EJB Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.2 No.3, Issue of December 15, 1999.

© 1999 by Universidad Católica de Valparaíso -- Chile Received August 20, 1999 / Accepted October 18, 1999.
RESEARCH ARTICLE

Visualization and Functional Analysis of a Maxi-K Channel (mSlo) Fused to

Green Fluorescent Protein (GFP)

Michael P. Myers *
Department of Pharmacology and Physiology
University of Rochester School of Medicine and Dentistry,
Rochester, New York, USA 14642-8711.
E-mail : [email protected]

Jay Yang
Department of Pharmacology and Physiology, and Department of Anesthesiology
University of Rochester School of Medicine and Dentistry,
Rochester, New York, USA 14642-8711.
E-mail : [email protected]

Per Stampe
Department of Pharmacology and Physiology
University of Rochester School of Medicine and Dentistry,
Rochester, New York, USA 14642-8711.
E-mail : [email protected]

We have constructed a fusion protein between mSlo (a Aequorea victoria is a powerful tool for biotechnology in
recombinant, high conductance, calcium-activated studying the expression of various proteins. GFP is easily
potassium channel or maxi-K), and GFP (green visualized when excited with UV light and its fluorescence
fluorescent protein). This construct represents a tag to does not depend on any exogenous compounds (Marshall et
not only monitor channel expression, but to locate the al., 1995). This protein, when expressed alone, appears
protein in living cells. The GFP was fused in frame to cytosolic, with no cellular targeting mechanisms of its own,
the carboxy-terminus of the mSlo core protein (mSlo- making it ideal to tag a protein of interest. Our fusion
GFP fusion protein). Expression of this fusion protein construct, called mSlo-GFP (for a review of GFP
in COS-7 cells resulted in robust fluorescence localized nomenclature see Gerdes and Kaether, (1996)), has the
near the cell membrane. Fluorescing cells that were green fluorescent protein fused in frame to the c-terminus
patch clamped exhibited whole cell currents with a of the channel. The result is a functional tagged potassium
direction consistent with potassium currents. channel. Our results demonstrate that the fusion protein
Conversely, non-fluorescing cells showed no significant can be expressed in COS-7 cells, harvested, and
whole cell currents. Excised inside out patches revealed reconstituted into lipid bilayers allowing detailed single
single channel currents and calculated conductances in channel analysis. In addition, we show that the fusion
the range of those expected for the maxi-K. The mSlo protein can be studied by patch clamp techniques as well.
and mSlo-GFP channels reconstituted into lipid bilayers
bound wild-type, recombinant CTX with high affinity Maxi-K channels belong to a class of calcium sensitive
and displayed a half-blocking concentration (KD ) of 7.4 potassium channels which possess a large single channel
and 7.6 nM, respectively (at +30 mV in 150 mM conductance (greater than ~ 200 pS in symmetrical 150 mM
equimolar KCl). This resulted in single channel KCl), thus they have been called BK (big potassium) or
evaluation of the functional inhibition of CTX on these maxi-K channels (Latorre et al., 1989). These high
clones. As newly constructed GFP chimeras emerge for conductance channels have been measured in numerous
the study of physiological processes in living organisms, tissues and are associated with various physiological
this work provides another area of insight illuminated processes, including the repolarization of action potentials in
by GFP. neurons, regulation of fluid secretion in exocrine cells, and
maintenance of contractile tone in smooth muscle
(McManus, 1991). Recent physiological work has linked
maxi-K channels to the mediation of vasorelaxant effects of
Green Fluorescent Protein (GFP), cloned from the jellyfish K+ channel openers in the porcine coronary artery
*Corresponding author
This paper is available on line at http://www.ejb.org/content/vol2/issue3/full/3
Myers, M., Yang, J., Stampe, P.

