extracellular N terminus, and an intracellular (S9-S10) C terminus
voltage-dependent ion channels with seven N-terminal transmembrane segments (S0-S6), an
channel, a distinct member of+ Large conductance voltage- and calcium-dependent K
Pratap Meera, Martin Wallner, Min Song, and Ligia Toro
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Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 14066–14071, December 1997
Large conductance voltage- and calcium-dependent K?channel,
a distinct member of voltage-dependent ion channels with seven
N-terminal transmembrane segments (S0-S6), an extracellular
N terminus, and an intracellular (S9-S10) C terminus
PRATAP MEERA*†, MARTIN WALLNER*†, MIN SONG*, AND LIGIA TORO*‡§
Departments of *Anesthesiology and‡Molecular and Medical Pharmacology, and§Brain Research Institute, University of California, Los Angeles, CA 90095
Edited by H. Ronald Kaback, University of California, Los Angeles, Los Angeles, CA, and approved October 7, 1997 (received for review August 1, 1997)
dependent K?(MaxiK) channels show sequence similarities
to voltage-gated ion channels. They have a homologous S1-S6
region, but are unique at the N and C termini. At the C
terminus, MaxiK channels have four additional hydrophobic
regions (S7-S10) of unknown topology. At the N terminus, we
have recently proposed a new model where MaxiK channels
have an additional transmembrane region (S0) that confers ?
subunit regulation. Using transient expression of epitope
tagged MaxiK channels, in vitro translation, functional, and
‘‘in vivo’’ reconstitution assays, we now show that MaxiK
channels have seven transmembrane segments (S0-S6) at the
N terminus and a S1-S6 region that folds in a similar way as
in voltage-gated ion channels. Further, our results indicate
that hydrophobic segments S9-S10 in the C terminus are
cytoplasmic and unequivocally demonstrate that S0 forms an
additional transmembrane segment leading to an exoplasmic
Large conductance voltage- and Ca2?-
High conductance voltage- and Ca2?-sensitive K?(MaxiK)
channels are expressed in a wide variety of tissues. Mammalian
MaxiK channels are characterized by their high sensitivity to
blockade by iberiotoxin (IbTx) and charybdotoxin. In neurons
and skeletal muscle they may be responsible for fast repolar-
ization of the action potential and modulate transmitter re-
lease (1–3). In smooth muscles they are crucial in regulating
contractility (4, 5). The MaxiK channel was first cloned from
Drosophila melanogaster (6, 7). Subsequently, it was cloned
from several mammalian species including humans (8–14) and
recently from Caenorhabditis elegans (15).
The N-terminal third of MaxiK channels shows sequence
homology to the voltage sensor and the pore region of
voltage-dependent ion channels. However, we have recently
given evidence that MaxiK channels carry a unique N-terminal
transmembrane segment (S0) that leads to an exoplasmic N
terminus. This additional transmembrane segment (S0) is
critical for ? subunit modulation (16).
The C-terminal region (after S6) carries four additional
hydrophobic, possibly membrane spanning regions (S7, S8, S9,
and S10). This region comprises about two-thirds of the total
length of the primary amino acid sequence. The last one third
(also called ‘‘tail’’), containing hydrophobic regions S9 and
S10, shows the highest sequence conservation among species.
This motif can be expressed as a separable domain, and has
been suggested to determine the Ca2?sensitivity (17). A series
of negative charges just before S10 is believed to participate in
Ca2?sensing and has been called the ‘‘Ca2?bowl’’ (15).
Voltage-dependent ion channels form a large family of
related structures that include K?, Na?, and Ca2?channels
and also cyclic nucleotide-gated channels, despite the fact that
the latter are not voltage activated (18, 19). Based on their
sequence similarity it is thought that all of them have the same
membrane topology: six membrane spanning regions with
intracellular N and C termini, extracellular linkers between
S1-S2 and S3-S4, and a pore loop between transmembrane
regions S5 and S6 that dips into the membrane from the
external side (20, 21). This membrane topology has been
confirmed in many studies (22–28).
Sequence analysis (7, 16) and the fact that MaxiK channels
practical absence of Ca2?(29–31) support the view that MaxiK
channels have a close functional and structural relationship
with voltage-gated ion channels. We have recently shown that
MaxiK channels share some of the conserved charged residues
critical in voltage-dependent gating (sensing and structural
residues) (16), not only in the S4 segment but also in S3 region,
with voltage-gated ion channels (32–34).
