Electrophysiological characterization of LacY
Juan J. Garcia-Celmaa, Irina N. Smirnovab, H. Ronald Kabackb,1, and Klaus Fendlera,1
aDepartment of Biophysical Chemistry, Max Planck Institute of Biophysics, D-60438 Frankfurt am Main, Germany; andbDepartments of Physiology and
Microbiology, Immunology, and Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles, CA 90095-7327
Contributed by H. Ronald Kaback, March 12, 2009 (sent for review January 8, 2009)
Electrogenic events due to the activity of wild-type lactose per-
mease from Escherichia coli (LacY) were investigated with proteo-
liposomes containing purified LacY adsorbed on a solid-supported
membrane electrode. Downhill sugar/H?symport into the proteo-
liposomes generates transient currents. Studies at different lipid-
to-protein ratios and at different pH values, as well as inactivation
by N-ethylmaleimide, show that the currents are due specifically to
the activity of LacY. From analysis of the currents under different
conditions and comparison with biochemical data, it is suggested
that the predominant electrogenic event in downhill sugar/H?
symport is H?release. In contrast, LacY mutants Glu-3253Ala and
Cys-1543Gly, which bind ligand normally, but are severely defec-
tive with respect to lactose/H?symport, exhibit only a small
electrogenic event on addition of LacY-specific substrates, repre-
senting 6% of the total charge displacement of the wild-type. This
activity is due either to substrate binding per se or to a conforma-
symport. We propose that turnover of LacY involves at least 2
electrogenic reactions: (i) a minor electrogenic step that occurs on
(ii) a major electrogenic step probably due to cytoplasmic release
of H?during downhill sugar/H?symport, which is the limiting step
for this mode of transport.
bioenergetics ? membrane proteins ? permease ?
solid-supported membrane ? transport
only a few functions, they catalyze uptake of nutrients, export of
toxic compounds, translocation of macromolecules, regulation
of cell turgor, and creation of electrochemical ion gradients
important for the function of other membrane proteins. Bacte-
rial homologues of mammalian transporters have also become
important, because they can be conveniently obtained, purified
readily in large amounts, and used for crystallization and struc-
ture determination. One of the best-characterized systems is the
lactose permease from Escherichia coli (LacY), structures of
which have been solved recently at atomic resolution (1–3).
Because of the wealth of biochemical and biophysical data
available for LacY (4–8), it represents an ideal model system for
the investigation of the basic principles and molecular details of
secondary active transport.
Among the many methods for functional characterization,
electrophysiology is arguably the most universal, because it does
not require labeled substrate. Also, it is an extremely sensitive,
highly time-resolved technique that allows direct measurement
of charge movement. Although it has been known for many years
that lactose/H?symport catalyzed by LacY is an electrogenic
reaction (9–11), despite numerous efforts, LacY has so far
resisted all attempts at electrophysiological analysis. Although
the lacY gene is expressed well in frog oocytes and other
eukaryotic cells, LacY remains in the cis-Golgi and the perinu-
clear membrane, and does not target to the plasma membrane
electrophysiological study of LacY by using purified, reconsti-
tuted proteoliposomes with solid-supported membrane (SSM)
based electrophysiology (12).
econdary active transporters in the bacterial plasma mem-
brane are of fundamental importance for the cell. To name
Downhill Sugar/H?Symport Generates Transient Currents. Proteoli-
posomes containing reconstituted LacY were immobilized on an
SSM-coated gold electrode (the sensor), and charge displace-
ment induced by downhill sugar/H?symport into the proteoli-
posomes was detected by capacitive coupling (13). Transport was
initiated at 1.5 s by a sugar concentration jump using rapid
solution exchange (Fig. 1). Approximately 40 ms later, sugar
reaches the surface of the SSM, and a transient current starts
abruptly. The time course of the signals is characterized by 2
distinct phases: a rapid rise to a maximum followed by a much
slower decay toward the baseline. Because the decay is not
exponential, it was quantified by the decay time from peak to
half-maximal current, ?1/2, which is ?50 ms for all LacY sub-
Because the amount of proteoliposomes adsorbed to an
individual sensor exhibits some variability, only current ampli-
tudes obtained from the same sensor are compared directly. As
shown, the maximal value of the peak current observed after a
50 mM concentration jump of lactose is 950 pA, and the peak
and melibiose are ?65% and ?50%, respectively, of that re-
corded with lactose. However, addition of 50 mM sucrose (Fig.