(Balwierczak et al., 1995), to the regulation of airway x 20 x 25 Angstroms in dimension, with a globular
cholinergic neurotransmission (Tagaya et al., 1995), to the structure formed by a three-turn alpha helix lying on an
control of the dynamics ofaqueous humor outflow in the antiparallel β sheet with three internal disulfide bonds
anterior of the human eye (Stumpff et al., 1997), and to (Bontems et al., 1991). Use of CTX has not only identified
second messenger systems contributing to the membrane residues in contact with the toxin, it marks residues in
conductance of retinal Müller cells (Bringmann et al., proximity to the receptor for CTX (Goldstein et al., 1994).
1997). These large conductance channels have also been When CTX is applied to preparations of native maxi-K
implicated in several diseases, including the pathogenesis channels, it blocks the channel in a bimolecular fashion,
of hypertension in spontaneously hypertensive rats (Asano binding to the external vestibule of the channel and
et al., 1995), the molecular mechanism of antiasthma physically occluding or “plugging” the outer entry to the
therapy (Barnes, 1995), and to the inherited susceptibility to conduction pore (Miller, 1995). Moreover, studies of CTX
non-insulin-dependent diabetes mellitus (NIDDM) (Ferrer binding have linked specific residues on the toxin to
et al., 1996). permeation sites in the pore, enriching our molecular
picture of the toxin and the nature of its receptor in
While a great deal of information has been accumulated potassium channels (Naini et al., 1996). Recently, CTX
from studying native maxi-K channels, the channel was and its close scorpion toxin analogue, agitoxin2, have been
difficult to clone and then to express. This has barred it used to show that prokaryotic K+ channels have the same
from the explosion of data provided by molecular biology pore structure as eukaryotic K+ channels (MacKinnon et al.,
that has increased our knowledge of other potassium 1998). This result is important, given that the crystal
channels. Maxi-K channels have been cloned and expressed structure of a prokaryotic K+ channel from Streptomyces
from the slowpoke locus (Adelman et al., 1992). This lividans has been revealed by Doyle et al. (1998). The
began the molecular characterization of the channel. The testing of this ground-breaking structure will require
first maxi-K mammalian clone from the mouse brain (mSlo) detailed toxin profiles, such as those presented here,
appeared in 1993 (Butler et al., 1993). Human analogues especially if we are to extrapolate the structure of a two-
(hSlo) of this channel have been cloned from a variety of transmembrane K+ channel to a six-transmembrane K+
sources including the brain (Dworetzky et al., 1994; Tseng- channel, such as Shaker, or to an even more complex ten-
Crank et al., 1994), arterial smooth muscle (McCobb et al., transmembrane K+ channel, such as the maxi-K described
1995), myometrium (Wallner et al., 1995), and pancreatic in this report. Preliminary data of these findings have been
islet cells (Ferrer et al., 1996). While structurally reported in abstract form (Myers et al., 1997).
homologous to the purely voltage-gated potassium channels
such as Shaker (both are characterized by six n-terminus Materials and Methods
hydrophobic membrane spanning domains and an
additional pore domain), the cloned DNA for maxi-K Molecular biology
channels is more than double the size, containing a large c-
terminus coding region believed to confer their calcium For the expression of GFP alone, Green Fluorescent Protein
sensitivity (Knaus et al., 1995; McCobb et al., 1995; cDNA (from the pGFP clone, Clontech, Palo Alto, CA) was
Adelman et al., 1992). This increased size may be one subcloned into expression vector pMT3 (from the
reason that causes the difficulty of channel expression. laboratory of Daniel D. Oprian, Brandeis University). The
Cloned maxi-K channels appear to express well in oocytes, pMT3 vector is pMT2 (Sambrook et al., 1989) with the Pst
yielding robust macropatch cell currents, but single channel I and Eco RI sites mutated to Kpn I and Not I, respectively.
analysis necessary for detailed kinetic analysis of CTX To express GFP as a fusion protein, the GFP was subcloned
block has not been forthcoming, especially in the into an expression vector of pMT3 containing the α subunit
mammalian clones (Butler et al., 1993; McCobb et al., of mSlo (mbr5) (Butler et al., 1993). The stop codon in
1995). Furthermore, studies measuring the affinity of CTX mSlo was mutated to a lysine residue (TTG), which allowed
to BK channels reconstituted into artificial bilayers such as for the translation of nine additional bases (CTCCCAGGA)
the one described here, have an advantage in that oocytes as at the c-terminus of mSlo before the sequence of GFP
an alternative expression system are now known to be began. As a result of the subcloning steps, fourteen base
contaminated with native BK channels (Krause et al., pairs were eliminated from the 3’ untranslated sequence of
1996). The initial characterization of the pharmacology of mSlo (CTATTTTTTTAAAG). A mismatch mutagenesis
cloned maxi-K channels was completed only recently strategy (Kammann et al., 1989) using PCR (polymerase
(Gribkoff et al., 1996), and our work adds to the analysis of chain reaction) was employed to produce the mutations of
the mSlo clone, particularly with regard to its interaction the stop codon and subcloning. DNA fragments were
with CTX. separated by agarose gel electrophoresis and purified using
QIAQUICK gel extraction kit (Qiagen, Chatsworth, CA).
CTX has been very useful in studies of ion channel Oligonucleotides used in this study were custom made by
structure and function. Its interaction with specific residues the Oligonucleotide Core Facility at the University of
in the pore region of ion channels makes it an ideal probe Rochester. To confirm the fusion construct, DNA
when relating specific amino acids to their physiological sequencing was carried out at the Oligonucleotide Core
role. Studies show CTX to be 37 amino acids in length, 20