In this study, we have used several experimental approaches
to analyze the membrane topology of MaxiK channels. We
expressed epitope tagged channels and used, in addition to
fluorescent labeled antibodies (Abs), Ab-coated magnetic
cytosolic nature of the tail region, we performed in vitro
translation experiments and employed an ‘‘in vivo’’ reconsti-
tution approach. We were able to record MaxiK currents after
a patch containing the membrane-bound ‘‘core’’ region was
introduced into the cytoplasm of an oocyte expressing the tail
structurally distinct members of the voltage-gated ion channel
family; their topology is highlighted by an extracellular N
terminus, and a seventh transmembrane domain at the N
terminus (S0). They also demonstrate that hydrophobic seg-
ments S9 and S10 are intracellular.
MATERIALS AND METHODS
Molecular Biology. Numbering in humanMaxiK (Hslo) and
Drosophila MaxiK (Dslo) is according to GenBank accession
numbers U11058 and JH0697, respectively (7, 13). However,
Dslo variant used is A1C2E1G3I0 (7). GCG programs (35)
were used for sequence analysis.
The c-myc epitope (AEEQKLISEEDL) was introduced
either with two complementary phosphorylated oligos at
unique restriction sites (HF1, NcoI; HF4, BsrGI), or by intro-
ducing the c-myc sequence into overlapping PCR primers
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
© 1997 by The National Academy of Sciences 0027-8424?97?9414066-6$2.00?0
PNAS is available online at http:??www.pnas.org.
This paper was submitted directly (Track II) to the Proceedings office.
Abbreviations: MaxiK, large conductance voltage- and calcium-
dependent K?channel; Hslo, human MaxiK; Dslo, Drosophila MaxiK;
IbTx, iberiotoxin; FITC, fluorescein isothiocyanate; Ab, antibody.
A commentary on this article begins on page 13383.
†P.M. and M.W. contributed equally to this paper.
(HF2 and HF3) (36, 37). For expression in COS-M6 cells,
constructs were cloned into the pcDNA3 vector (Invitrogen).
The predicted protein sequences of constructs used are as
follows. HF1, MGAEEQKLISEEDLV followed by Met-1 to
Leu-1113 from Hslo; HF2, GAEEQKLISEEDLG inserted
between Ser-70 and Ser-71 of Hslo; HF3, PGAEEQKLI-
SEEDLG inserted between Asn-136 and Pro-137 of Hslo;
HF4, MYTGAEEQKLISEEDLV inserted between Glu-535
and Met-536 of Hslo; HDP: Asn-258 to Gly-300 of Hslo
replaced by Asn-273 to Gly-314 from Dslo.
Hslo-tail and Dslo-tail constructs were made by PCR.
Hslo-tail: Met-679 to Leu-1113. Dslo-tail: Met-690 to Ser-
1175; His-691 was replaced by Asp to introduce an NcoI site
at the translational start.
Hslo-core carries Met-1 to Gln-675 and an additional (ran-
dom) 58 amino acids before the translation reaches a stop
codon. HS7-S8: Met-Val followed by Arg-443 to Pro-583 from
Hslo. DS7-S8: Met-Val followed by Met-456 to Pro-600 from
Dslo. All constructs were analyzed by restriction digestions.
Sequences amplified by PCR and ligation connections were
confirmed by sequencing. cRNA was transcribed using the
mMesSAGEmMACHINE kit (Ambion, Austin, TX).
Electrophysiology. Oocytes were injected with 5–10 ng of
cRNA and measured 2–5 days after injection. Currents were
measured in the inside-out or outside-out configuration of the
patch clamp technique in symmetrical 110 mM K?(105 mM
K?-methanesulfonate?5 mM KCl?10 mM Hepes, pH 7) (16,
29). The bath solution had in addition 5 mM N-(2-
hydroxyethyl)ethylenediamine triacetic acid (HEDTA) and
CaCl2, which was added to the desired free Ca2?. Free Ca2?
was measured with a Ca2?electrode.
In Vitro Translation and Protein Gels. cRNA was translated
(0.5–1 ?g in a 25 ?l reaction) with rabbit reticulocyte lysate in
presence of dog pancreatic microsomes (Promega) and [35S]-
methionine. Aliquots (5–10 ?l) were diluted with 100 ?l of
PBS (9 mM Na2HPO4?1.4 mM NaH2PO4?137 mM NaCl), 0.1
or 0.3 M Na2CO3(pH 11), and kept on ice for 30 min (38).