1), a sugar that is not a substrate of LacY, has no effect.
Sugar binding and transport catalyzed by LacY are inactivated
by alkylation of Cys-148 primarily (14–16). Consistently, no
electrical transient is observed after treatment with N-
ethylmaleimide (NEM; Fig. S1).
Varying Lipid-to-Protein Ratio (LPR). Electrogenic transport by a
reconstituted protein leads to transient currents in the capaci-
tively coupled system (13, 17). With wild-type LacY, downhill
sugar/H?symport into the proteoliposomes generates an inside-
positive potential, which acts to decelerate the downhill symport
reaction catalyzed by LacY, leading to transient currents. How-
ever, any conformational transition that displaces charged amino
acyl side chains or reorients electrical dipoles also represents an
electrogenic transition that may contribute to the transient
nature of the currents. Indeed, it has been shown with the
melibiose permease from E. coli (MelB) that melibiose binding
triggers an electrogenic conformational transition that is a major
component of the transient currents observed (18, 19). To
discriminate between downhill sugar/H?symport and electro-
genic conformational transition, experiments were performed
with proteoliposomes reconstituted at different LPR (wt/wt). As
shown by freeze–fracture electron microscopy (Fig. S2 A and B),
at LPRs of 10 or 5, liposomes with LacY particle densities of
?1,000 and ?4,500 particles per ?m2, respectively, are observed.
Author contributions: J.J.G.-C. and K.F. designed research; J.J.G.-C. performed research;
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/cgi/content/full/
May 5, 2009 ?
vol. 106 ?
no. 18 ?
An electrogenic conformational transition is expected to yield
identical time constants for decay of the transients at different
particle densities. In contrast, charging of the liposomal mem-
brane by downhill sugar/H?symport should lead to decreasing
decay time (lower ?1/2) at increasing protein density (i.e., lower
LPR). As shown in Fig. 2, increased LacY particle density clearly
leads to significantly faster decay. Thus, the transient currents
observed for wild-type LacY represent mainly charging of the
liposome membrane due to downhill sugar/H?symport activity.
Effect of pH.Theshapeandmagnitudeofthetransientsgenerated
by downhill lactose/H?symport strongly depends on pH (Fig. 3).
An overall increase in pH from 6.6 to 8.5 causes a 5-fold increase
in the magnitude of the peak current (Fig. 3; Table 1). The decay
time also depends on pH, because ?1/2decreases from ?103 to
46 to 27 ms, respectively, at pH 6.6, 7.6, and 8.5 (Fig. 3; Table 1).
This trend is anticipated, because higher electrogenic activity of
the transporter leads to faster charging of the liposome mem-
brane and a concomitant faster current decay (i.e., lower ?1/2)
(17). Therefore, the effect of pH on the amplitude and time
dependence of the transient currents is consistent with the
proposed assignment of the peak currents to the symport activity
Proteoliposomes adsorbed to an SSM surface are stable for
hours without loss of activity, allowing investigation of the effect
of pH on the kinetics of the transient currents induced at
different lactose concentrations (Fig. 3 Inset; Table 1). An
increase in pH from 6.6 to 8.5 generates a 5-fold increase in the
saturating peak current (Fig. 3; Table 1). Indeed, rates of efflux
(i.e., downhill lactose/H?symport in the opposite direction)
after a 50 mM sugar concentration jump at t ? 1.5 s. The traces in black, red,
and green correspond to concentration jumps of lactose (50 mM; ?Lactose),
lactulose (50 mM; ?Lactulose), melibiose (50 mM; ?Melibiose), and sucrose
(50 mM; ?Sucrose), respectively. The nonactivating solution (50 mM glucose)
pH 7.6 plus 1 mM DTT. All traces shown were recorded from 1 sensor.