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 Visualization and Functional Analysis of a Maxi-K Channel (mSlo) Fused to Green Fluorescent Protein (GFP)

Facility using the dideoxy chain termination method voltages in the cis chamber are expressed relative to
(Sanger et al., 1977). ground. The CTX experiments were all carried out at + 30
mV.
Whole cell and vesicle preparation
For patch clamp experiments, whole cell currents were
COS-7 cells (ATCC, Rockville, MD) were incubated in a evoked by voltage steps from -50 mV to +30 mV (with a
CO2 incubator in 100 mM tissue culture plates at 37 °C holding potential of -80 mV), and single channel currents
until they reached 70-80% confluency. The cells were then evoked in excised inside out patches at a holding potential
transfected using a DEAE-Dextran cationic-lipid method of +40 mV. The extracellular solution consisted of (in
(Oprian et al., 1987) using 2 µg of DNA per plate. Cells mM): 140 NaCl, 2.8 KCl, 1.0 MgCl2 , 10 HEPES, 10
were assayed for channel activity by patch clamp or glucose, pH 7.4 with 100 µM CaCl2 , and the pipet solution
harvested into vesicles for reconstitution into lipid bilayers consisted of (in mM): 140 KCl, 2 MgCl2 , 1.1 EGTA, 0.1
48 to 72 hours after transfection. For lipid bilayer CaCl2 , 5 HEPES, and 5 K2 -ATP.
preparation, the cells were scraped off the culture plates and
collected into ice cold 10 mM sodium phosphate buffer, pH Data acquisition and analysis
7.0, containing 150 mM NaCl. The cells were resuspended
in a high pH solution (150 mM KCl, 2mM MgCl2 , 5 mM The cis and trans chambers were connected with electrodes
EGTA, pH 10.6 with ammonium hydroxide) and broken up to an Axopatch 200A patch clamp amplifier (Axon
by being taken up in a 20 ml syringe 4 times through a high Instruments, Foster City, CA) in capacitive feedback
gauge needle (2 times with a 22 gauge, then 2 times with a configuration. The lowpass cutoff filtering frequency was
25 gauge). The broken cells were sonicated 30 secs to form set at 1 kHz. The analog output signal was digitized by a
lipid vesicles (Branson model 450 sonicator, duty cycle Data Acquisition Processor (DAP) 3200e, (Microstar
50%, output 4). The vesicles were purified on a step Laboratories) at 100 kHz. Open and closed times were
sucrose gradient (40% and 20% sucrose cushion, interface computed by an on-board processor (486DX) on-line at 100
between these layers contains the vesicles). The purified kHz with a threshold set at 50% of the whole open-level
vesicles containing the channels were resuspended in current. At the same time, single-channel currents were
isotonic sucrose buffer (10 mM MOPS and 250 mM sampled at 1 kHz. Cumulative open probability (Po ) was
sucrose, pH 7.4), quick frozen on dry ice, and stored at - computed from the total open and closed times. During the
80°C. recording, Po vs. number of transitions was displayed after