Microsomes bearing translated proteins were collected by
centrifugation (1 hr at ?20,000 ? g at 4°C). Pellets were
washed two times with PBS (100 ?l). Supernatant proteins
(100 ?l) were precipitated with cold acetone (200 ?l), centri-
fuged, and dissolved in sample buffer (0.125M Tris, pH
6.8?20% glycerol?4% SDS?2% 2-mercaptoethanol). SDS?
PAGE was carried out on 12–15% gels. After electrophoresis,
gels were stained in 30% methanol, 10% acetic acid supple-
mented with 0.1% Coomassie brilliant blue R-250 and
destained in the same solution without dye before soaking for
30 min in Amplify (Amersham). Gels were dried and exposed
to x-ray films.
Cell Transfections. Cos-M6 cells were transfected using the
DEAE-Dextran (Pharmacia) method as described (39). Cells
were replated on poly-L-lysine-coated petri dishes 24 hr after
transfection. For cell permeabilization the cells were fixed with
4% paraformaldehyde in PBS for 20 min at 4°C followed by
three or four washes with 0.2% Triton X-100 in PBS at room
Immunostaining. Cells were incubated with a 1:200 dilution
of anti-c-myc (clone 9E10) mAb (PharMingen) for 1 hr. Excess
primary antibody was removed by two washes with PBS. Cells
?l of sheep anti-mouse IgG coated Dynabeads (Dynal, Lake
Success, NY) or 1:500 dilution of fluorescein isothiocyanate
(FITC)-labeled secondary antibody. Excess secondary anti-
body (beads or FITC-labeled Ab) was removed by washing
with PBS before imaging. Cells with beads were imaged with
a microscope mounted video camera connected to a frame
grabber board. Cells stained with FITC were imaged with
fluorescence (Axiovert 135) and confocal microscopes (LSM
Hydrophobicity Analysis and Topology Models of MaxiK
Channels. Transmembrane regions are usually characterized
by the abundance of hydrophobic residues in stretches of ?20
amino acids. Fig. 1A shows the hydrophobicity pattern of the
mammalian and the Drosophila MaxiK channels. With respect
to the C terminus, this analysis reveals that: (i) the overall
hydrophobicity of the C-terminal regions S7–S10 is consider-
ably lower than that of transmembrane regions S0, S1, and S6,
and (ii) the hydrophobicity of regions S8 and S9 in Dslo is low
and they may be too short to span the membrane. Therefore,
assuming that Hslo and Dslo fold in the same way, it seems
rather unlikely that these regions are membrane spanning.
Similar to Dslo, the recently cloned C. elegans homolog (15)
shows a low hydrophobicity in regions S8 and S9 and also
carries the additional hydrophobic region S0 (not shown).
The membrane topology of MaxiK channels has been pro-
dependent ion channels, but with four additional hydrophobic
regions at the C terminus with uncertain topology (Fig. 1B).
Some authors (7, 21) have chosen to draw the C terminus
(downstream from S6) as entirely cytoplasmic, presumably
because of the relatively low hydrophobicity. In addition, this
part is likely to contain the Ca2?binding site(s) that has (have)
to be on the cytoplasmic side of the membrane. Others have
channel topology. (A) Hydrophobicity analysis of Hslo and Dslo using
GCG-program PEPPLOT (window size of nine amino acids). Hydro-
phobic regions (S0 to S4) are according to the model in C (16).
Numbers at arrows indicate amino acids that were removed from the
protein sequence. Bars above S8, S9, and S10 in Dslo correpond to 16
a lipid bilayer of 2.5 nm thickness). (B) Previous model with intra-
cellular N terminus, and unknown topology of the C terminus (S7-
S10). (C) Proposed membrane topology of MaxiK channels. (B and C)
Flags, numbered from 1–4 in both models, mark positions where c-myc
epitopes were introduced. ?, consensus sites for N-linked glycosylation
(NXS?T) in bovine slo, regardless of being extra- or intracellular.
Hydrophobicity analysis and alternative Models for MaxiK
Neurobiology: Meera et al.Proc. Natl. Acad. Sci. USA 94 (1997)14067
shown these regions as membrane spanning in their proposed
models (40) (Fig. 1B). However, considering that each of these
four additional hydrophobic regions may or may not span the
membrane, there are many other alternative models. Fig. 1C
shows our recently proposed model with seven transmembrane
regions, an exoplasmic N terminus and an intracellular C
Glycosylation is generally an indication that the glycosylated
part of the protein is exoplasmic, i.e., faces the lumen of
vesicles or the extracellular face of the plasma membrane.
