J Neurophysiol 107: 5–11, 2012.
First published October 5, 2011; doi:10.1152/jn.00728.2011.
Prestin in HEK cells is an obligate tetramer
Richard Hallworth1 and Michael G. Nichols2
1
Department of Biomedical Sciences, Creighton University School of Medicine, and 2Department of Physics, Creighton
University, Omaha, Nebraska
Submitted 4 August 2011; accepted in final form 2 October 2011
hair cell; electromotility; outer hair cell
PRESTIN is a 744-amino acid membrane protein found in the
lateral wall plasma membrane of cochlea outer hair cells
(OHCs), and is a member of the Slc26a family of anion
transporters. Its importance to mammalian hearing, and OHC
survival, has been revealed by knockout and knockin studies in
mice (Cheatham et al. 2004; Dallos et al. 2008; Liberman et al.
2002), although its role in hearing is far from clear.
Evidence for the oligomerization of prestin comes from
several sources. Freeze fracture images of the OHC lateral wall
plasma membrane revealed a dense, regularly spaced array of
10- to 12-nm round particles (He et al. 2010; Kalinec et al.
1992; Koppl et al. 2004). These particles, plausibly located in
the OHC lateral wall plasma membrane, are thought to be
prestin. However, the particles are 4 –5 times the expected size
of a 744-amino acid membrane protein. A Western blot analysis of prestin purified from expression systems suggested
dimer and tetramer states, with the tetramer state being more
easily dissociated (Zheng et al. 2006). However, another study
suggested that dimers were the normal configuration of prestin
and some other Slc26 family members (Detro-Dassen et al.
2008). Förster resonance energy transfer (FRET) analyses of
prestin coupled to fluorescent proteins also suggested the
existence of homooligomeric interaction, but did not provide
Address for reprint requests and other correspondence: R. Hallworth, Dept.
of Biomedical Sciences, Creighton Univ., 2500 California Plaza, Omaha, NE
68178 (e-mail:[email protected]).
www.jn.org
any information on stoichiometry (Currall et al. in press;
Gleitsman et al. 2009; Greeson et al. 2006; Navaratnam et al.
2005; Wu et al. 2007). A low-resolution electron density map
of purified prestin exhibited fourfold symmetry, consistent
with a tetramer (Mio et al. 2008). Overall, the experimental
results have been equivocal on the question of stoichiometry.
Alternate approaches to the question are clearly needed.
Our approach takes advantage of recent advances in the
detection and analysis of single-molecule fluorescence. Detection of single-molecule fluorescence requires very high sensitivity detection methods with low background noise, such as
photomultipliers or cooled electron-multiplying charge-coupled device cameras. Single-molecule detection, imaging, and
tracking have been applied to numerous questions, including
the mobility of neurotransmitter and odorant receptors (Heine
et al. 2008; Jacquier et al. 2006), protein conformation change
(Harms et al. 2003), and protein turnover (Leake et al. 2006).
A more relevant application is the application of single-molecule sequential bleaching to the stoichiometry of ion channel
subunit oligomerization (Harms et al. 2001; Ji et al. 2008;
Ulbrich and Isacoff 2007). Briefly, single fluorescently coupled
molecules are isolated and exposed to continuous excitation.
The fluorescence is proportional to the number of active
fluorophores in the molecule, i.e., the number of subunits. If
the number is small, discrete equal-amplitude step decreases in
fluorescence (bleaching) are observed until all fluorophores are
bleached and the fluorescence is indistinguishable from background levels. The step count corresponds to the number of
monomers in the molecule. The technique has been applied to
L-type calcium channels (Harms et al. 2001), cyclic nucleotide-gated sodium channels (Ulbrich and Isacoff 2007), calcium release-activated calcium channels (Ji et al. 2008), and a
voltage-gated proton channel (Tombola et al. 2008).
In this study, we applied the sequential bleaching method to
the problem of prestin stoichiometry. We first established that
we could measure single-molecule fluorescence by imaging a
preparation of known single fluorescent molecules. We used
streptavidin-Alexa Fluor 488 bound to biotin-coated coverslips
as our test preparation.