Transient currents obtained with wild-type LacY proteoliposomes
exchange protocol and the nonactivating solution were as described in Fig. 1,
but the activating solution contained 40 mM glucose plus 10 mM lactose.
Therefore, the difference between the test and nonactivating solutions rep-
proteoliposomes reconstituted at a LPR of 10 (?1,000 particles per ?m2; Fig.
S2A) or at a LPR of 5 (?4,500 particles per ?m2; Fig. S2B) were activated with
is decreased almost 5-fold from a ?1/2? 53 ? 2 ms at an LPR of 5 (black trace)
to a ?1/2? 260 ? 2 ms at an LPR of 10 (gray trace).
concentration jumps at different pH values. The traces were successively
recorded on the same sensor after equilibration was reached and are, there-
fore, directly comparable. To equilibrate the pH across the proteoliposome
membrane after changing the pH of the solutions, the immobilized proteo-
liposomes are incubated for ?20 min at the new pH. Subsequent lactose
concentration jumps produced constant currents indicating that the pH value
had indeed equilibrated. The nonactivating solution contained 50 mM glu-
or 6.6 (light gray trace) plus 1 mM DTT. (Inset) Dependence of peak currents
plus 50 ? x mM glucose to maintain a constant sugar concentration. The
value plus 1 mM DTT, and the pH was equilibrated across the proteoliposome
membrane. The peak currents recorded at pH 8.5 for each lactose concentra-
tion jump were fitted with a hyperbolic function, and all data obtained with
that sensor (every lactose concentration at the 3 pH values) were expressed as
fraction of maximum value at pH 8.5 (Ipeak
analysis, the complete dataset was recorded on 3 different sensors, and the
averaged values and errors (SE) are shown. From the hyperbolic fits, apparent
K0.5values with SE were obtained at every pH (Table 1).
Effect of pH on the transient currents generated after 50 mM lactose
max). This normalization procedure
Table 1. Kinetic parameters of the transient currents measured
for wild-type LacY
pH Efflux, % Peak current, %
27 ? 0.6
46 ? 2
103 ? 7
6.9 ? 0.7
5.3 ? 0.5
4.3 ? 0.86
For comparison the effect of pH on the initial rates of [14C]lactose efflux is
in Fig. 3.
www.pnas.org?cgi?doi?10.1073?pnas.0902471106 Garcia-Celma et al.
from right-side-out membrane vesicles (20), or proteoliposomes
reconstituted with purified LacY (21, 22), exhibit a similar
dependence on pH (Table 1). The half saturating concentration
increases only slightly with the pH (Fig. 3 Inset; Table 1). At pH
7.6, a K0.5of 5.3 ? 0.5 mM is obtained, which is close to that of
3.1 ? 0.7 mM determined for downhill lactose/H?influx in
proteoliposomes reconstituted with purified LacY (23).
Transient Currents in Mutants. To dissect the overall electrogenic
response, mutants of LacY that bind ligand but do little or no
lactose/H?symport were used. Mutant E325A is specifically
defective in all steps involving H?release from LacY but
catalyzes exchange and counterflow at least as well as wild-type
(24, 25). E325A LacY was reconstituted into proteoliposomes at
an LPR of 5 and a particle density comparable with that of the
wild-type preparation (?3,500 particles per ?m2; Fig. S3A).
