms by
which the binding of neurotransmitter triggers channel opening
and desensitization are still unfolding, and our understanding of
the protein motions underlying these processes is limited.
pLGICs are composed of five identical or homologous subunits
arranged pseudosymmetrically around a central ion-conducting
channel. Our current structural knowledge of these proteins comes
from cryo-EM structures of the Torpedo nAChR in a presumed
unliganded closed state (4 A˚resolution) and liganded open state
(6.2 A˚resolution) [2,3], high-resolution crystal structures of the
extracellular binding domains of the nAChR a1 and a7 subunits
[4,5], crystal structures of full-length prokaryotic pLGIC homologs
from Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus (GLIC)
solved in presumed closed and open channel conformations [6–8],
respectively, and a recent crystal structure of a glutamate-activated
chloride channel (GluCl) in an open channel conformation from C.
elegans . In general, each subunit can be divided into two parts:
an extracellular binding domain (ECD) folded into a b-sandwich
core and a transmembrane channel domain (TMD) consisting
of four a-helical membrane-spanning segments (M1 to M4).
PLOS Biology | www.plosbiology.org1November 2013 | Volume 11 | Issue 11 | e1001714
Neurotransmitter binding occurs at sites located at interfaces
between subunits in the ECD (reviewed in ), and the M2 helices
of each of the subunits form the ion-conducting channel. In each
subunit, flexible loops (loop 2, loop 7, loop 9, and the M2–M3
loop) connect the binding domain to the channel domain (Figure 1)
and play a critical role in coupling binding site movements to the
Comparison of ELIC and GLIC structures suggests that
channel activation is associated with an anticlockwise concerted
twist of each ECD b-sandwich and a radial tilting of the pore
lining M2 a-helices away from the channel axis [7,10]. Rea-
rrangements in the flexible loops that form the interface between
the ECD (loop 2, loop 7, and loop 9) and the TMD (M2–M3 loop)
(Figure 1) are also observed. Some studies, however, suggest
that the GLIC structure may correspond to a desensitized state
[11,12] and the ELIC structure to an ‘‘uncoupled’’ nonfunctional
conformation [11,13]. Thus, whether the motions inferred by
comparing the static structures of two related (but with only
18% sequence identity) proteins solved in detergent micelles in
uncertain functional states accurately predict the dynamic gating
motions of a pLGIC in its native environment is unknown.
In this study, we used site-directed spin labeling (SDSL) electron
paramagnetic resonance (EPR) spectroscopy and functional
GLIC channels reconstituted into liposomes to examine if, and
how far, the loops at the ECD/TMD gating interface move during
proton-dependent gating transitions from the resting to desensi-
tized state. SDSL EPR spectroscopy is a powerful method for
monitoring the structure and dynamics of membrane proteins in
conditions closely resembling the proteins’ native environment
[14,15]. In SDSL EPR, a cysteine residue is introduced at a site
of interest, and a sulfhydryl-specific nitroxide reagent (typically 1-
nate spin label, MTSL) is covalently attached to the free sulfhydryl
as a paramagnetic probe to create the R1 side chain (Figure S1A).
Backbone and side chain mobility can be detected with the
continuous wave (CW) method, and distances and distance
changes between pairs of probes can be measured by double
electron electron resonance (DEER) spectroscopy (up to ,60 A˚)
. SDSL EPR spectroscopy is the ideal complement to high-
resolution static snapshots of crystal structures and has been
used successfully to study the dynamic motions of the voltage-
gated K+channel [17–19], other membrane proteins (e.g., the
mechanosensitive channel of small conductance, MscS , and
rhodopsin ) and recently, GLIC [22,23].
Here, we show that proton-dependent GLIC gating from
resting to desensitized conformation induces a large inward
movement of loop 9 towards the channel lumen and an immo-
bilization of loop 2, which is accompanied by substantial
rearrangements of the intra- and intersubunit interface between
the ECD and TMD. No appreciable proton-induced motions
in the M2–M3 loop in the TMD were detected, demonstrating the
conformation of this critical loop is similar in resting (closed,
unliganded) and desensitized (closed, liganded) states. Proton-
induced motions in GLIC were absent when the protein was in
detergent micelles, indicating that lipids are required for functional
gating transitions and suggesting that the detergents used for
protein solubilization and crystallization may influence the
conformations captured in the crystal structures. In general,
residue positions and the proton-induced motions in functional
GLIC protein embedded in lipid differ from those predicted based
on the crystal structures of GLIC and ELIC obtained in detergent
micelles, suggesting the GLIC crystal structure does not corre-
spond to a desensitized conformation and that ELIC is not a
suitable model for the structure of the M2–M3 loop of GLIC in
the resting, closed state.
Functional Characterization of Mutant GLIC Protein
To study proton-induced motions in loops forming the ECD/
TMD interface of GLIC by EPR spectroscopy, we generated a
cys-free GLIC mutant by replacing the lone native cysteine, C26,
with alanine (Figure 1A,B). We then individually mutated K32
(loop 2), T157 (loop 9) and K247 and P249 (M2–M3 loop) to
cysteine in the mutant C26A background (Figure 1A,B). We
expressed wild-type and mutant proteins in Xenopus laevis oocytes
and measured proton-induced currents using two-electrode
voltage clamp (Figure S1B). All of the mutants formed functional
channels with wild-type GLIC properties (pH50=5.260.1, Hill
We also measured currents elicited by pH50 concentra-
tions before and after reaction with the sulfhydryl-specific MTSL
to determine if the wild-type cysteine (C26) and the introduced
cysteines could be labeled by MTSL. For C26, K32C, T157C,
and P249C, treatment with 1 mM MTSL for 2 min inhibited
pH50currents (30%–70%), demonstrating that the cysteines were
accessible to modification with MTSL (Figure S1 and Table S1).
The MTSL modification shifted the pH50to more acidic values
but did not alter maximal proton-activated currents (data not
shown). For the mutants C26A and K247C, MTSL treatment
had no effect on subsequent proton-activated currents. For
K247C, treatment with the bulkier sulfhydryl-modifying reagent,
MTSEA-biotin, inhibited proton-induced currents. To test
whether MTSL modified K247C, we applied MTSL prior to
MTSEA-biotin. MTSL blocked the ability of MTSEA-biotin to
inhibit proton-induced currents, indicating that MTSL labels
K247C but has no functional effect on channel activation.
We then expressed wild-type and mutant GLIC proteins in E.
coli, purified the proteins in n-Dodecyl-b-D-maltoside (DDM), and
Ligand-gated ion channels reside in the membranes of
nerve and muscle cells. These proteins form channels that
span the membrane, where they transduce chemical
signals into changes in electrical excitability. Neurotrans-
mitters bind to the extracellular surface of these proteins
to trigger global structural rearrangements that open the
channel, allowing ions to flow across the cell membrane. In
the continued presence of neurotransmitters, the channels
desensitize and close. Channel opening and closing
regulate muscle contraction and signaling in the brain,
and defects in these channels lead to a variety of diseases.
While crystal structures have provided frozen snapshots of
these proteins in presumed closed and open channel
states, little is known about how the channels desensitize
and move during actual signaling events. Here, we applied
a technique to investigate the structure and local
dynamics of proteins known as site-directed spin labeling
to a prototypical ligand-gated channel, GLIC. We directly
quantified ligand-induced motions in regions at the
boundary between the binding domain (loops 2 and 9)
and the channel domain (M2–M3 loop). We show that a
large movement of loop 9 and an immobilization of loop 2,
which rearranges the interface between the binding and
channel domains, accompanies GLIC channel gating
transitions into a desensitized state. These data provide
new insights into the protein movements that underlie
electrochemical transmission of signals between cells.