We then used a preparation of membranes of human embryonic kidney cells (HEK cells) containing prestin coupled Cterminal to the enhanced green fluorescent protein (eGFP). We
observed that the average step count was close to 2.7. As a
positive control, we performed identical measurements on
HEK cells synthesizing the A3 subunit of the human cyclic
nucleotide-gated sodium channel (CNGA3, from cone photoreceptors), again coupled C-terminal to eGFP. CNG proteins
form tetrameric ion channels in rod and cone photoreceptors
composed of A and B subunits (Liu et al. 1996). The B
subunits appear to be modulatory, while the A subunits form
0022-3077/12 Copyright © 2012 the American Physiological Society
5
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Hallworth R, Nichols MG. Prestin in HEK cells is an obligate
tetramer. J Neurophysiol 107: 5–11, 2012. First published October 5,
2011; doi:10.1152/jn.00728.2011.—The unusual membrane motor
protein prestin is essential for mammalian hearing and for the survival
of cochlear outer hair cells. While prestin has been demonstrated to be
a homooligomer, by Western blot and FRET analyses, the stoichiometry of self association is unclear. Prestin, coupled to the enhanced
green fluorescent protein, was synthesized and membrane targeted in
human embryonic kidney cells by plasmid transfection. Fragments of
membrane containing immobilized fluorescent molecules were isolated by osmotic lysis. Diffraction-limited fluorescent spots consistent
in size with single molecules were observed. Under continuous
excitation, the spots bleached to background in sequential and approximately equal-amplitude steps. The average step count to background
levels was 2.7. A binomial model of prestin oligomerization indicated
that prestin was most likely a tetramer, and that a fraction of the green
fluorescent protein molecules was dark. As a positive control, the
same procedure was applied to cells transfected with plasmids coding
for the human cyclic nucleotide-gated ion channel A3 subunit (again
coupled to the enhanced green fluorescent protein), which is an
obligate tetramer. The average step count for this molecule was also
2.7. This result implies that in cell membranes prestin oligomerizes to
a tetramer.
6
PRESTIN IS A TETRAMER
functional tetrameric ion channels in the absence of B subunits
(Bonigk et al. 1993; Kaupp et al. 1989). We found the same
average step count for this molecule. We therefore conclude
that prestin in membranes is also an obligate tetramer.
MATERIALS AND METHODS
y⫽
A
w 兹2 ␲
冉
exp ⫺
(x ⫺ xc)2
2w2
冊
y⫽
A1
w 兹2 ␲
冉
exp ⫺
(x ⫺ xc)2
2w2
A2
2w兹2␲
冉
exp ⫺
(x ⫺ 2xc)2
8w2
冊
(2)
where A1 and A2 are positive constants, and xc and w have the same
meanings as before.
For each ROI, the step count was determined by inspection,
informed by the average step size xc as determined above. The
fluorescence in most ROIs reached background fluorescence levels, as
determined by sampling the fluorescence of ROIs adjacent to the
point, within the 800 frames. Only records in which background levels
were reached were analyzed. All points meeting the above criteria
were included in the analysis. The distribution of step counts was
plotted in histogram form. The resulting histogram was fitted to a
binomial sampling distribution as follows:
y⫽A
(1)
where xc is the average step amplitude (in counts), w is the standard
deviation, and A is a positive constant.
In some experiments, 0.1 ␮g/ml streptavidin-Cy2 (Invitrogen) was
used in place of the Alexa Fluor 488 conjugate.
Plasmids and transfection. A plasmid encoding gerbil prestin in
frame with eGFP was a gift from Peter Dallos (Northwestern Univ.).
A plasmid encoding human CNGA3 (isoform 1), coupled C-terminal
to eGFP, was a gift from Jacqueline Tanaka (Univ. of Pennsylvania).
Cells (HEK-293) were cultured on glass-bottomed cultured dishes
(MatTek, Ashland, MA) precoated with rat-tail collagen (BD Biosciences, Bedford, MA). Transfections of 60 – 80% confluent cells were
performed using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. Lipofectamine was removed after 6 – 8 h.
Osmotic lysis method. Dishes of cells were observed 12–24 h after
transfection. Cells were lysed using the method of Ziegler et al.
(1998), as adapted by Murakoshi et al. (2006), with modifications as
冊
⫹
冉
n!
x ! (n ⫺ x) !
冊
pxq(n⫺x)
(3)
where p is a probability, q ⫽ 1-p, A is a positive constant, and n is the
integer sample size. The average of the binomial sampling distribution, ␮, is np. For a sample size of n, the probability p was then
calculated as ␮/n.
RESULTS
Size determination of diffraction limited fluorescence points.