Concentration jumps of 50 mM lactose, lactulose, or melibiose
produce transient currents with virtually identical kinetics and
negligible differences in magnitude (Fig. 4A), which are abol-
ished after treatment with NEM (Fig. S4A). However, the
transient currents are ?5-times smaller than those observed for
lactose with wild-type LacY (Table 2), and exhibit mono-
exponential decays (? ? 10 ms) followed by a shallow negative
phase (? ? 300 ms). Notably, the nonexponential decay of the
wild-type is characterized by ?1/2, whereas here the time constant
? is used. The negative component represents discharge of the
liposome membrane after rapid charge translocation. This phe-
nomenon is common for the capacitively coupled system (13),
and indicates absence of significant steady-state charge transport
across the liposome membrane (i.e., downhill sugar/H?symport
Mutant C154G binds sugar as well as wild-type and exhibits
extremely low, but significant, transport activities (26–29). Pro-
of ?3,500 particles per ?m2(Fig. S3B). Concentration jumps
with 50 mM lactose, lactulose, or melibiose generate transient
currents of comparable magnitude as E325A LacY (compare
S4B). In contrast to E325A, the magnitude and kinetics of the
peak currents recorded with C154G LacY depend on the sugar
used (Fig. 4B). Concentration jumps of 50 mM lactose or
lactulose trigger transient currents that decay mono-exponen-
tially, with time constants of ?20 ms. Interestingly, a 50 mM
melibiose concentration jump generates the largest peak current
with a significantly faster exponential decay (? ? 10 ms) followed
by a small negative phase.
From the transient currents measured, the kinetics of the true
transport currents generated by the mutants can be recon-
structed by using an iterative least-squares deconvolution algo-
rithm (Fig. S5 A and B). This operation requires a transfer
function for the specific measurements determined as described
(30). The transfer function is the derivative of the substrate
concentration rise at the surface of the SSM, and corresponds to
the time resolution of the measurement (15 ms) (13, 30).
However, significantly faster processes (k ? 200 s?1; see Fig. S5)
can be resolved with a least-square deconvolution algorithm
(30). With this deconvolution procedure, the time constants for
the underlying charge displacement are determined. For E325A
LacY, regardless of the sugar used, the transient currents are
indistinguishable from the transfer function, indicating that
charge translocation is too fast to be resolved by the measure-
ments. In this case, we can only estimate a lower limit for the rate
constant (k) of the process as ?200 s?1from the iterative
least-squares algorithm (Fig. S5A). For C154G LacY, the rate
constants for 50 mM concentration jumps of lactose or lactulose
are 53 ? 5 s?1or 72 ? 5 s?1, respectively, but the k for a 50 mM
concentration jump of melibiose is too rapid for accurate
measurement (k ? 200 s?1).
Wild-Type LacY. Reconstitution of LacY using the procedure
described previously (21, 31, 32) results in proteoliposomes with
?85% of the LacY molecules in the right-side-out orientation
(i.e., with the periplasmic side facing the exterior of the proteo-
liposomes) (33). Therefore, application of a substrate concen-
tration jump corresponds to substrate transport in the physio-
logical direction. All transported sugars trigger positive transient
currents, in agreement with the displacement of positive charge
(H?) into the proteoliposomes, as a result of downhill sugar/H?
symport. Of all sugars tested, lactose generates the largest
transient current, consistent with it being the most efficiently
transported substrate. The currents depend on pH and substrate
concentration, and are blocked by alkylation with NEM. Also,
good correlation is observed between kinetic parameters deter-
change protocol and composition of the solutions was the same as described
for Fig. 1. The baseline is represented in blue. (A) E325A LacY was reconsti-
tuted into liposomes, and activated with 50 mM concentration jumps of
lactose (50 mM; ?Lactose), lactulose (50 mM; ?Lactulose), or melibiose (50
mM; ?Melibiose) at pH 7.6. All traces exhibit virtually identical kinetics and
only small differences in magnitude with an exponential decay toward the
baseline (? ? 10 ms) followed by a negative phase (? ? 300 ms). (B) Transient
currents obtained with C154G LacY proteoliposomes after 50 mM sugar
?Lactose or 50 mM ?Lactulose decay mono-exponentially toward the base-
line, with time constants of ?20 ms, whereas the transients observed with 50
mM ?Melibiose exhibit the largest peak current and a significantly faster
exponential decay (? ? 10 ms) followed by a small negative phase.
Transient currents obtained with LacY mutants. The solution ex-
Garcia-Celma et al.PNAS ?