SDSL EPR Reveals Gating Motions in pLGICs
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labeled them with MTSL. To test whether the purified GLIC
proteins were functional, we reconstituted mutant C26A into
liposomes formed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phos-
phospho-(19-rac-glycerol) (PG), and recorded single-channel cur-
rents in planar lipid bilayers also formed with PE:PG (Figure 2A).
PE and PG are major components of the inner cell membrane of
most bacteria, and PE:PG bilayers are good models for the
bacterial membrane . The purified C26A mutant reconstitut-
ed into PE:PG liposomes produced proton-elicited single-channel
currents with unitary conductance of 13.660.6 pS, which is
comparable to the 8 pS value reported for wild-type GLIC in
HEK293 cells .
We next tested whether the addition of cholesterol or cardiolipin
along with PE and PG would affect GLIC single-channel
properties. Cholesterol is essential for eukaryotic pLGIC function
[26–30], and was recently shown to increase GLIC current
desensitization rates . Cardiolipin is an anionic lipid typically
found in the bacterial cell membrane, and previous studies
have reported that anionic lipids can modulate eukaryotic
pLGIC function [29,31–33]. We reconstituted mutant C26A into
liposomes formed with PE:PG:cholesterol at a 3.4:1.3:1 molar
ratio or PE:PG:cardiolipin at a 5.8:2.3:1 molar ratio, and recorded
single-channel currents in planar lipid bilayers formed with the
same lipids (Figure 2A). The single-channel conductance of C26A
mutant GLIC was not altered by cholesterol (12.860.5 pS) or
cardiolipin (10.061.6 pS). The open dwell time in the presence
of cholesterol (10.1560.08 ms, at 2100 mV) was similar to that
in PE:PG (11.8360.06 ms, at 2100 mV), whereas in the pre-
sence of cardiolipin, it was slightly increased (19.6460.06 ms, at
2100 mV). Overall, the data demonstrate that purified GLIC
C26A mutant protein reconstituted in PE:PG liposomes is
functional and that cholesterol and cardiolipin have little effect
on the GLIC single channel properties measured.
We also confirmed that the purified reconstituted cysteine
mutant GLIC proteins were functional. Single-channel currents
recorded in PE:PG bilayers for K32C, T157C, and P249C mutant
protein (Figure 2B) had single channel conductances from
14.860.4 pS (T157C) to 5.860.8 pS (P249C), comparable to
mutant C26A and wild-type GLIC . In addition, we injected
purified, reconstituted, MTSL-labeled protein directly into Xenopus
oocytes to verify their functionality [34,35]. We recorded
significantly larger proton-dependent induced currents (approxi-
mately 300 nA–1 mA) from oocytes injected with mutant C26A,
K32R1, T157R1, K247R1, and P249R1 as compared to
noninjected oocytes (70 nA) (Figure 2C). Overall, the data
demonstrate that the purified mutant GLIC proteins reconstituted
into liposomes were functional and proton-sensitive.
Figure 1. Location of loop 2, loop 9, and M2–M3 loop in GLIC. (A) Crystal structure of GLIC (PDB entry 3EHZ) with residues C26, K32, T157,
K247, and P249 shown in space-fill. The fifth subunit on the backside was removed for clarity. (B) Close-up view of an intersubunit interface
highlighting the region between the extracellular and transmembrane domains and the sites spin-labeled (space-fill). (C) Positions of loop 2 and M2–
M3 loop in GLIC (3EHZ, cyan) and ELIC (2VL0, red) in aligned structures. Relative to ELIC, GLIC loop 2 is shifted inward towards the channel pore-lining
M2 helix (labeled), whereas GLIC M2–M3 loop is shifted outward (arrows). (D) Positions of loop 9 in GLIC (cyan) and ELIC (red) in aligned structures.
Relative to ELIC, GLIC loop 9 is shifted inward toward the M2 helix (arrow).
SDSL EPR Reveals Gating Motions in pLGICs
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CW EPR Spectroscopy Reveals Proton-Induced
We initially recorded the CW spectra of MTSL-labeled
wild-type GLIC (C26R1), at pH 7.6 and pH 4.6 (Figure 3). The
shape of the CW spectrum reflects the mobility of the R1 side
chain, which ultimately depends on packing of its surroundings.
Therefore, proton-induced changes in the CW spectrum reflect
structural rearrangements that alter the local environment around
R1 (i.e., neighboring side chain motions and backbone flexibility).
The CW EPR measurements were collected at room temperature
over an hour (steady-state conditions). Based on our proton-
concentration response curves, at pH 7.6, the channels will
predominantly be in an unliganded, resting conformation. At
pH 4.6, the channels will predominantly be in a desensitized
conformation. At both pH values, the CW spectra of C26R1
showed the spin labels were largely immobile (Figure 3), indicating
a tightly packed environment near the C26R1 side-chain,
consistent with its buried location on b-strand 1. Switching to
pH 4.6 had no effect on the shape of the C26R1 CW spectrum,
indicating that probe mobility did not change, which suggests the
local environment near the probe is the same or it rearranged into
an equally packed conformation. The CW spectrum of an MTSL-
treated C26A mutant showed virtually no signal (Figure S2),
demonstrating the absence of spin-labeled protein contaminants.
To detect proton-induced conformational rearrangements in
loop 2, loop 9, and the M2–M3 loop, we recorded the CW spectra
of the MTSL-labeled GLIC mutants K32R1, T157R1, K247R1,
and P249R1 at pH 7.6 and pH 4.6 (Figure 3). In the ECD,
the CW spectra of K32R1, located in loop 2, showed two distinct
EPR spectral components, mobile and immobile (indicated by
arrows in Figure 3), likely associated with two alternative rotameric
spin-label conformations . At pH 7.6 (closed, resting state),
a greater proportion of the spin probes were in a mobile
conformation, whereas at pH 4.6 (desensitized) a greater propor-
tion were immobile, indicating a proton-induced structural
rearrangement occurred that resulted in a more densely packed
environment near the spin probe. Proton-induced changes were
also detected in the CW spectra of T157R1 (Figure 3), located in
loop 9. The low field regions of the spectra were entirely different
at the two pH values, indicating a completely new motional
environment, with the spin probes predominantly immobile at
pH 7.6 (resting) and mostly mobile at pH 4.6 (desensitized). For
both K32R1 and T157R1, switching to pH 4.6 did not result in
significant spectral broadening or changes in center resonance
line amplitude, indicating that the observed differences reflect
changes in probe mobility and not intersubunit dipolar spin–
spin interactions. Changes in spin probe mobility are plotted in
Figure 4 and were calculated by measuring the inverse width of
Figure 2. Purified GLIC reconstituted into liposomes is functional. (A) Single-channel currents of purified GLIC C26A mutant protein
reconstituted into PE:PG, PE:PG:cholesterol, and PE:PG:cardiolipin liposomes were recorded in planar lipid bilayers composed of the same lipids.