To assure ourselves that we were able to detect single fluorescent molecules, we used streptavidin-Alexa Fluor 488 applied
to biotin-coated slides, as described in MATERIALS AND METHODS.
Extensive (30 –90 s) prebleaching of the slide reduced the
fluorescence to multiple discrete points, as illustrated in Fig.
1A. Discrete fluorescent points were readily identifiable, as
seen in Fig. 1B. The time course of average fluorescence in the
ROI was determined frame by frame using 5 ⫻ 5 pixel ROIs,
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Observation of Alexa Fluor 488 fluorescence. Polyethylene glycolcoated glass coverslips with a low density of covalently attached
biotin were obtained from MicroSurfaces (Austin, TX). Coverslips
were coated with 0.1 ␮g/ml streptavidin-Alexa Fluor 488 (Invitrogen,
Carlsbad, CA) in high-purity water for 2–5 s, rinsed in high-purity
water exposure, drained, and mounted for observation on an Olympus
IX-70 inverted fluorescence microscope via a 100 magnification, 1.40
numerical aperture objective. Excitation and observation were performed using a mercury excitation source filtered by a standard
fluorescein filter set. Areas for observation were first bleached to near
darkness before acquiring images to minimize the number of fluorescent spots. Each streptavidin molecule had between two and four
covalently linked Alexa Fluor 488 molecules, according to the
manufacturer.
Sequences of fluorescence images were acquired using an Andor
Technology (Belfast, N. Ireland) DU-897E cooled, back-thinned,
electron-multiplying charged-coupled device camera (quantum efficiency ⬎0.9). Images were acquired typically for 2,000 frames at 0.18
s/frame over a 128 by 128 pixel image field equivalent to 19.2 by 19.2
␮m, with the camera electronics cooled to ⫺60°C and the electronmultiplication gain set to between 50 and 80. Images were acquired in
a darkened room with the microscope shrouded by a darkroom cloth.
Measurements were made at room temperature.
Analysis of image sequences was performed using a procedure
developed in MatLAB (The MathWorks, Natick, MA). Acquired
images were smoothed using two passes of a three-point low-pass
spatial filter. A 5 pixel by 5 pixel region of interest (ROI) (750 nm by
750 nm) was selected to encompass putative single-molecule fluorescent points. The summed fluorescence (in arbitrary units) was calculated for each image in a sequence using the ROI as a mask. The
corner points (0, 0; 0, 5; 5, 0; 5, 5) were set to zero because they
contribute little to the signal from a single fluorescent molecule, which
appeared to have a radius of ⬃700 nm. The summed ROI fluorescence
in each frame was then divided by 21 to obtain the average fluorescence, and the values were plotted and stored in temporal order.
Step decreases in fluorescence were selected only if the decreases
occurred over one or at most two frames. Step amplitudes were
measured using the average fluorescence values before and after the
step. The step amplitudes were accumulated in histogram form, and
the histogram was fitted to a Gaussian distribution:
follows. Cells were exposed to a hypoosmotic buffer in the cold for 30
min. The buffer contained 4 mM PIPES and 30 mM KCl (pH 6.2, 80
mOsm). Cells were then subjected repeatedly to a stream of the same
buffer delivered via a blunted 28-gauge hypodermic needle. Dishes
were allowed to come to room temperature before observations were
made. No intact cells could be observed on the culture dish after this
treatment.
Observation of eGFP fluorescence. Presumed membrane fragments
containing fluorescence were observed on the stage of an Olympus
IX-70 inverted fluorescence microscope. Excitation and imaging were
as before except that during acquisition a neutral density filter attenuated the excitation to retard bleaching. Images were acquired typically for 800 frames at 0.2 s/frame over a 128 by 128 pixel image
field, with the camera electronics cooled to ⫺70°C and the electronmultiplication gain set to between 80 and 200.
Analysis of single-molecule eGFP fluorescence. ROIs containing
putative single molecule fluorescence were distinguished from random noise by following the ROI over sequences of images. Consecutive pairs of images were averaged and the resulting images spatially
filtered as before. Background subtraction was performed by estimating the background fluorescence of each image as a function of time,
using a 9 pixel by 9 pixel ROI placed away from potential single
molecules. Putative single-molecule fluorescent points were analyzed
as described earlier. A fraction (0.8) of the background fluorescence
was subtracted from the ROI fluorescence by time function, so that the
values remained positive.