May 5, 2009 ?
vol. 106 ?
no. 18 ?
mined in our electrophysiological measurements and the values
previously obtained from downhill sugar/H?symport in proteo-
liposomes (23). All these observations strongly support the
contention that the transient currents reflect specifically the
electrogenic activity of LacY.
Because the decay time constant of the currents strongly
depends on the number of transporters incorporated into the
proteoliposomes, and decreases at high transport activity, it is
concluded that this phase reflects charging of the liposomes as
a result of downhill lactose/H?symport. A comparable depen-
dence of the decay time constant on the electrogenic transport
activity of bacteriorhodopsin was described for purple mem-
brane adsorbed to a planar lipid bilayer (17). Likewise, increas-
ing the amount of reconstituted Na?/H?exchanger in proteo-
liposomes adsorbed to the SSM resulted in a faster decay of the
transient currents (34). In both cases the measured peak currents
have been attributed to the continuous transport activity of the
corresponding transporter, and the same applies to LacY.
It is interesting to compare the LacY currents with those
observed with MelB, at similar time resolutions. With MelB,
biphasic current patterns are observed decaying with time
constants of ?1? 17 ms and ?2? 380 ms (18). The fast phase ?1
is due to an electrogenic conformational transition triggered by
melibiose binding, whereas the slow phase ?2 is related to
downhill sugar/Na?symport activity of MelB. With LacY, a fast
initial current phase is not observed. This deficiency is especially
apparent in the current recorded at low transporter density,
which decays slowly with a ?1/2 ? 260 ? 2 ms (Fig. 2). Such
behavior can only be explained if the initial electrogenic reac-
tions are slow or if charge translocation occurs late in the LacY
reaction cycle. As discussed below, sugar binding occurs with a
rate constant of ?50 s?1, ruling out a slow initial step. Although
this argument is strictly valid for the mutants only, the fact that
the mutants are fully capable of sugar binding indicates that the
same rate constants apply also to the wild-type. Together, the
data indicate that the major electrogenic step in wild-type LacY
occurs late in the reaction cycle.
LacY Mutants. The transient currents of the mutants E325A and
are 5–10 times smaller (Table 2) and considerably faster than the
transients observed with wild-type LacY. Smaller transient
currents could be interpreted as residual downhill sugar/H?
symport with reduced turnover in E325A and C154G. However,
this residual symport would lead not only to smaller current
amplitude, but also to slower decay (17). A good example for this
behavior is the transient currents observed with wild-type LacY
at pH 6.6, which are ?5 times smaller, and decay 5 times slower
observed with both mutants at pH 7.5 decay even faster than the
transients observed with the wild-type at maximal activity (pH
8.5). This observation indicates that the currents observed with
E325A and C154G LacY are not generated by sugar/H?sym-
port, but rather represent sugar binding induced electrogenic
A comparison between mutants and wild-type (Table 2) must
take into account the amount of reconstituted protein in the
membrane of the adsorbed proteoliposomes on the sensor.
Freeze fracture electron microscopy shows a comparable trans-
porter density for both mutants and the wild-type. The inte-
grated charge of C154G and E325A LacY of ?5 pC (Table 2)
represents the charge translocated in a single turnover by all
transporters on the SSM electrode. By comparison, the trans-
located charge per turnover of wild-type LacY is much larger;
particle density of wild-type and mutant proteoliposomes, it is
concluded that substrate binding to the mutants induces a charge
displacement corresponding to the transport of the equivalent of
only ?6% of an elementary charge across the membrane.
Although smaller, these transient currents can be used to esti-
mate binding rate constants for the different sugars and mutants.
They range between 50 and ?200 s?1, and depend on the nature
of substrate and mutant. Recent stopped-flow experiments
reveal that the binding of p-nitrophenyl ?,D-galactopyranoside
(?-NPG) to C154G/V331C LacY in detergent micelles is a 2-step
process: a binding step followed by a slower conformational
change with a rate constant of 238 s?1detected with fluorescent-
labeled protein (35). Clearly, sugar binding triggers a confor-
mational change with a rate constant similar to that observed in
the electrophysiological experiments, which may be responsible
for the charge translocation observed. Unfortunately, a direct
comparison of rate constants is not possible because of the
strong interaction of ?-NPG with the membrane that generates
large electrical artifacts.