Bovine MaxiK channel ?-subunits are not glycosylated when
purified from tissues (41). In the model shown in Fig. 1B, three
consensus sequences for N-linked glycosylation in bovine
MaxiK channel ?-subunits and Hslo would be extracellular. In
our model (Fig. 1C) only one of these sites, located in the short
loop (only three amino acids) between transmembrane regions
to the membrane are likely to be restricted in their accessibility
for glycosylating enzymes. Therefore, the model in Fig. 1C is
consistent with the finding that bovine MaxiK channel ?-
subunits are not glycosylated. Furthermore, this model is
strongly supported by our previous experimental evidence
showing that: (i) truncation clones of Hslo and Dslo, carrying
only the hydrophobic segment S0, behave like integral mem-
brane proteins and exhibit the expected glycosylation pattern
and (ii) the addition of an N-terminal cleavable signal se-
quence to Hslo and Dslo did not alter their functional behavior
MaxiK Channels Have an Extracellular N Terminus. To
unequivocally distinguish between the two possible folding
models of MaxiK channels at the N terminus, we introduced
c-myc epitope tags into critical positions in Hslo (flags 1–3 in
Figs. 1 B and C and 2A) and tested for antibody binding under
permeabilizing and nonpermeabilizing conditions. This ap-
proach exploits the fact that Abs cannot cross cell membranes.
Therefore, Abs binding under nonpermeabilizing conditions is
a demonstration that the epitope is extracellular. Intracellular
epitopes become accessible only after cell permeabilization.
Epitope tagged constructs that showed functional expres-
sion were used for immunolabeling studies. Functional expres-
sion was considered as an indication that the epitope insertion
did not lead to changes in the overall membrane topology.
Constructs HF1, HF2 (Fig. 2A), and HF4 (see Fig. 4A) are
similar to wild-type channels in their Ca2?and voltage sensi-
tivities (Fig. 2B). Only HF3 (Fig. 2A) deviates from the
wild-type Hslo channels; it shows a lower voltage sensitivity at
all Ca2?concentrations tested (Fig. 2B). The behavior of HF3
(c-myc epitope between S1 and S2 in our model), is qualita-
tively similar to Shaker K?channels with an epitope insertion
between transmembrane segments S1 and S2, which also
require higher voltages for activation (28). This similarity
supports the view that this region in Shaker K?and MaxiK
channels is structurally and functionally equivalent.
We used two different methods to detect primary (anti
c-myc) antibody binding: (i) FITC-labeled antibodies (42), and
(ii) magnetic beads coated with secondary antibodies. The
latter is a modification of a procedure developed for visual
identification of individual transfected cells for electrophysi-
ological experiments (43). The beading method has some
advantages: it is inexpensive, sensitive and does not require a
fluorescence microscope. Labeling of extracellular epitopes
with beads under nonpermeablizing conditions was more
sensitive and gave clearer results for clone HF3 (see Fig. 2)
than using fluorescent-labeled Ab. However, under permeabi-
lizing conditions detection of Ab binding with beads was
difficult to distinguish from background staining. This may be
due to restricted accessibility to Ab binding caused by the
Fig. 2A shows representative images of antibody-labeled
cells expressing constructs HF1, HF2, and HF3. Cells express-
ing HF1 and HF3 are decorated with the beads under non-
observed in cells expressing HF2, which carries the epitope tag
in the intracellular linker between S0 and S1 of our model.