every 1,000 transitions. Patch Clamp data were also
Electrophysiology obtained using an Axopatch 200 amplifier (5 kHz low pass
filter) and currents were recorded using pCLAMP 5.2
For single channel analysis, the vesicles containing the software. Rate constants for association and dissociation of
recombinant channels were reconstituted into lipid bilayers CTX and Ba 2+ were calculated from the statistical
and characterized. The recording chamber was made out of distributions of the blocked and unblocked dwell times, as
acetyl copolymer (Patriot Plastics, Londonderry, NH). It previously described (Anderson et al., 1988). A
includes two open wells (one with a glass side called the cis pentium/150 MHz Personal Computer and a custom written
chamber and another behind it called the trans chamber) program were employed to control the experiment, and to
separated by a thin wall with a 250 µm hole drilled through store and analyze the data.
it. A forming solution of 100 µl of N2 dried lipid (1-
palmitoyl, 2-oleoyl-phosphotidylethanolamine/1-palmitoyl, Detection of expressed fluorescence
2-oleoyl-phosphotidylcholine, 10 mg/ml, (Avanti Polar
Lipid, Alabaster, Al), 7:3 respectively and 50 µl n-decane The cells were imaged for analysis in one of two ways: 1)
(Sigma) was used to prepaint the chamber. The cis To obtain higher resolution in imaging the fusion construct,
chamber contained 650 mM KCl, 10 mM HEPES, 100 µM the cells were fixed in 4% paraformaldehyde and washed in
Ca2+, pH 7.4. The trans chamber contained an identical 0.05 M phosphate buffer. The cells were air dried and
solution, except its KCl concentration was 50 mM. mounted for observation on a Nikon microphot
Vesicles from the COS cell prep were added to the cis fluorescence microscope (figure 1). All cells were imaged
chamber and after incorporation of a single maxi-K using a Hoffman Modulated contrast objective. The cells
channel, the cis side of the chamber was perfused with 150 were excited with UV light (mercury lamp), and observed
mM KCl and KCl was added to the trans chamber resulting in the green wavelength (with a Fitc filter cube). 2) Similar
in an isotonic KCl concentration of 150 mM for all results were obtained using confocal microscopy (data not
recordings. To measure calcium activation and sensitivity shown) at the Adherent Cell Analysis and Sorting (ACAS)
to block, stock solutions of CaCl2 , BaCl2 , and recombinant Facility of the University of Rochester. They were
CTX were pipetted into the appropriate chamber when trypsinized and transferred to quartz plates for imaging.
needed. The CTX used was purified to its active form after The cells were excited at a wavelength of 395 nm and their
cleavage from a fusion protein in E. Coli (Park et al., 1991). relative fluorescence was recorded at 488 nm.
The trans chamber was connected to ground and all the