Representative single-channel current traces (left), and current-voltage relationships with single-channel conductance values (right) are shown. Open-
dwell times tofor the GLIC C26A mutant were 11.8360.06 ms when reconstituted into PE:PG liposomes, 10.1560.08 ms into PE:PG:cholesterol
liposomes, and 19.6460.06 ms into PE:PG:cardiolipin liposomes, respectively. (B) Single-channel currents of purified GLIC mutants (K32C, T157C, and
P249C) reconstituted into PE:PG liposomes were recorded in planar lipid bilayers composed of the same lipids. Representative single-channel current
traces (left) and current-voltage relationships with single-channel conductance values (right) are shown. (C) Currents induced by pH jumps from
uninjected Xenopus laevis oocytes and ooctyes injected with purified, single cysteine MTSL-labeled GLIC protein reconstituted into PE:PG liposomes
(C26A, K32C, T157C, K247C, and P249C). Currents from GLIC-protein injected oocytes were significantly larger than those from uninjected oocytes.
SDSL EPR Reveals Gating Motions in pLGICs
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the central line, DH021: an increase in DH021indicates an
increase in motion, whereas a decrease in DH021indicates a
decrease in motion [16,17,37].
We also collected CW spectra of T157R1 reconstituted in
PE:PG:cholesterol and in PE:PG:cardiolipin at pH 7.6 and
pH 4.6 (Figure 5) to test the effects of lipids on proton-induced
motions in GLIC. In the presence of cholesterol, the CW spectra
were essentially indistinguishable from the spectra of T157R1
reconstituted in PE:PG, indicating that cholesterol had no effect
on the proton-induced structural rearrangements near loop 9.
In the presence of cardiolipin, there was a marked decrease in
the population of spin probes that switched to the more mobile
conformation at pH 4.6 (Figure 5), suggesting that cardiolipin
hinders proton-induced motions near loop 9.
In the TMD, the CW spectra of K247R1 and P249R1, located
in the M2–M3 loop, also revealed the spin labels were motionally
restricted, indicating a sterically packed environment (Figure 3).
For K247R1, we observed an additional slight decrease in mobility
upon switching to pH 4.6 and no changes in mobility for P249R1.
The lack of significant proton-induced changes in probe mobility
was unexpected, since motions in the M2–M3 loop have been
suggested to play an important role in coupling agonist binding to
channel gating [7,38–40]. To ensure that we were maximally
activating the MTSL-labeled GLIC protein, we also collected CW
Figure 3. CW EPR spectra reveal proton-induced gating movements. Comparison of X-band CW EPR spectra of spin-labeled GLIC wild-type
and mutant protein at pH 7.6 (black, closed state) and pH 4.6 (blue, desensitized state). Spectra were recorded at room temperature over 100 G. Pairs
of data were recorded on the same spectrometers and under identical conditions. Immobile and mobile components in the low-field region of the
K32R1 spectrum are indicated by arrows. The low-field regions of the K32R1, T157R1, and K247R1 spectra are enlarged to highlight the proton-
induced changes. (Top left) Close-up view of GLIC crystal structure with spin-labeled positions C26, K32, T157, K247, and P249 shown in space-fill.
SDSL EPR Reveals Gating Motions in pLGICs
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spectra at pH 3.0 (Figure S3). Upon changing the pH to 3.0, probe
mobility for K247R1 decreased slightly more, whereas no changes
were seen for P249R1, as judged by the inverse central line width
DH021(Figure 4). In general, no significant changes in the overall
line shape of the CW spectra at pH 3.0 compared to pH 4.6 or
pH 7.6 were observed (Figure S3).
Measuring Distances and Proton-Induced Distance
Changes Using DEER Spectroscopy
Using DEER spectroscopy, distances in the range of 18 to
60 A˚between paramagnetic centers in a membrane protein can
be measured [41–43]. Because GLIC is a homopentamer, two
distances are expected at each labeled position: one between
spin probes on adjacent subunits, another between probes on
nonadjacent subunits (Figure 6, Figure S4A) with a theoretical
nonadjacent and adjacent distance ratio of 1.6 expected. We
measured the distances between probes in GLIC at pH 7.6, which
stabilizes the closed state, and at pH 4.6, which favors desensitized
states, to test if, and how far, the loops at the ECD/TMD gating
interface (e.g., K32R1, T157R1, K247R1, and P249R1) move
during proton-dependent gating transitions. Currently, a high-
resolution structure of GLIC is only available in an apparently
open channel conformation, and little is known about the process
of desensitization at the structural level. While there are uncer-
tainties in assigning functional states to the ELIC and GLIC
crystal structures, comparing ELIC (PDB entry 2VL0) and GLIC
(PDB entry 3EHZ) solved in apparently closed and open channel
conformations, respectively [6,7,10], suggests that loops 2 and 9
move inward toward the channel lumen (,1.7 A˚for K32 relative
to ELIC’s L29, and ,5 A˚for T157 relative to ELIC’s D158),
whereas the M2–M3 loop moves ,6 A˚outward away from the
channel lumen (K247 relative to ELIC’s R254 and P249 relative
to ELIC’s P256) with channel activation (Figure 1C,D; Table 1;
see Materials and Methods and Figure S4B for displacement
calculations). By comparing our experimental DEER distances
obtained from functional protein in lipids to those predicted from
the crystal structures, we can begin to assess the conformational
states to which these structures correspond.
We initially examined C26R1, which is located on b-strand 1 in
the ECD, for intersubunit distances by DEER spectroscopy.
Figure 6 shows the background subtracted dipolar evolution
fit using Tikhonov regularization, a model-free approach. The
interspin DEER-derived distances were 22 A˚and 35 A˚at pH 7.6
(adjacent and nonadjacent subunits, respectively), and 22 A˚and
34 A˚at pH 4.6 (Figure 6, Table 1). Similar distance distributions
were obtained when we fit the data using 2-Gaussian or 2 Rice3D
model-based approaches. The nonadjacent:adjacent distance
ratios were 1.6, in excellent agreement with the theoretical
value for a homopentameric labeled protein. The DEER-derived
interspin distances were slightly shorter than the Cb–Cbdistances
(Table 1) measured in the crystal structures of ELIC and GLIC.
The absence of detectable pH-induced distance changes indicates
either a lack of motion or a concerted rigid-body motion for the
For K32R1, in loop 2, the experimental distances were 20 A˚
and 28 A˚at pH 7.6 (adjacent and nonadjacent subunits,
respectively), and 19 A˚and 27 A˚at pH 4.6 (Figure 6, Table 1).
Figure 4. Proton-induced changes in spin probe mobility,
DH021. For each GLIC mutant (C26R1, K32R1, T157R1, K247R1, and
P249R1), the inverse width of the central line of the CW spectra, DH021,
is plotted at pH 7.6 (black), pH 4.6 (blue), and pH 3.0 (red, K247R1 and
P249R1 only). An increase in DH021reflects increased R1 mobility,
whereas a decrease reflects decreased mobility.
Figure 5. Effects of lipids on proton-induced gating motions. X-
band CW EPR spectra of T157R1 reconstituted into PE:PG (top),
PE:PG:cholesterol (middle), and PE:PG:cardiolipin (bottom) liposomes
at pH 7.6 (black, resting state) and pH 4.6 (blue, desensitized). Proton-
induced changes in T157R1 mobility in the presence of cholesterol were
indistinguishable from those of T157R1 reconstituted in PE:PG, whereas
cardiolipin hindered gating-induced changes in T157R1 mobility.
SDSL EPR Reveals Gating Motions in pLGICs
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The nonadjacent:adjacent distance ratios were 1.4, which are
smaller than the 1.6 theoretical value, suggesting that the probe
locations were not perfectly symmetrical. The small (less than 1 A˚)
proton-dependent change in the interprobe center distances
suggests that there is little to no proton-induced displacement of
For T157R1, in loop 9, the interspin DEER-derived distances
were 30 A˚ and 49 A˚ at pH 7.6 (adjacent and nonadjacent
subunits, respectively), and 19 A˚and 31 A˚at pH 4.6 (Figure 6).