Electrogenic Steps in the LacY Reaction Cycle.Combiningtheresults
obtained with wild-type LacY and the mutants, there is clear
evidence for a major electrogenic step late in the reaction cycle,
and a rapid electrogenic reaction during or immediately after
sugar binding with relatively low electrogenicity (6% of an
elementary charge). It seems unlikely that the latter is a unique
property of the E325A and C154G mutants. Most probably, this
electrogenic event also takes place in the wild-type, but is
completely masked by the 20-fold greater charge transport
activity of the wild-type, and is only observed under conditions
where sugar/H?symport is blocked, as in the mutant proteins.
Based on previous observations on the kinetics of LacY (20,
22, 36, 37), and on these electrophysiological findings, we
propose that the main electrogenic step corresponds to depro-
tonation of wild-type LacY in the inward-facing conformation.
In this context, it is notable that: (i) structural, biochemical, and
biophysical data strongly support the contention that wild-type
and C154G LacY are predominantly in an inward-facing con-
reconstituted with purified LacY is strongly influenced by the
voltage across the membrane, whereas exchange is completely
voltage independent (21); thus, the main electrogenic step is
related to protonation/deprotonation of LacY in either the
inward- or outward-facing conformations or the return of the
empty carrier; and (iii) the shape of the transient currents
indicates that the main electrogenic step in wild-type LacY
occurs late in the reaction cycle (see above). Considering these
points, it is likely that the inward-facing deprotonated trans-
porter is the initial state, and that deprotonation corresponds to
the main electrogenic step of the transport cycle. Following this
of the transport cycle, in agreement with the conclusions ob-
tained from the analysis of the transient currents. Because
downhill lactose/H?symport is specifically inhibited 3- to 4-fold
Table 2. Characteristics of LacY wild-type and mutant
Particles per ?m2
Peak current, pA
960 ? 190
96 ? 19
102 ? 67
5 ? 2
211 ? 50
4.4 ? 0.4
The number of particles per ?m2was estimated from freeze–fracture
images (Fig. S2 and Fig. S3). The peak currents refer to the transients after 50
1 and 4. The total translocated charge (Q) is obtained from numerical inte-
gration of the currents. Charge translocation per turnover of the wild-type
was estimated by using the current of 960 pA and an estimated turnover rate
of 10 s?1.
www.pnas.org?cgi?doi?10.1073?pnas.0902471106Garcia-Celma et al.
in deuterium oxide, deprotonation is probably not only the main
electrogenic step, but is also rate limiting (22).
With C154G LacY, all transport reactions, including equilib-
rium lactose exchange, are almost abolished (16, 38), indicating
that the substrate translocation step is blocked. In the absence of
substrate, this mutant is in an inward-facing conformation, and
paralyzed in an open conformation on the periplasmic side, but
E325A LacY exhibits no active transport whatsoever; however,
in contrast to C154G LacY, mutant E325A catalyzes exchange
and counterflow at least as well as wild-type LacY, indicating
that this mutant is permanently protonated (i.e., H?dissociation
is blocked in E325A LacY but binding is normal) (24, 25, 28). In
both mutants, a rapid but weakly electrogenic reaction with
similar magnitude and kinetic properties is seen, which agrees
with our notion that periplasmic H?binding is not responsible
for the charge translocation observed, and that the rapid initial
charge displacement of minor electrogenicity is associated with
sugar binding. Possibly, the rapid conformational transition after
sugar binding (35) leads to rearrangement of charged residues
within LacY (3, 40), which may account for this phenomenon.
Materials and Methods
Construction of Mutants and LacY Purification. Construction of mutants
and purification of the His-tagged proteins were carried out as described (7).