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Myers, M., Yang, J., Stampe, P.

Figure 1. COS-7 cells transfected with Green Fluorescent Protein (GFP) and the mSlo-GFP fusion protein.

Top panels: Section of COS-7 cells transfected with GFP-pMT3 showing one cell with intense fluorescence. The left panel
is a bright field image of the cells, the middle panel is a pseudo color image of the relative level of fluorescence, the right
panel is a layered image of the first two panels, with scale bar (all images created on the same page in Adobe Photoshop).
Bottom panels: Section of COS-7 cells transfected with mSlo-GFP-pMT3 showing one cell with intense localized
fluorescence. Panels are the same as above (note a cell process from another cell in the upper right corner).

Results voltage dependent (figure 3, A and B). The opening of the

tagged channel was also voltage dependent with an average
Visualization of GFP and expressed channels channel conductance of 272 pS (figure 3, D and E). mSlo
was calcium sensitive in the µM range (figure 3C), which
To find the optimal conditions for transfection, the fusion closely matches the calcium sensitivity of mSlo-GFP
protein was used to transfect COS-7 cells under differing (figure 3F). The calcium activation data was least-squares
conditions of cell density, concentrations of DNA to lipid fitted with the Hill equation (KD = 13.9 µM, n = 2.9, Vmax =
reagent, and transfection times. When GFP was expressed 94, and KD = 11.2 µM, n = 2.5, Vmax = 84 for mSlo and
alone in our cloning vehicle (plasmid vector GFP-pMT3), mSlo-GFP, respectively). Although only one distribution of
conventional fluorescent microscopy revealed cells labeled the voltage dependence is displayed for typical mSlo and
with intense fluorescence throughout the cell (figure 1, top mSlo-GFP channels, both the calcium and voltage
panels). When GFP was fused to the maxi-K channel sensitivity varied from channel to channel. Wu et al.
(plasmid vector mSlo-GFP-pMT3), the same microscopy (1996), describe the calcium variation as a clustered
revealed robust fluorescence localized near the cell distribution of calcium sensitivities in maxi-K channels
membrane (figure 1, bottom panels). After transfection from avian nasal gland cells. To determine any differences
with mSlo-GFP-pMT3, fluorescent cells that were patch- in voltage or calcium sensitivity, the open probability of
clamped (4 out of 5), displayed typical outward going several single channel experiments under identical
voltage gated maxi-K currents (figure 2, A and B). COS-7 conditions of voltage and free calcium concentration were
cells that were not fluorescing showed no currents (4 out of averaged together. The mean open probabilities of mSlo
4). and mSlo-GFP channels (63.9 ± 4.5% and 52.8 ± 12%, n =
10 and 9, respectively) were not significantly from each
Characterization of channels reconstituted into other at +30 mV and 100 µM Ca 2+. Thus, the addition of
lipid bilayers green fluorescent protein to the mSlo channel had very
little, if any, effect on its functional characteristics
Currents through mSlo channels reconstituted into lipid described above.
bilayers had an average conductance of 265 pS and were

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 Visualization and Functional Analysis of a Maxi-K Channel (mSlo) Fused to Green Fluorescent Protein (GFP)

Figure 2. Currents recorded from a fluorescing COS-7 cell transfected with mSlo-GFP-pMT3

A: Whole cell currents were evoked by voltage steps from -50 mV to +30 mV (with a holding potential of -80 mV). The
extracellular solution consisted of (in mM): 140 NaCl, 2.8 KCl, 1.0 MgCl2 , 10 HEPES, 10 glucose, pH 7.4 with 100 µM
CaCl2 , and the pipet solution consisted of (in mM): 140 KCl, 2 MgCl2 , 1.1 EGTA, 0.1 CaCl2 , 5 HEPES, and 5 K2 -ATP. B :
Multiple single channel currents (arrows denote each channel level) recorded from an excised inside out patch of a
fluorescing COS-7 cell. Currents of about 16 pA were evoked (solutions were the same as above, with a holding potential
of +40 mV).

Figure 3. Currents through mSlo and mSlo-GFP channels from transfected COS-7 cell membranes reconstituted
into lipid bilyers.

A: The current-voltage relationship of mSlo channels, with the solid line representing a linear fit of 8 single channel
experiments (n=5 for each data point). The standard error of the mean (SEM) is less than the width of the points and the
average channel conductance was 265 pS. B : Voltage dependence of a typical single mSlo channel. The solid line
represents a least squares fit of the Boltzman distribution. C: Single channel current (mSlo) activation by free Calcium
added to the internal side of the channel in 150 mM symmetrical KCl. The data was least-squares fitted with the Hill
equation (KD = 13.9 µM, n = 2.9, Vmax = 94). D: The current-voltage relationship of mSlo-GFP channels, with the solid line
representing a linear fit of 9 single channel experiments (n=5 for each data point, except for +60 mV, where n =1). The
standard error of the mean (SEM) is less than the width of the points and the average channel conductance was 272 pS. E:
Voltage dependence of a single mSlo-GFP channel in 150 mM symmetrical KCl. The solid line represents a least squares fit
of the Boltzman distribution. F: Single channel current (mSlo-GFP) activation by free Calcium added to the internal side of
the channel in 150 mM symmetrical KCl. The data was least-squares fitted with the Hill equation (KD = 11.2 µM, n = 2.5,
Vmax = 84).