The nonadjacent:adjacent distance ratios were 1.6, in excellent
agreement with the theoretical value for a homopentameric
labeled protein. At pH 4.6, we collected data out to a shorter
dipolar evolution time (solid blue line) to increase the quality of the
data. When we collected data out to the same evolution time as the
pH 7.6 sample (dotted blue line), intersubunit distances longer
than 31 A˚were not observed. Upon switching to pH 4.6, the
interprobe distance changed more than 17 A˚for the nonadjacent
distance, indicating that the probe attached to loop 9 undergoes a
large proton-induced inward movement toward the channel
lumen, with a displacement of 9.2 A˚(Figure S4).
Since T157R1 is a good reporter of proton-induced conforma-
tional motions, we examined the ability of GLIC to undergo these
rearrangements in detergent micelles. For T157R1 in DDM
micelles, the interspin DEER-derived distances were 31 A˚and
49 A˚at pH 7.6 (adjacent and nonadjacent subunits, respectively),
and 30 A˚and 49 A˚at pH 4.6 (Figure 7). The distances were
nearly identical at both pH values and matched the distances
obtained from GLIC reconstituted into PE:PG liposomes at
pH 7.6—that is, in the resting, closed channel state (Figure 6 and
Table 1). The data suggest that DDM inhibits proton-induced
motions in GLIC and locks GLIC in a conformation resembling
the resting state.
For K247R1, in the M2–M3 loop, at pH 7.6 only one interspin
distance of 30 A˚ was obtained using Tikhonov regularization
(Figure 6). These data were also fit using 2-Gaussians and 2 Rice3D
model-based approaches, which also resulted in only one distance
(i.e., two distances of the same value resulted). The interspin
distance does not correlate with either of the Cb–Cbdistances in
the ELIC crystal structure (13.8 A˚adjacent, 22.3 A˚nonadjacent)
but is comparable to the nonadjacent Cb–Cbdistance in GLIC
(33.7 A˚). At pH 4.6, we detected two distances of 18 A˚and 30 A˚
(adjacent and nonadjacent subunits, respectively) similar to Cb–Cb
distances measured in the GLIC crystal structure (Table 1). The
apparent absence of any proton-induced change in the nonadja-
cent 30 A˚ distance suggests that K247R1 occupies the same
location at both pH values, which is consistent with the very
modest changes we observed in the CW EPR spectra upon
switching to pH 4.6 (Figure 3). We cannot, however, rule out the
possibility that the interspin 30 A˚distance measured at pH 7.6 is
between adjacent subunits. Given the short phase memory time
Figure 6. Proton-induced distance changes revealed by DEER
spectroscopy. (Top panel) Top-down view of GLIC crystal structure
shown in ribbon representation with T157 in spacefill, black lines depict
distances between adjacent and nonadjacent residues. (Left panels)
Background subtracted Q-band DEER-refocused echo intensity (grey
lines) is plotted versus evolution time for each spin-labeled position at
pH 7.6 (resting state) and pH 4.6 (desensitized state) and fit using
model-free Tikhonov regularization (pH 7.6, black lines; pH 4.6, blue
lines). Pairs of data were recorded on the same spectrometers and
under identical conditions. (Right panels) The corresponding interspin
distance distributions are plotted at pH 7.6 (black) and pH 4.6 (blue)
with the mean distances for each peak labeled. For T157R1 and K247R1
samples at pH 4.6, we also collected data out to the same dipolar
evolution times as the pH 7.6 samples (dotted blue lines).
SDSL EPR Reveals Gating Motions in pLGICs
PLOS Biology | www.plosbiology.org7 November 2013 | Volume 11 | Issue 11 | e1001714
for this position (Tm=0.6 ms), a nonadjacent distance of approx-
imately 49 A˚(expected for a 30 A˚adjacent distance) is beyond the
range that can be reliably measured [41–43].
For P249R1, also in the M2–M3 loop, the interspin distances
were 20 and 30 A˚(adjacent and nonadjacent subunits, respec-
tively) at pH 7.6, and 20 A˚ and 28 A˚ at pH 4.6 (Figure 6),
indicating little to no proton-induced changes in distances,
consistent with the lack of proton-elicited changes seen in the
CW EPR spectra (Figure 3). Overall, the data suggest that the
M2–M3 loop is in a similar position in both the resting and
desensitized GLIC conformational states. This result is in contrast
to its approximately 6 A˚outward displacement away from the
channel lumen predicted by comparing the crystal structures of
ELIC and GLIC .
Estimating Distances by Computer Modeling
We also used computer modeling to evaluate how well the ELIC
and GLIC crystal structures predict our experimental DEER data.
We built a homology model of GLIC based on the ELIC crystal
structure and used the PRONOX program to estimate the
distances between spin labels in the GLIC model (Table S2). The
computed distances were then compared to our experimental
DEER distances. Using standard conditions in the program, no
distances were computed for C26R1, T157R1, and P249R1 due
to clashes (i.e., MTSL did not fit using favored rotamer
conformations), and the average interspin distances computed
for K32R1 and K247R1 were shorter than our experimental data.
When we relaxed conditions and allowed additional MTSL
rotamer conformations, the program still could not compute
distances for C26R1 and P249R1 and the distance for T157R1
was shorter than our experimental data. In general, the modeling
suggests the ELIC structure obtained in detergent micelles, at least
for these positions, is not a good model for the resting state of
GLIC embedded in lipids. We also used the PRONOX program
to estimate the distances between spin labels using the crystal
structure of GLIC (PDB entry 3EAM) as the input (Table S2).
Using standard or relaxed conditions, no distances were computed
for C26R1. For T157R1 and K247R1, the estimated distances
were similar to our experimental DEER distances obtained at
pH 4.6. The computed distances for K32R1 and P249R1 were
much shorter than the experimental DEER distances at pH 4.6,
suggesting the GLIC crystal structure, at least at these positions,
does not correspond to a desensitized conformation.
The structural rearrangements underlying how pLGICs tran-
sition between closed, open, and desensitized states are still
unclear. While high-resolution crystal structures of pLGICs in
apparently closed and open channel conformations [6,7,9,10]
have provided insights into possible activation mechanisms,
whether these static protein structures, solved in detergent
micelles, accurately capture the conformational gating transitions
that a functional pLGIC undergoes when embedded in a lipid
bilayer is unknown. Using SDSL EPR spectroscopy and functional
GLIC channels reconstituted in liposomes, we measured protein
motions associated with GLIC gating under conditions that
promote conformational transitions from closed to desensitized
states. We focused on the loops forming the interface between the
ECD and the TMD, specifically loops 2 and 9 in the ECD and the
Table 1. Summary of distances.
pH 7.6pH 4.6
Residue Short (A˚) Width (A˚)Long (A˚)Width (A˚) Short (A˚)Width (A˚)Long (A˚) Width (A˚)
C26R1225 355 225 3450
K32R1206 287 194 277 0.7, inward
L29ELIC/K32GLIC 16.1 26 14 22.7 1.7, inward
T157R1 305 499 196 317 9.2, inward
D158ELIC/T157GLIC 27.644.721.7 35.1 5, inward
K247R1303 185 3060
R254ELIC/K247GLIC13.8 22.3 20.833.7 6, outward
P249R1 206 305 207 2870
In roman type, adjacent (short) and nonadjacent (long) interspin distances and corresponding peak widths measured by DEER spectroscopy at pH 7.6 and pH 4.6 for
GLIC mutants reconstituted into PE:PG liposomes. In italics, adjacent (short) and nonadjacent (long) interresidue Cb–Cbdistances for C26, K32, T157, K247, and P249
obtained from GLIC (PDB entry 3EHZ, pH 4.6 distances), and for aligned residues I23, L29, D158, R254, and P256 in ELIC (PDB entry 2VL0, pH 7.6 distances). Last column
shows the calculated intrasubunit displacement d values (see Materials and Methods and Figure S4).