Similar results were observed with fluorescent-labeled sec-
ondary Ab. Confocal images did not show any labeling of
nonpermeabilized cells expressing HF2. Labeling of cells
expressing HF1 and HF3 was restricted to the cell surface in
nonpermeabilizing conditions; whereas under permeabilizing
conditions, cells expressing all three constructs were profusely
Although we did not notice any differences in functional
expression levels between clones HF1 and HF3, the antibody
labeling either with beads or fluorescein was always stronger
in cells expressing HF1. After the permeabilizing treatment,
the staining intensities of HF1 and HF3 were similar. There-
fore, the difference in antibody binding to nonpermeabilized
cells may be due to steric hindrance by the lipid bilayer that
additional membrane spanning segment (S0). (A) Immunocytochem-
istry of c-myc tagged MaxiK channels (HF1, HF2 and HF3) expressed
in COS-M6 cells. Cells were incubated with anti-c-myc mAb under
nonpermeabilizing and permeabilizing conditions. Antibody binding
was visualized using beads coated with secondary antibodies or with
FITC-labeled secondary antibodies. Confocal images (two right pan-
els) are from sections at the middle of the cells. Experiments were
performed at least three times for each construct (also in Fig. 4) with
similar results. (B) Functional expression of c-myc tagged clones in
oocytes measured in inside-out patches. (Left) Voltage activation
curves at 10 ?M intracellular Ca2?, [Ca2?]i. Values for half activation
potentials (V1?2) in 10 ?M [Ca2?]iare (in mV): 12 ? 18 (n ? 64) for
wild-type Hslo (Wt); 5 ? 9 (n ? 3) for HF1; 14 ? 8 (n ? 6) for HF2;
98 ? 7 (n ? 4) for HF3; 13 ? 6 (n ? 3) for HF4 (see Fig. 4). (Right)
V1/2as a function of [Ca2?]i.
MaxiK channels have an exoplasmic N terminus with an
14068Neurobiology: Meera et al. Proc. Natl. Acad. Sci. USA 94 (1997)
disappears when the membrane is dissolved by the permeabi-
lizing treatment. A similar observation has been reported for
epitope tags introduced close to the transmembrane regions of
the N-methyl-D-aspartate receptor (42), and of Shaker K?
The fact that HF2 was only labeled after permeabilization is
consistent with the view that this epitope is intracellular. We
deem unlikely that the HF2 epitope is extracellular, and
hindered to Ab-labeling due to closeness to the membrane,
because the epitope was inserted well away from flanking
transmembrane segments (S0 and S1).
The labeling of HF1 and HF3 under nonpermeablizing
conditions proves that MaxiK channels possess an exoplasmic
N terminus and that the linker between homology region S1
and S2 is outside, as in Fig. 1C. The inaccessibility of the
epitope in clone HF2 is consistent with the notion that this
region is intracellular. These results are incompatible with the
previous model (Fig. 1B), in which the epitopes in HF1 and
HF3 should be accessible only after cell permeabilization, and
the epitope in HF2 should be labeled without permeabiliza-
Localization of the Pore Region. To confirm that the linker
between homology regions S5 and S6 in MaxiK channels faces
the extracellular milieu and forms part of the pore region, as
in other K?channels, we took advantage of the different ‘‘pore
pharmacology’’ of mammalian (Hslo) and Drosophila (Dslo)
MaxiK channels. Mammalian MaxiK channels are blocked by
low concentrations (Kd? 1 nM) of externally applied IbTx
(Fig. 3A) (13). In contrast, Drosophila channels are completely
insensitive to IbTx concentrations up to 100 nM (Fig. 3B). To
for this difference, we constructed a chimera (HDP) where we
replaced the S5-S6 linker of the human channel with the
corresponding part of the Drosophila channel and measured
IbTx block in the outside-out configuration of the patch clamp
technique. The chimeric construct HDP (Hslo with Dslo pore)
is completely insensitive to 100 nM IbTx (Fig. 3C). This result
confirms the view that the S5-S6 linker determines IbTx
binding and forms the MaxiK channel pore loop entering the
membrane from the external side.
Membrane Topology of the C-Terminal Regions S7 and S8.
MaxiK channels carry a long C terminus that has four addi-
tional hydrophobic, possibly membrane spanning regions,
termed S7, S8, S9, and S10. However as shown in Fig. 1A, the
hydrophobicity of these regions is relatively low. To determine
if S7 and S8 are membrane spanning, we introduced a c-myc
epitope tag between hydrophobic regions S7 and S8 (HF4)
(Fig. 1B and Fig. 4). This construct was functional and had
similar Ca2?and voltage sensitivities when compared with
wild-type Hslo (see Fig. 2B). In immunolabeling experiments
under nonpermeablizing conditions, we could not detect bead-
ing or fluorescent staining. Only after permeabilization a
strong labeling was observed (Fig. 4A). This result suggests
that the epitope is intracellular, but is not conclusive since
detergent treatment used for permeabilization could have
exposed a previously hidden extracellular epitope. Therefore,
to further test the intracellular nature of this region (S7 and
S8), we carried out in vitro translation experiments in presence
of microsomes of both Dslo and Hslo. Membrane spanning
regions are inserted into microsomal membranes during the in
vitro translation and can be separated from soluble proteins by
centrifugation; high pH treatment is used to exclude a periph-
eral membrane association (38). Interestingly, upon in vitro
translation of both Dslo (DS7-S8) and Hslo (HS7-S8) clones
carrying S7 and S8 (including 40 N-terminal and 25 C-terminal
adjacent amino acids) resulted in about 50% of the protein in
the pellet fraction, and the residual 50% in the soluble fraction
(Fig. 4B). These results are inconclusive and may be due to the
limitations of this in vitro approach; proteins may assume
proper folding and their native membrane orientation only in
the context of a functional domain. Thus, at present the
membrane topology of this region remains uncertain.