On the first analysis pass, the step decrease magnitudes were
determined as before and were plotted in histogram form. The distribution of step sizes was fitted to the sum of two Gaussian functions as
follows:
PRESTIN IS A TETRAMER
7
Figure 2A3 shows an example of the observation of repetitive
on-off fluorescence steps, of equal amplitude. This phenomenon, called blinking, is characteristic of small-molecule fluorophores such as the Alexa Fluor dyes (Aitken et al. 2008).
The amplitudes of 95 step fluorescence decreases from a
single experiment are plotted in histogram form in Fig. 2B. The
histogram was well fit by a Gaussian distribution (Eq. 1, R2 ⫽
0.86). The fit implies that the step decreases were unitary
events, subject to measurement error, which is consistent with
the assumption that the steps are the bleaching events of single
fluorescent molecules. We experimented with larger (7 ⫻ 7
and 9 ⫻ 9 pixel) ROIs, but the signal-to-noise ratio of the
records was lower.
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Fig. 1. Determination of the diffraction-limited single-molecule fluorescence point
size. A: representative image field of a streptavidin-Alexa Fluor 488-labeled
biotin-coated coverslip after extensive prebleaching. Imaging conditions were as
set out in MATERIALS AND METHODS. The image of 128 ⫻ 128 pixels represents
19.2 ⫻ 19.2 ␮m2 at the coverslip. B: higher magnification field of the same image
showing representative placement of regions of interest (ROIs) (5 ⫻ 5 pixel,
750 ⫻ 750 nm) demarcating putative points of single-molecule fluorescence.
as shown in Fig. 1B (750 nm at the specimen), and plotted as
a function of time. Examples of the time course of fluorescence
in ROIs are shown in Fig. 2A. Figure 2A1 shows the most
common observation, of a discrete step decrease in fluorescence that occurred over 1 or 2 frames. Fig. 2A2 shows an
example of an occasional observation, of two consecutive,
approximately equal-amplitude step decreases in fluorescence.
Fig. 2. Evidence for imaging of single fluorescent molecules. The red lines are
the segments that were averaged to estimate the step size. A: representative time courses of fluorescence from the same experiment as Fig. 1. The scale
bars represent 5 s (time) and 100 counts (amplitude). A1: typical step decrease
in fluorescence. A2: occasional observation of 2 approximately equal step
decreases. A3: blinking by a single molecule. B: histogram of 95 observed
fluorescence step decreases in a single experiment. The fitted Gaussian distribution parameters are xc ⫽ 87.49, w ⫽ 37.48, A ⫽ 467.13 (R2 ⫽ 0.86).
C: histogram of 339 observed fluorescence decrease steps in a single experiment using streptavidin-Cy2 instead of streptavidin-Alexa Fluor 488 (3 coverslips). The fitted Gaussian distribution parameters are xc ⫽ 205.99, w ⫽
71.53, A ⫽ 3345.77 (R2 ⫽ 0.96).
J Neurophysiol • doi:10.1152/jn.00728.2011 • www.jn.org
8
PRESTIN IS A TETRAMER
Fig. 3. Punctate fluorescent points observed in HEK cell membranes after
hypoosmotic lysis, showing representative ROIs demarcating putative points
of single-molecule fluorescence. The image of 128 ⫻ 128 pixels represents
19.2 ⫻ 19.2 ␮m2 at the culture dish.
We repeated the experiment using streptavidin-Cy2 instead
of streptavidin-Alexa Fluor 488. The histogram of 339 step
amplitudes from one such experiment is shown in Fig. 2C.
Again, the histogram was well fit by a Gaussian distribution
(R2 ⫽ 0.96).
Sequential bleaching of prestin. We first examined the
fluorescence of HEK cells synthesizing prestin-eGFP. We
observed points of fluorescence consistent in size with single
molecules, as determined above, as well as larger points that
may have been macromolecular assemblies. However, many of
the points were mobile, and some appeared to undergo endocytosis, which made analysis of bleaching events unreliable.
We therefore removed the cells by osmotic swelling and
lysis, as described in MATERIALS AND METHODS. Inspection of the
culture dish under transmitted light revealed no intact, attached
cells. However, UV excitation revealed points of fluorescence,
again consistent in size with single molecules, which were not
mobile (Fig. 3), as well as larger points as before.