Figure 7. Detergent prevents proton-induced GLIC gating
motions. (Right panel) Interspin distance distributions from model-
free Tikhonov fits of X-band DEER data from GLIC T157R1 purified in
detergent (DDM) micelles at pH 7.6 (black) and pH 4.6 (blue). (Left
panel) The background-corrected dipolar evolution data at pH 7.6 and
pH 4.6 (grey lines) and the Tikhonov fits (pH 7.6, black lines; pH 4.6,
blue lines). The interspin DEER-derived distances are similar at both pH
values, indicating that detergent-solubilized GLIC does not undergo
proton-mediated gating motions.
SDSL EPR Reveals Gating Motions in pLGICs
PLOS Biology | www.plosbiology.org8November 2013 | Volume 11 | Issue 11 | e1001714
M2–M3 loop in the TMD, which previous studies have shown to
be important for coupling agonist binding to channel gating
[39,40,44,45]. Comparing the crystal structures of ELIC and
GLIC suggests that these loops undergo structural rearrangements
during activation, with loops 2 and 9 moving inward toward the
channel lumen, whereas the M2–M3 loop moves outward
(Figure 1C,D). Whether and if these loops move during
desensitization is unknown.
Here, we show that proton-dependent GLIC channel gating
transitions into a desensitized state induces substantial rearrange-
mentsof the intra-and intersubunitinterfacebetweenthe ECDand
TMD. The biggest change occurred in loop 9, where T157R1
underwent a large (,9.2 A˚) proton-induced inward movement
toward the channel lumen (Figure 6). The displacement was
accompanied by concurrent rearrangements in its surrounding
tertiary contacts, with the CW spectra (Figure 3) revealing a densely
packed environment in the resting state (pH 7.6) that becomes less
packed in the desensitized state (pH 4.6). The DEER-derived
interspin distances for T157R1 at pH 7.6 are longer than the Cb–
Cbdistances in the ELIC crystal structure and the intersubunit
distances estimated computationally, whereas the DEER distances
at pH 4.6 are shorter than the Cb–Cbdistances in the GLIC crystal
structure and the distances estimated computationally (Table 1,
Table S2). Thus, the resulting proton-mediated 9.2 A˚ inward
displacement of loop 9 measured by DEER spectroscopy is larger
than the predicted motion based on comparing the ELIC and
GLIC crystal structures. Since the residues in loop 9 in ELIC and
GLIC differ substantially [7,10], it is not surprising that the
magnitude of the DEER-derived displacement measured in a single
protein, GLIC, does not match the displacement calculated by
comparing the structures of two different proteins. Also, the loop 9
displacement measured by DEER spectroscopy in a functional
protein reconstituted in liposomes may differ from the motion
observed when the protein is constrained in a crystal lattice in
detergent. In support of this latter possibility, our DEER data
demonstrate that the proton-mediated motion of T157R1 is lost in
DDM micelles compared to T157R1 reconstituted in PE:PG
liposomes (Figures 6 and 7). This finding is consistent with the loss
of channel function observed for eukaryotic nicotinic acetylcholine
receptors solubilized in DDM  and supports the idea that
detergent-solubilization of membrane proteins can affect structural
dynamics and result in conformational ambiguity of the crystal
structures solved in the presence of detergents (reviewed in ).
Our DEER spectroscopy experiments measured the intersubu-
nit distances of T157R1 in GLIC in a resting, closed state (pH 7.6)
and in a desensitized, closed state (pH 4.6). Currently, our
experiments cannot distinguish whether loop 9 moves in the open
state and remains displaced during desensitization or whether its
movement occurs specifically in the desensitized state. Nonethe-
less, the data directly demonstrate that a large inward motion of
loop 9 occurs during GLIC gating transitions, which results
in substantial rearrangements of the intersubunit interface. We
predict that a similar inward motion of loop 9 in eukaryotic
pLGICs occurs during agonist-mediated channel gating transi-
tions. By measuring changes in cysteine accessibility, disulfide
bond formation, and attached fluorophore emissions, agonist-
induced local rearrangements near loop 9 have been detected in
nicotinic acetylcholine receptors [47,48], GABAAreceptors [49–
52], glycine receptors , and serotonin-type 3 receptors .
Proton-mediated structural rearrangements in the local protein
environment near K32R1 in loop 2 were also observed. CW EPR
spectroscopy (Figure 3) revealed K32R1 is in a more densely
packed environment in the desensitized state (pH 4.6) compared
to the resting state (pH 7.6), suggesting that during channel
activation to desensitization loop 2 becomes less mobile. Since our
DEER measurements at pH 7.6 and 4.6 demonstrate loop 2
undergoes minimal proton-induced displacement (Figure 6), the
decrease in K32R1 mobility likely arises primarily from an
increase in its surrounding tertiary interactions. Residues in loop 7,
loop 9 (adjacent subunit), M2–M3 loop, and the pre-M1 are in
close proximity to loop 2, and functional studies in eukaryotic
pLGICs have shown that a network of electrostatic and hydro-
phobic interactions between these regions and loop 2 play a role in
coupling binding to gating [45,55]. Our data suggest that increases
in these interactions and a resulting immobilization of loop 2
accompany GLIC channel gating transitions into the desensitized
Proton-induced conformational rearrangements near the M2–
M3 loop in the TMD were minimal, at least for the two positions
we examined, K247R1 and P249R1. In the resting closed channel
state (pH 7.6), the EPR spectra (Figure 3) showed the spin-probes
at both positions were motionally restricted, reflecting a sterically
packed environment near the probes. Upon switching to pH 4.6
or 3.0 (desensitized state), only a modest decrease in K247R1
mobility was observed, whereas no change in P249R1 mobility
was seen (Figures 3 and 4). Moreover, our DEER data indicate
that there were essentially no proton-mediated changes in inter-
subunit distances between probes at these positions (Figure 6).
Overall, our data suggest the M2–M3 loop does not undergo
Based on the crystal structures of ELIC and GLIC, the M2–M3
loop is predicted to move ,6 A˚(Cb–Cb) outward away from the
channel lumen during activation (Figure 1C and Table 1). One
possible explanation for the difference between our data and the
structure-based predictions is that in the desensitized state, which
we are preferentially monitoring at pH 4.6, the M2–M3 loop
adopts a conformation that resembles its conformation in the
resting state. Consistent with this idea, photolabeling of a residue
in the M2–M3 loop of the nicotinic acetylcholine receptor delta
subunit is state-dependent with robust labeling seen only in open
and fast desensitized states and little to no labeling in the resting
and slow desensitized states . In a cysteine accessibility study,
modification of cysteines introduced into the GLIC M2 helix and
the M2–M3 loop were faster at pH 5.0 than pH 7.5, indicating a
proton-mediated increase in accessibility . Again, a possible
explanation for the difference between their data and ours is that
we are monitoring desensitized states at pH 4.6, whereas in their
study the channels are submaximally activated at pH 5.0 and in a
mixture of open and possibly resting and desensitized conforma-
tions. Overall, our SDSL EPR data suggest that the M2–M3 loop
is in a relatively packed environment in the resting state that is
relatively unchanged in the desensitized state. A recent work 
suggests residues in the middle of M2 move inward to occlude the
channel during desensitization, whereas residues in the extracel-
lular end of M2 remain displaced outward. One might expect then
that the M2–M3 loop, which is attached to M2, would remain
displaced outward. Our data suggest that this is not the case.