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Myers, M., Yang, J., Stampe, P.

a single exponent, 5.26 ± 0.923 msec for mSlo, and 6.07 ±

1.27 msec for mSlo-GFP (see table 1). These values are
consistant with those recently reported for wild type mSlo
(Sullivan et al., 1997). The rate constants describing the
open and shut times of both channels were nearly identical
for the wild type mSlo and mSlo-GFP fusion channels.
Thus adding GFP to the channel does not appear to have
any significant effect on the gating of the channel.

Figure 4. Closed dwell time histograms of mSlo and

mSlo-GFP

The top histogram shows the distribution of 23,500 shut

times in a control experiment with no CTX added to the
external side of the channel mSlo. The bottom histogram
shows the distribution of 22,500 shut times in a control
experiment with no CTX added to the external side of the
channel mSlo-GFP. The histogram data were overlayed
with the probability density function. Figure 5. Open time histograms of mSlo and mSlo-GFP.

The top histogram shows the distribution of 23,500 open

Statistical distribution of mSlo and mSlo-GFP
times in a control experiment with no CTX added to the
dwell times
external side of the channel mSlo. The bottom histogram
shows the distribution of 10,500 open times in a control
To further test if the gating of the channel was unchanged,
experiment with no CTX added to the external side of the
the statistical distributions of the shut and open times of
channel mSlo-GFP. The histogram data were overlayed
control records were analyzed using the probability density
with the probability density function.
function (representative distributions of shut and open
dwell times are shown in figures 4 and 5. respectively).
CTX block of mSlo and mSlo-GFP
Rate constants from 4 channels for each clone with similar
open probabilities were averaged together (see table 1). We
Structural features of the mSlo-GFP fusion channel, namely
were able to resolve 3 exponents to describe the distribution
the outer vestibule, were explored using CTX and Barium,
of the shut times of the channels. The average shut time rate
respectively, as probes. CTX block of typical single
constants ± SEM, were: τc1 = 0.453 msec ± 0.103, τc2 = 4.30 channels (figure 6) revealed similar functioning of the two
msec ± 0.540, τc3 = 19.1 msec ± 2.95 for mSlo, and τ c1 = channels. The KD for CTX block for the fused channel was
0.544 msec ± 0.101, τ c2 = 4.43 msec ± 0.962, τ c3 = 16.5 7.5 nM compared to 7.3 nM for mSlo (see table 2). Ba 2+
msec ± 2.95 for mSlo-GFP, respectively (see table 1). The was also used to probe the channels in a single experiment
distribution of the open dwell times were well described by one ach clone (see figure 6). The Barium block of

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 Visualization and Functional Analysis of a Maxi-K Channel (mSlo) Fused to Green Fluorescent Protein (GFP)

Figure 6. Single channel assay of mSlo (left traces) and mSlo-GFP (right traces) with Barium and charybdotoxin
(CTX).

Channels were expressed in COS-7 cell membranes and incorporated into lipid bilayers. Channel openings are upward, and
the small arrow to the left of each trace denotes the closed state. All traces represent single channel currents in symmetrical
150 mM solutions at +30 mV. Top traces represent control experiments with no CTX. An expanded time scale is shown in
the Traces second from the top represent a portion of the trace above with a 100 fold expanded time scale. From 23 minutes
of recorded data for CTX block of mSlo-GFP, the burst time constant was 3.12 sec and the blocked time constant was 13.1
sec, which corresponds to an on rate of 12.8 x 106 s -1 M -1 and an off rate of 76.3 x 10-3 s -1, respectfully. These data give a
CTX equilibrium constant KD of 6.0 nM for this channel. These data closely match the single channel diplayed for mSlo,
which has a CTX equilibrium constant KD = 6.7 nM. For overall kinetic parameters from a total of 12 experiments, see
table 1. The Barium block of mSlo-GFP shows a blocked time constant of 4.56 sec, almost exactly equal to mSlo (4.64 sec).

Figure 7. Concentration dependence of CTX Block of mSlo.

At each CTX concentration, records were taken for 25-45 min, and mean dwell times were calculated for the active (bursts,
solid circles) state and the blocked (open circles) state. Each point represents the mean dwell time (± s.e.m.) calculated
from 3 to 5 separate single channels. Each line represents a linear fit of the data. Note that the point at which the lines cross
is equal to the KD (7.4 nM for mSlo).