Purified proteins (10–15 mg/mL) in 50 mM NaPi/0.02% n-dodecyl-beta-D-
Reconstitution of Proteoliposomes. Reconstitution of purified wild-type or the
E325A and C154G mutants was carried out with E. coli phospholipids (Avanti
Polar Lipids) by using dodecyl maltoside/octyl glucoside dilution, followed by
1 cycle of freeze–thaw/sonication (41, 42). Purified wild-type LacY and lipo-
somes were mixed at an LPR of 10 or 5 (wt/wt), as indicated. Mutant E325A or
C154G was reconstituted at an LPR of 5. Before use, the samples were thawed
on ice and gently sonicated for 2–5 s. Reconstitution was verified in all cases
by freeze–fracture electron microscopy.
SSM Measurements. SSM measurements were performed as described previ-
of 1 mg/mL was allowed to adsorb for 1 h to an octadecanethiol/
exchange protocol consisted in 3 phases of duration 1.5, 2, and 1.5 s, respec-
tively. The nonactivating solution flows through the cuvette during the first
and third phases (from t ? 0 to 1.5 s, and from t ? 3.5 to 5 s), whereas the
activating solution flows during the second phase (from t ? 1.5 to t ? 3.5 s).
A valveless diverted fluidic geometry was chosen to apply the different
solutions (30) at a flow rate of 0.46 mL/s. The nonactivating solution always
at a concentration of 50 mM, unless stated otherwise (Fig. 3). All solutions
with a current amplifier set to a gain of 109-1010V/A and low pass filtering set
to 300–1,000 Hz.
ACKNOWLEDGMENTS. We thank Lina Hatahet and Andre Bazzone for excel-
lent assistance in the laboratory, Ernst Bamberg for helpful discussions and
support, and Winfred Haase for the excellent freeze–fracture micrographs of
the reconstituted liposomes. This work was funded by Deutsch Forschungs-
gemeinshaft SFB 807 (to K.F.); National Institutes of Health Grants DK051131,
DK069463, GM073210, and GM074929; and National Science Foundation
Grant 0450970 (to H.R.K.).
1. Abramson J, et al. (2003) Structure and mechanism of the lactose permease of Esche-
richia coli. Science 301:610–615.
2. Guan L, Mirza O, Verner G, Iwata S, Kaback HR (2007) Structural determination of
wild-type lactose permease. Proc Natl Acad Sci USA 104:15294–15298.
3. Mirza O, Guan L, Verner G, Iwata S, Kaback HR (2006) Structural evidence for induced
fit and a mechanism for sugar/H?symport in LacY. EMBO J 25:1177–1183.
4. Guan L, Kaback HR (2006) Lessons from lactose permease. Annu Rev Biophys Biomol
5. Kaback HR, et al. (2007) Site-directed alkylation and the alternating access model for
LacY. Proc Natl Acad Sci USA 104:491–494.
6. Majumdar DS, et al. (2007) Single-molecule FRET reveals sugar-induced conforma-
tional dynamics in LacY. Proc Natl Acad Sci USA 104:12640–12645.
7. Smirnova I, et al. (2007) Sugar binding induces an outward facing conformation of
LacY. Proc Natl Acad Sci USA 104:16504–16509.
8. Zhou Y, Guan L, Freites JA, Kaback HR (2008) Opening and closing of the periplasmic
gate in lactose permease. Proc Natl Acad Sci USA 105:3774–3778.
9. West IC (1970) Lactose transport coupled to proton movements in Escherichia coli.
Biochem Biophys Res Commun 41:655–661.
into Escherichia coli. Biochem J 127:P56.
Escherichia coli membrane vesicles: Further evidence for the involvement of histidine
residue(s). Biochemistry 21:5805–5810.
12. Seifert K, Fendler K, Bamberg E (1993) Charge transport by ion translocating mem-
brane proteins on solid supported membranes. Biophys J 64:384–391.
13. Schulz P, Garcia-Celma JJ, Fendler K (2008) SSM-based electrophysiology. Methods
14. Trumble WR, Viitanen PV, Sarkar HK, Poonian MS, Kaback HR (1984) Site-directed
15. Bieseler B, Prinz H, Beyreuther K (1985) Topological studies of lactose permease of
Escherichia coli by protein sequence analysis. Ann N Y Acad Sci 456:309–325.