Whether the GLIC M2–M3 loop moves substantially during
proton-induced channel opening and then moves back to a
position similar to that adopted in the resting state during
desensitization is unclear. The motions inferred from static crystal
structures of two different proteins in uncertain functional
conformations might not accurately reflect gating motions of a
functional protein embedded in a lipid membrane. The inter-
subunit distances we measured at pH 7.6 in the resting state
for both K247R1 and P249R1 are substantially different from
the Cb–Cb distances in the crystal structure of ELIC for the
structurally aligned residues R254 and P256 and from the
SDSL EPR Reveals Gating Motions in pLGICs
PLOS Biology | www.plosbiology.org9 November 2013 | Volume 11 | Issue 11 | e1001714
intersubunit distances that we estimated computationally, sug-
gesting that the ELIC structure is not a good model for the
conformation of GLIC’s M2–M3 loop in the resting state. In the
recently crystallized locally closed GLIC structures, the three
conformations of the M2–M3 loop are different than the M2–
M3 loop conformation in ELIC . In addition, comparing
one of the locally closed channel GLIC structures (LC2
conformation, PDB entry 3TLS) with GLIC in an apparently
open channel state (PDB entry 3EAM) suggests channel closing/
opening can occur without significant rearrangements of the
MTSL labeling and/or detergent solubilization and membra-
ne-reconstitution could lock the receptor in a nonactivatable
‘‘uncoupled’’ state, which abolishes its ability to undergo
conformational transitions in response to pH changes. Previous
work on nicotinic acetylcholine receptors have shown that lipid
composition and choice of detergent are critically important for
maintaining optimal receptor functionality [32,33,46,58]. Since
we record robust proton-elicited currents in bilayers with our
purified PE:PG lipid reconstituted GLIC protein, and when we
directly inject purified MTSL-labeled lipid reconstituted protein
into oocytes (Figure 2), these possibilities seem unlikely. More-
over, in a recent study, the ability of GLIC to undergo proton-
dependent gating transitions was maintained following its
reconstitution in a variety of different lipids , consistent with
Both cholesterol and anionic lipids are well-known modulators
of pLGIC function , and it was recently shown that cholesterol
modulates GLIC gating kinetics, speeding its desensitization .
Our data using CW EPR spectroscopy showed that cholesterol,
when added to PE:PG liposomes, had no effect on the proton-
induced structural rearrangements near loop 9 (Figure 5). In
addition, cholesterol had little effect on GLIC single channel
conductance or open-dwell time. On the other hand, adding
cardiolipin to PE:PG liposomes reduced probe mobility at pH 4.6
compared to PE:PG alone or PE:PG:cholesterol, suggesting that
cardiolipin inhibits proton-induced motions near loop 9 (Figure 5).
In a recent report, GLIC protein appeared slightly more rigid
when reconstituted into membranes formed by E. coli lipids (i.e., a
PE:PG:cardiolipin mixture) as compared to reconstitution in
asolectin or phosphocholine , consistent with our finding that
cardiolipin decreases GLIC mobility.
In summary, using SDSL EPR spectroscopy, we present new
information about the structural changes associated with ligand-
induced gating motions in the prokaryotic pLGIC GLIC using a
functional protein reconstituted into a native-like lipid environ-
ment. We provide direct experimental evidence that structural
rearrangements of the intra- and intersubunit interface between
the ECD and TMD accompany pLGIC gating transitions from
closed to desensitized states. Specifically, in the ECD, proton-
induced gating transitions from closed to desensitized states
decrease local side-chain interactions with loop 9, which increases
loop 9 mobility and results in a large inward movement of loop 9,
whereas loop 2 becomes more immobilized. These data suggest
that desensitization not only involves structural changes in the M2
channel helix to block ion conduction  but also entails motions
in the ECD that likely change the network of interactions between
residues in loop 2, loop 7, loop 9, preM1, and the M2–M3 linker.
In the resting state, the M2–M3 loop in the TMD domain is
relatively immobile and in a packed environment, and remains in
nearly the same position in the desensitized state. The position of
P249R1 in the M2–M3 loop in the desensitized state is
substantially different than that observed in the GLIC apparently
open channel structure, suggesting the crystal structure is not in a
desensitized conformation. Currently, a resting, closed channel
state structure of GLIC is not available. Our DEER data provide a
first glimpse of the positions of GLIC residues in the resting state
and suggest that the ELIC structure is not a good model for the
resting state. These findings advance our understanding of the
molecular mechanisms underlying pLGIC gating.
Materials and Methods
Cloning and Mutagenesis
The DNA sequence encoding GLIC (residues 44–359) was
extracted by PCR amplification from G. violaceus cells (ATCC), and
subcloned in vectors pUNIV  for two-electrode voltage clamp
experiments, and pET-26b (Novagen) for expression in E. coli.
GLIC DNA sequence was preceded in pUNIV by the DNA
sequence encoding the signal peptide of the GABAAreceptor b2
subunit to promote cell surface expression. pET-26b incorporates
an N-terminal pelB signal sequence for potential periplasmic
localization. In addition, DNA sequence for maltose-binding
protein (MBP) followed by a ,20 amino acid linker containing a
consensus sequence for thrombin cleavage was cloned following
the pelB signal and N-terminal to GLIC. GLIC mutants were
created using the QuikChange site-directed mutagenesis kit
(Stratagene). Mutations were confirmed by DNA sequencing.
Two-Electrode Voltage Clamp Recordings in Xenopus
Capped cRNAs encoding WT and mutant GLIC were
transcribed in vitro using the mMessage mMachine T7 kit
(Ambion). Single X. laevis oocytes were injected with 27 nL of
cRNA (50–100 ng/mL/subunit). Injected oocytes were incubated
at 16uC in ND96 (5 mM HEPES pH 7.4, 96 mM NaCl, 2 mM
KCl, 1 mM MgCl2, 1.8 mM CaCl2) supplemented with 100 mg/
ml of gentamycin and 100 mg/mL of bovine serum albumin for 2–
5 d before use for electrophysiological recordings. Oocytes were
perfused continuously with ND96 at pH 7.4 at a flow rate of
5 mL/min, while being held under two-electrode voltage clamp at
260 mV in a bath volume of 200 mL. Borosilicate glass electrodes
(Warner Instruments) used for recordings were filled with 3 M KCl
and had resistances of 0.4 to 1.0 MV. Electrophysiological
data were collected using Oocyte Clamp OC-725C (Warner
Instruments) interfaced to a computer with an ITC-16 A/D
device (Instrutech) and were recorded using the Whole Cell
Program, version 4.0.9 (kindly provided by J. Dempster, Univer-
sity of Strathclyde, Glasgow, UK). Proton-induced currents were
measured by perfusing ND96 buffered at pH 6.5–3.8. For
pH 5.0–3.8 HEPES was replaced with 5 mM Na Citrate as the
buffering agent. For pH 6.5–6.0 5 mM MES was used as the
buffering agent. GraphPad Prism 4 was used for data analysis
and fitting. pH response data were fit to the equation I=
Imax/(1+10(pH-pH50)*n), where I is the peak response at a given
pH, Imax is the maximum amplitude of current, pH50 is the
pH inducing half maximal response, and n is the Hill coefficient.