Image sequences of the fluorescent points were acquired as
described in MATERIALS AND METHODS, with background subtraction. ROIs containing putative single molecules were analyzed
as follows and as depicted in Fig. 4, for three experiments. We
analyzed only points for which the fluorescence decreased to
background levels, estimated as described in MATERIALS AND
METHODS. Figure 4A is an example of a typical point that
decreased to the background fluorescence level in four discernable steps. We also observed numerous points for which the
fluorescence decreased in three steps (Fig. 4B1, 4B2). We also
observed some points with two steps (Fig. 4C) and some with
only one step (Fig. 4D).
Step amplitudes were measured as described and accumulated in histogram form (Fig. 5A1, B1, C1). Histograms were
best fit by the double Gaussian distribution (Eq. 2) with only a
Fig. 4. Representative time courses of fluorescence of single-molecule ROIs in
the isolated membrane of a prestin-transfected cell. A: example of 4 steps. B1
and B2: examples of 3 steps. Arrow in record B1 indicates on-blink. Arrow in
record B2 indicates transient dwell at an intermediate level. C: example of 2
steps. Arrow indicates transient off-blink. D: example of single step. In all
records, the red lines represent the average fluorescence in that time range used
to measure the step amplitude. The scale bars represent 10 s (time) and 100
average counts per pixel (amplitude).
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small fraction of the steps of amplitude 2xc (i.e., A2⬍⬍A1). We
interpreted this as evidence that we were observing mainly a
single population of bleaching events, with the occasional
near-simultaneous pairing of events.
We then determined the step count for each point, using the
single-step amplitude xc derived from the amplitude histogram
to resolve any ambiguities. The step counts were accumulated
and displayed in histogram form (Fig. 5A2, B2, C2). The
average step counts in the three experiments were 2.69, 2.71,
and 2.71. The step count histogram for each experiment was
then fit to a binomial distribution (Eq. 3). As shown in Fig. 5, the fits
were remarkably similar, with sample size n ⫽ 4, and the
probability p between 0.65 and 0.68. This occurred even
though the electron-multiplying gains and the attenuation of
the excitation source were different for the three experiments.
Sequential bleaching of CNGA3. For comparison, we transfected cells with a plasmid expressing a C-terminal construct of
the human CNGA3 ion channel coupled C-terminal to eGFP, a
known tetramer. We performed the same experiment and
analyses on these cells. The results (Fig. 6) were almost
identical to those observed for prestin-eGFP. The average step
counts were 2.59 and 2.74, and the binomial distribution fits
were n ⫽ 4, p ⫽ 0.65 and 0.68.
PRESTIN IS A TETRAMER
9
DISCUSSION
The binomial model predicts that prestin is a tetramer. The
success of the binomial model in fitting the step count distribution of this experiment suggests a simple model to explain
the results. In the binomial sampling model, small samples of
size n are withdrawn from a large pool. Here, each molecule
represents a sample and n is the stoichiometry. In the binomial
model, not all units are good, only a fraction p. The remainder,
q ⫽ 1-p, are defective. The distribution of observation of good
items in repeated sampling is given by Eq. 3. In our experiment, good is interpreted as able to fluoresce. The optimum
sample size n to fit the data was four, which implies that the
molecules, prestin and CNGA3, are tetramers. Since CNGA3
is a known tetramer, our model predicts that prestin is also a
tetramer.
The value of p in this model, between 0.65 and 0.68, implies
that only 65– 68% of eGFP molecules were able to fluoresce,
the remainder being dark. This explains why we observed a
range of step counts in our sample, from four to one, which is
consistent with a binomial sampling process with p ⬍ 1. As is
clear from Figs. 5 and 6, we occasionally observed points with
step counts greater than four. We interpret these points as
having contained more than one prestin molecule, and therefore did not include them in the analysis.
Alternative explanations of the results. We considered several other models that might fit the results. Dimer and trimer
configurations were rejected because of the high frequency of
points in which the step count was greater than three. Random
association of monomers into oligomers would produce a
uniform distribution of step counts, unlike our observations. As
mentioned in the previous section, we observed a small number
of points with greater than four steps. We examined the
possibility that prestin is a pentamer (i.e., n ⫽ 5) and included
them in a re-analysis. However, the analysis suggested that p is
⬃0.5, i.e., that about half of the eGFP molecules were dark,
which we considered to be unlikely, especially since p was
0.65– 0.7 for the positive control CNGA3, for which the
average step count was identical to that of prestin.