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Myers, M., Yang, J., Stampe, P.

mSlo-GFP showed a blocked time constant of 4.56 sec, characterized (Trouet et al., 1997), further demonstrating
compared to a blocked time constant of 4.64 sec for mSlo. that GFP can mark cells while not affecting channel
behavior.
Concentration dependence of CTX block of mSlo
Our analysis of the time constants and distributions for the
To investigate the concentration dependence of CTX block mSlo and mSlo-GFP fusion channels revealed that the
of mSlo, the channel was assayed with the toxin at several normal gating of the channel was apparently unaffected by
different toxin concentrations (see figure 7). As seen with addition of GFP to the c-terminus. Our time constants are
native maxi-K channels, the interaction of CTX with mSlo consistent with those previously reported for mSlo.
was bimolecular. The blocked dwell times were Sullivan et al. (1997), describe the open times with one
exponentially distributed and the average blocked time, τb , exponent (τo = 58 msec), and were able to resolve 4
was independent of toxin concentration (figure 7, open exponents to describe the shut times of mSlo: τc1 = 0.59
circles). Increasing the concentration of CTX has no effect msec, τc2 = 2.5 msec, τc3 = 9.4 msec, and τc4 = 238 msec at
on the off rate. The burst times were also exponentially +20 mV in 200 µM Ca 2+. Their experimental conditions
distributed and the on rate increased with increased toxin (designed to analyze calcium sensitivity) were very
concentration (figure 7, closed circles). As the different from ours, making a comparison difficult.
concentration of CTX was increased, the on rate increased
linearly. While attaching GFP to the carboxy-terminus of mSlo had
no effect on channel function, results from an amino-
Discussion terminus attachment to hSlo were quite different. Tagging
the amino-terminus of hSlo with GFP apparently alters its
We have used GFP to estimate expression of maxi-K calcium sensitivity (Meyer and Fromherz, 1999). No such
channels prior to reconstitution into lipid bilayers and to affect on calcium sensitivity seen in our carboxy-terminus
locate transfected cells for patch clamping. The fusion construct could be a clue that only the amino-terminus of
protein has allowed us to monitor transfection conditions the protein is involved in sensing calcium. A more detailed
necessary to express the α subunit of mSlo. Given that examination of the calcium sensitivity of our tagged protein
single channel recordings of mSlo are rare (Muller et al., will need to be done before a final conclusion can be made.
1996) yet essential for a detailed evaluation of CTX block The amino-terminus tagged hSlo result also suggests that
including its interaction with the subunit (Strobaek et al., care should be taken before extrapolating the results
1996), a functional reporter gene to monitor expression in presented here to other channels.
various expression systems and conditions is a powerful
tool. In this study, we have demonstrated that the mSlo Another result of this study has been a single channel look
channel expression and protein targeting, kinetics, and at the functional inhibition of CTX on the α subunit of
functional properties of the outer vestibule are all mSlo. Our KD value of 7.4 nM for mSlo and 7.6 nM for
unchanged by GFP fusion at the carboxy-terminus.
mSlo-GFP are on the order of the inhibition constant
recently reported for the other CTX sensitive BK clone
Our halo like location of the protein (figure 1, bottom hSlo (Gribkoff et al., 1996). The EC50 for CTX of hSlo in
panels) has also been seen in a preliminary report of the Gribkoff et al. study (estimated from inhibition of two-
another tagged potassium channel (John et al., 1997), and electrode voltage clamped current from oocytes) is 30.8 nM
suggests that the channels are inserted into the plasma and 7.9 nM for Iberiotoxin (IbTX), a close structural
membrane. Therefore, sub-cellular localization of ion homologue of CTX. The EC50 of IbTX (also estimated
channels now appears possible. As expression of various from inhibition of two-electrode voltage clamped current
constructs are undertaken to elucidate ion channel structure from oocytes) in mSlo was estimated to be 9.1 nM in the
and function, it has been suggested that tagging of these Gribkoff et al. (1996) study. In addition, we demonstrate
clones will help us to distinguish between non-functional here that CTX block of mSlo is bimolecular, showing that
channels and those whose biogenesis was arrested the clone follows the interaction of CTX with native maxi-
(Makhina et al., 1997). Thus this new construct represents K channels (Miller et al., 1985). The Gribkoff et al. (1996)
a tag to not only monitor channel expression, but to locate study did not assess the concentration dependence of CTX
the protein in living cells. A preparation of transfected cells block on mSlo. They did find a bimolecular interaction for
can now be checked for efficiency before they are IbTX with both hSlo and mSlo channels which corresponds
reconstituted simply by evaluating the fluorescence of the to our finding. Their result of the interaction of CTX with
cells. The fluorescence observed is quite stable, and we hSlo was less definitive. A functional inhibition of CTX on
demonstrate here that cells with no fluorescence do not hSlo single channel current may reveal a more detailed
express channels. The patch clamp and lipid bilayer results, answer. The green fluorescent protein (GFP) from the
as well as the fluorescent images suggest that the GFP is jellyfish Aequorea victoria has become an important
indeed fused to the channel and that the channel is fully reporter molecule for monitoring gene expression. With this
functional. Moreover, a functional delayed rectifier K+ work, an RCK1 chimera (Trouet et al., 1997), and
channel clone (RCK1) has recently been fused to GFP and preliminary reports of other potassium channel GFP fusion