16. van Iwaarden PR, Driessen AJ, Lolkema JS, Kaback HR, Konings WN (1993) Exchange,
efflux, and substrate binding by cysteine mutants of the lactose permease of Esche-
richia coli. Biochemistry 32:5419–5424.
17. Bamberg E, et al. (1979) Photocurrents generated by bacteriorhodopsin on planar
bilayer membranes. Biophys Struct Mech 5:277–292.
18. Ganea C, Pourcher T, Leblanc G, Fendler K (2001) Evidence for intraprotein charge
transfer during the transport activity of the melibiose permease from Escherichia coli.
charge translocation in the melibiose permease from Escherichia coli. Biochemistry
20. Kaczorowski GJ, Kaback HR (1979) Mechanism of lactose translocation in membrane
vesicles from Escherichia coli. 1. Effect of pH on efllux, exchange, and courterflow.
1. Effect of pH and imposed membrane potential on efflux, exchange, and counter-
flow. Biochemistry 22:2524–2531.
22. Viitanen P, Garcia ML, Foster DL, Kaczorowski GJ, Kaback HR (1983) Mechanism of
Escherichia coli is fully functional. Proc Natl Acad Sci USA 81:1629–1633.
24. Carrasco N, Antes LM, Poonian MS, Kaback HR (1986) lac permease of Escherichia coli:
Histidine-322 and glutamic acid-325 may be components of a charge-relay system.
25. Carrasco N, et al. (1989) Characterization of site-directed mutants in the lac permease
of Escherichia coli. 2. Glutamate-325 replacements. Biochemistry 28:2533–2539.
26. Menick DR, Sarkar HK, Poonian MS, Kaback HR (1985) cys154 Is important for lac
permease activity in Escherichia coli. Biochem Biophys Res Commun 132:162–170.
27. Menick DR, Lee JA, Brooker RJ, Wilson TH, Kaback HR (1987) Role of cysteine residues
in the lac permease of Escherichia coli. Biochemistry 26:1132–1136.
28. Sahin-Toth M, Kaback HR (2001) Arg-302 facilitates deprotonation of Glu-325 in the
transport mechanism of the lactose permease from Escherichia coli. Proc Natl Acad Sci
29. Smirnova IN, Kaback HR (2003) A mutation in the lactose permease of Escherichia coli
that decreases conformational flexibility and increases protein stability. Biochemistry
30. Garcia-Celma JJ, et al. (2008) Rapid activation of the melibiose permease MelB immo-
bilized on a solid-supported membrane. Langmuir 24:8119–8126.
31. Foster DL, Garcia ML, Newman MJ, Patel L, Kaback HR (1982) Lactose-proton symport
by purified lac carrier protein. Biochemistry 21:5634–5638.
functional lactose carrier from Escherichia coli. J Biol Chem 256:11804–11808.
the lac carrier protein from Escherichia coli. 2. Binding studies with membrane vesicles
and proteoliposomes reconstituted with purified lac carrier protein. Biochemistry
34. Zuber D, et al. (2005) Kinetics of charge translocation in the passive downhill uptake
mode of the Na?/H?antiporter NhaA of Escherichia coli. Biochim Biophys Acta
35. Smirnova IN, Kasho VN, Kaback HR (2006) Direct sugar binding to LacY measured by
resonance energy transfer. Biochemistry 45:15279–15287.
36. Kaczorowski GJ, Robertson DE, Kaback HR (1979) Mechanism of lactose translocation
delta mu H?. Biochemistry 18:3697–3704.
37. Robertson DE, Kaczorowski GJ, Garcia ML, Kaback HR (1980) Active transport in
membrane vesicles from Escherichia coli: The electrochemical proton gradient alters
the distribution of the lac carrier between two different kinetic states. Biochemistry
Garcia-Celma et al. PNAS ?
May 5, 2009 ?
vol. 106 ?
no. 18 ?