Advantages of this approach. The advantages of this approach are numerous. Prestin molecules are in the lipid plasma
membrane and are therefore closer to their native state, unlike
Western blots or electron density mapping, in which the
molecules are in aqueous media (Westerns) or none at all
(electron density mapping). The molecules are also isolated
from each other, unlike FRET analyses, which required high
densities for adequate signal levels.
The HEK membrane is not necessarily identical to OHC
lateral wall membrane, which has unusually large cholesterol
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Fig. 5. Histograms of average step size (A1, B1, C1) and step
counts (A2, B2, C2) from 3 experiments. The fit parameters
are shown.
10
PRESTIN IS A TETRAMER
Fig. 6. Histograms of average step size (A1, B1) and step
counts (A2, B2) from 2 experiments using CNGA3-eGFP.
The fit parameters are shown.
subunits. Wang et al. (2010) argued on numerical grounds that
each subunit operates independently. However, Detro-Dassen
et al. (2008) found that prestin molecules formed in cells
coexpressing two prestin mutations with different electrical
properties had intermediate electrical properties in the aggregate. This, they suggested, indicates that one subunit can
influence another, which implies cooperativity. It is likely to
require single-molecule methods, such as the one demonstrated
here, to resolve this important structure-function question.
ACKNOWLEDGMENTS
We thank Max Ulrich and Ehud Isacoff for initial encouragement and
advice, Peter Dallos and Jacqueline Tanaka for plasmid constructs, and David
Z. Z. He and Heather Jensen-Smith for comments on the manuscript. We thank
Fang Xiang for statistical advice.
Fig. 7. Time course of average step size and binomial parameter p in a
simulated prebleaching experiment. The details are in DISCUSSION. Fit parameters are as follows: average step size, slope ⫽ ⫺0.72, R ⫽ ⫺0.89; p, ␶ ⫽
18.49 ⫾ 4.73, R2 ⫽ 0.99.
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levels (de Monvel et al. 2006; Rajagopalan et al. 2007).
However, the electrical properties of prestin in HEK cells, even
when coupled to eGFP, are essentially the same as in the OHC
(Deak et al. 2005; Santos-Sacchi and Navarrete 2002), thus the
configuration of prestin in HEK cell membranes is likely to be
representative of its natural state.
Sources of dark eGFPs. Our binomial model predicts that
the molecules are tetramers, based on the optimum fit with n
equal to 4. However, it also predicts a larger-than-expected
proportion of dark eGFP molecules (1-p). Previous work has
suggested that at least 20% of eGFPs are dark (Ulbrich and
Isacoff 2007). Our method requires some focusing on the
fluorescent points, because the preparation is not level over the
dish and possibly the membranes are themselves uneven,
which was not the case with the Ulbrich and Isacoff experiment. The excitation intensities used in our experiments were
greatly attenuated to delay bleaching events and thereby facilitate the analysis. However, the excitation intensities could not
be reduced further, or the bleaching events themselves became
undetectable. It is likely that some bleaching of eGFP occurred
before recording could begin, which may account for some part
of the 30 –35% dark eGFP molecules observed.
We confirmed this speculation by re-analyzing one of the
experiments, the one depicted in Fig. 5C. We compared the
results of the analysis, assuming n ⫽ 4, with the same analysis
but starting at 10 s, 20 s, or 50 s after the acquisition had begun.
This procedure simulated the effects of prebleaching the fluorophores. Our model predicts that the probability p should
decrease with prebleach duration, but the average step size
should be unaffected. As shown in Fig. 7, this was indeed the
case. The average step size remained remarkably constant,
while the p value decreased with a single exponential time
course.
Prestin mechanisms. The results of this study do not directly
address the mechanism of prestin conformation change. Nor do
they help decide the question of cooperativity between prestin
PRESTIN IS A TETRAMER
GRANTS
This work was supported by a grant from the State of Nebraska LB 692
fund to R. Hallworth and National Institutes of Health (NIH) Grant GM085776 to M. G. Nichols. Research was conducted in a facility constructed
with support from Research Facilities Improvement Program C06 RR17417-01 from the National Center for Research Resources of the NIH.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: R.H. and M.G.N. conception and design of research;
R.H. performed experiments; R.H. analyzed data; R.H. and M.G.N. interpreted
results of experiments; R.H. prepared figures; R.H. drafted manuscript; R.H.
and M.G.N. edited and revised manuscript; R.H. and M.G.N. approved final
version of manuscript.
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