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 Visualization and Functional Analysis of a Maxi-K Channel (mSlo) Fused to Green Fluorescent Protein (GFP)

Table 1. Time constants and open probabilities of mSlo and mSlo-GFP channels

Channel type τc1 (msec) τc2 (msec) τc3 (msec) τo (msec) Po (%) n

mSlo 0.453 ± 0.103 4.30 ± 0.54 19.1 ± 2.9 5.26 ± 0.92 64.2 ± 2.1 4

mSlo-GFP 0.544 ± 0.101 4.43 ± 0.96 16.5 ± 4.4 6.07 ± 1.27 65.2 ± 2.0 4

Analysis of dwell time histograms (representative histograms are shown in figures 4 and 5) measured at + 30 mV in
symmetrical 150 mM KCl and 100 µM Ca 2+. Closed time histograms were fitted with three exponentials (τc1 - τc3), and
open time histograms were fitted with one exponential (τo ). Each value represents the mean ± SE of the number of
measurements reported (n), each representing control recordings of a single channel in a separate bilayer.

Table 2. Kinetic parameters for CTX block of mSlo clones

Channel type k off (x10-3 s -1) k on (x106 s -1 M -1) KD (nM) n

mslo 74 ± 7 10 ± 0.6 7.4 7

mSlo-GFP 82 ± 14 11 ± 1.3 7.6 5

rat skeletal muscle* 62 ± 3 7.0 ± 1.1 8.8 9

Association and dissociation rate constants of CTX, kon and koff, and the dissociation constant KD , were measured at + 30
mV on single maxi-K channels. Each value represents the mean ± SE of the number of measurements reported (n), each
employing 100-246 blocking events of a single channel in a separate bilayer. *(Stampe et al., 1994)

proteins (John et al., 1997; Makhina et al., 1997; Veyna- mSlo-GFP will hopefully shed some light on the limited
Burke et al., 1997), as well as calcium channel GFP information we have about these critical proteins.
chimeras (Grabner et al., 1997; Zhou et al., 1997), a new
list of GFP constructs will have to be added to the existing Acknowledgments
categories (Gerdes and Kaether, 1996), namely ion
channels. Since we have demonstrated here that only The authors would like to thank Lawrence Salkoff for the
fluorescent COS-7 cells express the clone, patch clamping use of mSlo, Peter Shrager for the use of the Nikon
only fluorescent oocytes should increase the chances of fluorescent microscope, and Ted Begenisich for help in
recording from BK clonesand not endogenous maxi-K improving the manuscript. A special thanks goes to Ian
channels. Although we have not expressed and imaged Vabnick for help in recording the fluorescent images and
mSlo-GFP in Xenopus laevis oocytes in this study, a recent for the support of the members of the Papazian Lab at
report of another GFP fused K+ channel demonstrated that UCLA.
the fluorescent protein (constructed with the same
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