The functional effect of modifying substituted cysteines with
nate spin label (MTSL) was evaluated in oocytes using two-
electrode voltage clamp. Proton-induced currents were measured
at pH 5.0 until peak current amplitudes varied by ,5%. Oocytes
were then treated with 1 mM MTSL at pH 7.4 for 2 min,
washed for 5 min, and proton-induced currents were measured
again at pH 5.0. Extent of modification was quantified as
(12Iafter MTSL/Ibefore MTSL)*100%. WT and C26A were incubated
with 100 mM MTSL.
SDSL EPR Reveals Gating Motions in pLGICs
PLOS Biology | www.plosbiology.org 10November 2013 | Volume 11 | Issue 11 | e1001714
Protein Expression and Purification
E. coli BL21(DE3) strain cells (Invitrogen) were transformed
with the pET-26b vector encoding the GLIC constructs. Cells
were cultured in LB medium at 37uC to OD600,1.0–1.4, and then
expression was induced overnight at 20uC with 0.2 mM isopropyl
b-D-1-thiogalactopyranoside (IPTG). Cells were harvested and
lysed with an EmulsiFlex C-5 homogenizer (Avestin) in 20 mM
Tris-HCl pH 7.6, 150 mM NaCl (buffer B1) supplemented
with 1 mM PMSF, 2 mM pepstatin-A, and 2 mg/mL leupeptin
as protease inhibitors. The lysate was cleared by centrifugation
at 18,000 rpm for 30 min at 4uC, and then the pellet was
resuspended in B1 with 2% n-dodecyl-b-D-maltoside (DDM,
Anatrace) and gently agitated overnight at 4uC for protein
extraction from cell membranes. Solubilized pellet was cleared
by ultracentrifugation at 45,000 rpm with a 50.2 Ti rotor
(Beckman) for 45 min at 4uC and purified by affinity chromatog-
raphy with amylose resin (New England Biolabs). Amylose resin
with bound MBP-GLIC was washed with 10 volumes of B1 with
0.1% DDM followed by 10 volumes of B1 with 0.02% DDM
(buffer B2), and then the fusion protein was eluted in B2
supplemented with 20 mM maltose. MBP-GLIC was concentrat-
ed in Amicon Ultra-4 (100 KDa molecular weight cutoff)
concentrator tubes (Millipore) and subjected to size exclusion gel
filtration in a Superose6 GL10/300 column (GE Healthcare)
previously equilibrated in B2. Fractions of the peak corresponding
to pentameric MBP-GLIC (,400 kDa) were combined and
treated with MTSL and thrombin under gentle agitation at 4uC
overnight. In detail, protein was first treated with 5-fold molar
excess of DTT for 5 min at room temperature, and then 2- to 60-
fold molar excess of MTSL (Toronto Research) was added to
specifically label the unique cysteines, followed by 1 U of thrombin
(bovine, plasminogen-free, Calbiochem) per 100 mg of pentameric
MBP-GLIC. The digested product was applied to amylose resin
for a second round of affinity chromatography to purify the
cleaved, MTSL-labeled GLIC from the excess spin label and
MBP. GLIC was subjected to a final gel filtration, and peak
fractions corresponding to the pentameric form of the protein
(,180 kDa) were combined and concentrated to 3–6 mg/mL,
flash frozen in liquid nitrogen, and stored at 280uC.
Reconstitution of Purified GLIC Into Liposomes
Purified GLIC protein was reconstituted into liposomes formed
with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE)
(PG) at a PE:PG=2.7:1 molar ratio, or with PE:PG:cholesterol
at a 3.4:1.3:1 molar ratio. Lipid mixtures were prepared at a
concentration of 20 mg/mL in buffer B1, sonicated, and mixed
with GLIC purified in DDM, typically at 6,000-fold molar excess.
After a 3 h incubation at 4uC, the protein:lipid mixture was
diluted 2-fold in buffer B1 containing 10% glycerol and incubated
overnight at 4uC. To remove DDM, Biobeads (BioRad) were
added for 8–10 h and then removed. Finally, the Biobead-free
solution was ultracentrifuged at 100,000 rpm, and the pellets of
GLIC reconstituted into liposomes were stored at 280uC.
Two-Electrode Voltage Clamp Recording of Xenopus
leavis Oocytes Injected with Purified GLIC Reconstituted
Pellets of purified GLIC mutants reconstituted into liposomes
were thawed on ice. The amount of protein in a pellet was
estimated by assuming 70% reconstitution efficiency. The pellets
were resuspended in buffer B1 to a protein concentration of
approximately 1–2 mg/mL and were subjected to two rounds of
freeze-thaw. The proteoliposome solution was somewhat viscous
and to facilitate protein injection into the oocytes, the diameter of
the glass injection pipet was adjusted to about 5–10 mm. Protein-
injected oocytes were incubated for 5–8 h at 16uC before
recording. Two-electrode voltage clamp of oocytes injected with
lipid reconstuted GLIC protein was performed in the same
manner as oocytes injected with GLIC cRNA. pH-induced
currents from uninjected oocytes were used as controls.
Single-Channel Recordings in Planar Lipid Bilayers
For preparation of planar lipid bilayers, lipid mixtures of PE:PG
(5.8:2.3:1) were prepared in n-decane at a concentration of
20 mg/mL. Planar lipid bilayers were painted with a glass rod
across a 150 mm aperture in a Delrin cup. In order to create both
an ionic and a pH gradient, the trans chamber was filled with
150 mM NaCl at pH 7.6, whereas the cis chamber (where the
protein was added to) was filled with 450 mM NaCl at pH 5.2.
Prior to adding the GLIC K32R1, T157R1, and P249R1 protein
to the chamber, the protein was treated with 10 mM dithiothre-
itol (DTT) to remove the majority of the spin label. Once the
protein was incorporated into the planar lipid bilayer, the pH of
the cis chamber was dropped to 4.6 by adding 10% (v:v) of 1 M
Na Citrate. Single-channel currents were recorded using an
Axopatch 200B amplifier (Axon Instruments), filtered with an 8-
pole low-pass Bessel filter (Frequency Devices) set at 100 Hz, and
digitized at a rate of 4 kHz with a Digidata 1440A interface
(Axon Instruments). Data acquisition and analysis were per-
formed with pClamp10.2.
Continuous wave (CW) EPR spectroscopy was carried out at
room temperature on a Bruker ELEXSYS 500 X-band spectrom-
eter equipped with a superhigh Q (SHQ) cavity (Bruker Biospin).
Upon change in pH to 4.6 or 3.0, the proteolipid samples were
freeze-thawed to ensure even distribution of the protons inside and
outside the vesicles. Spectra were then recorded over 100 G under
nonsaturating conditions with a 100 kHz field modulation of 1 G.
Samples were typically 20 mL in volume and contained in a glass
capillary. Protein concentrations were typically 30 mM.
The DEER spectroscopy experiments were carried out at the
Ohio Advanced EPR Laboratory at Miami University using a
Bruker ELEXSYS 580 Q-band spectrometer equipped with
a Bruker EN5107D2 dielectric resonator or at the National
Biomedical EPR Center using a Bruker ELEXSYS 580 X-band
spectrometer equipped with a Bruker 3 mm split-ring cavity.
Samples were typically 10 mL for Q-band and 25 mL for X-band
at a concentration of 35–50 mM, contained 20% deuterated
glycerol as a cryoprotectant, were flash frozen using a dry ice-
acetone slurry, and run at 80 K. A four-pulse DEER sequence
 was used with two-step phase cycling. The dipolar evolution
data were analyzed for distance distributions using DeerAnaly-
sis2011 software  and model-free Tikhonov regularization as it
gave the best fit to the background-corrected data. Distribution
curves obtained from model-free Tikhonov regularization were
then fit to Gaussian shapes using Peak Fitter (T. O’Haver,
MATLAB File Exchange) to obtain the mean peak center distance
values. Rice3D and Gaussian analyses of the dipolar evolution
data yielded similar results as the model-free Tikhonov regular-
ization analysis. All DEER data distributions shown are the
result of model-free Tikhonov regularization. Pairs of data were
recorded on the same spectrometers and under identical
SDSL EPR Reveals Gating Motions in pLGICs
PLOS Biology | www.plosbiology.org11 November 2013 | Volume 11 | Issue 11 | e1001714
Calculation of Minimum Displacement d
The minimum displacement d for a spin probe is calculated
using the formula:
where ACand AOare the DEER-determined distances between
probes in adjacent subunits (indicated as ‘‘short’’ in Table 1) at
pH 7.6 and 4.6, respectively, and NCand NOare the distances
between probes in nonadjacent subunits (indicated as ‘‘long’’ in
Table 1) at pH 7.6 and 4.6, respectively (see Figure S4B). The
formulas are also used to calculate the displacement of a residue in
GLIC relative to the structurally aligned amino acid in ELIC using
the crystal structures. In this case, ACand AOare the Cb–Cb
distances separating pairs of equivalent residues in adjacent
subunits in ELIC and GLIC, respectively, whereas NCand NO
are the distances separating pairs of equivalent residues in
nonadjacent subunits in ELIC and GLIC, respectively. In this
method the displacement d depends on distances calculated within
each crystal structure (i.e., the intrinsic coordinates), which is a
more reliable method than positioning the two structures on top of
each other and measuring distances between aligned residues.
Homology Modeling and Computational Modeling of
MTSL on GLIC
A closed state homology model of GLIC based on the crystal
structure of ELIC (PDB entry 2VL0) was built using Modeller
as described by Ghosh and co-workers . The PRONOX
pronox/pronox.html) was used as described by Hatmal and
colleagues  to estimate the distances between spin labels using
our GLIC homology model and the crystal structure of GLIC (PDB
entry 3EAM) as inputs. In general, the PRONOX distances were
estimated using the standard approach. For some positions, we
used the fine search option to help remove clashes. All distances are
from N to N atoms. The computed PRONOX distances calculated
for the GLIC homology model and the GLIC crystal structure were
compared to our experimental DEER distances measured at
pH 7.6 and pH 4.6, respectively. Note, the DEER distances are
obtained from lipid-embedded functional GLIC protein, while the
PRONOX distances are based on static X-ray crystal structures of
two different proteins in detergent micelles.
in oocytes. (A) Chemical structure of MTSL and the R1 side
chain that is created upon reaction of MTSL with cysteine. (B) pH
dose-response curves for wild-type and mutant GLIC receptors
expressed in Xenopus laevis oocytes. All mutants formed functional
channels. (C) Representative currents induced by pH 5.0 buffer
from oocytes expressing C26A and T157C before and after 2 min
application of 100 mM and 1 mM MTSL, respectively. MTSL
significantly reduced proton-mediated current amplitude for
T157C, indicating that MTSL covalently modified the introduced
cysteine at this position.
Functional characterization of GLIC mutants
GLIC mutant receptor. No significant EPR signal is observed,
indicating the absence of spin-labeled protein contaminants. (Left)
CW EPR spectrum from MTSL-treated C26A
Expanded view of GLIC crystal structure with C26 shown in
mutant receptors. Comparisons of CW EPR spectra of
K247R1 (top right) and P249R1 (bottom right) GLIC mutants
reconstituted into PE:PG liposomes at pH 7.6 (black), pH 4.6
(blue), and pH 3.0 (red). For K247R1, pH 3.0 induced an
additional slight decrease in probe mobility compared to
pH 4.6. Expanded view of GLIC crystal structures are shown
with spin-labeled positions K247 (top left) and P249 (bottom left)
CW EPR spectra of K247R1 and P249R1 GLIC
Because GLIC is a homopentamer, two distances are expected
at each pH: one between spin probes on adjacent subunits,
another between probes on nonadjacent subunits. (B) Schematic
diagram illustrating the proton-induced displacement, d, of the
spin probe in a single subunit based on the DEER data. ACand
AOare the DEER-determined distances for adjacent subunits at
pH 7.6 and 4.6, respectively; NCand NOare the distances for
nonadjacent subunits at pH 7.6 and 4.6, respectively; rCand rO
are the radii of circles circumscribing the pentagons. To take
into account a general quaternary twisting, d is derived assuming
a rotation h of one pentagon relative to the other. For simplicity,
we assume the two pentagons lie on the same plane. The
resulting equation can be derived using basic geometry and
trigonometry. The simplest case, no rotation (i.e., h=0),
provides the minimum displacement a spin probe undergoes
Calculating spin probe displacement, d. (A)
fication of WT and mutant GLIC channels. pH50 is
pH value that elicited 50% of the maximal proton-induced
current. nHis the Hill coefficient. Data are mean 6 SEM from
n experiments. MTSL modification is defined as (12Iafter MTSL/
Ibefore MTSL)*100%, where Iafter MTSLand Ibefore MTSLare currents
elicited by pH50proton concentration after and before exposure to
MTSL, respectively. Data are mean 6 SEM from n2 experiments.
Values significantly different from C26A, *p,0.01, **p,0.001.
Summary of pH responses and MTSL modi-
placed in a GLIC homology model based on the ELIC
crystal structure (PDB entry 2VL0) and in the GLIC
crystal structure (PDB entry 3EAM). ND: Using standard
conditions in the program, no distances were computed for
C26R1, T157R1, and P249R1 due to clashes (i.e., PRONOX
could not place MTSL at the position). *Using relaxed conditions,
Pronox could still not place MTSL at these positions and no
distances were computed. **Using relaxed conditions, intersubunit
distances of 25 A˚(adjacent) and 40 A˚(nonadjacent) for T157R1
We are grateful to Dr. Hector Valdivia (University of Michigan) for use of
his planar lipid bilayer set-up and for insightful suggestions. We thank Dr.
Michelle Capes for her help with planar lipid bilayer single-channel
SDSL EPR Reveals Gating Motions in pLGICs
PLOS Biology | www.plosbiology.org12November 2013 | Volume 11 | Issue 11 | e1001714
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: CDD SMH
CSK CC. Performed the experiments: CDD BG SMH JMR VAG GMD
AMS KS CSK. Analyzed and discussed the data: CDD CSK CC.
Contributed reagents/materials/analysis tools: CSK CC. Wrote and edited
the paper: CDD CSK CC. Made mutants: CDD VAG SMH BG.
Conducted two-electrode voltage clamp recordings in oocytes: SMH BG.
Purified the protein: CDD JMR VAG GMD AMS. Reconstituted GLIC
protein into liposomes, recorded and analyzed single-channel currents in
planar lipid bilayers: CDD. Collected and analyzed EPR data: CSK. Built
homology model of GLIC: BG KS. Used computer modeling to estimate
interspin distances: KS. Supervised the project: CC.
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