MaxiK Channels C-Terminal Tail Region (Containing S9
and S10) Is Cytosolic. Wei et al. (17) showed that the tail
region can be functionally expressed as a separable domain in
Xenopus oocytes. We have reproduced these results (Fig. 5B),
and tested the tail region clones of Hslo and Dslo for mem-
brane insertion by in vitro translation in the presence of
microsomes (Fig. 5A). The majority of the protein was found
channels. (A) IbTx sensitivity of Hslo. IbTx (100 nM) applied to the
external side of an outside-out patch, completely blocks ionic currents.
Holding potential ? 0 mV; test potential ? 80 mV. (Right): Time
course of IbTx blockade at different IbTx concentrations. (Insert)
Dose-response curve, Kd ? 0.72 nM, Hill coefficient near one. (B)
Dslo currents are insensitive to 100 nM IbTx. (C) Chimeric construct
HDP carries the pore of Dslo in the Hslo backbone. Current traces
show that the pore of Dslo makes Hslo insensitive to 100 nM IbTx.
Linker between S5 and S6 forms the pore region of MaxiK
of Hslo epitope tagged between S7 and S8 (HF4). Visualization with
both magnetic beads and FITC-labeled antibodies. (B) In vitro trans-
lation of HS7S8 and DS7S8 containing hydrophobic regions S7 and S8.
P, pellet or microsomal fraction; S, soluble fraction; M, protein
Topology of S7 and S8 regions. (A) Immunocytochemistry
Neurobiology: Meera et al. Proc. Natl. Acad. Sci. USA 94 (1997)14069
in the soluble fraction (Fig. 5A), suggesting that the tail region
containing S9 and S10 segments is cytosolic. The slight amount
of protein associated with the microsomal fraction is probably
not due to membrane association since it was also observed for
luciferase, a soluble protein that was used as a control (not
shown). The decrease in signals in the soluble fraction after
high pH treatment (0.1 and 0.3 M Na2CO3) seems to be due
to the increased tendency of protein aggregation (seen as a
smear at higher molecular weight) after Na2CO3treatment.
In addition, we performed experiments in an in vivo system.
We injected cRNA for Hslo core (that includes S0-S8) and tail
into separate oocytes to investigate the independent folding
and localization of the tail protein by an electrophysiological
technique that we named ‘‘cross-cramming’’ (Fig. 5C). The
core region (Fig. 5C) as well as the tail region (not shown) by
themselves do not form functional channels. However, func-
tional channels assembled within minutes when patches ex-
cised from core expressing oocytes were crammed into tail
injected oocytes (Fig. 5C, n ? 11). This formation of functional
channels was observed in all experiments performed. The
efficiency of assembly seems to be low. It has to be kept in
mind, however, that under these experimental conditions
(voltage, cell-attached mode) the reconstituted channels are
facing the low intracellular calcium concentration, where the
open probability of wild-type channels is below 10% (29).
Similar current levels were obtained when core and tail are
coexpressed in the same oocyte (Fig. 5B). The above results
suggest that the tail domain folds into its native conformation
independently from the core region. Because, integral mem-
brane proteins are restricted in their diffusion when studied
with the patch clamp technique, the most natural way to
explain these findings using cross-cramming is that the tail
region forms a cytosolic protein in its native conformation. To
our knowledge, the concept of testing the folding of a protein
by electrophysiological methods is the first of its kind.
The fact that an epitope at the N terminus is accessible for
antibody binding in nonpermeabilized cells (Fig. 2), unequiv-
ocally demonstrates that MaxiK channels have an exoplasmic
N terminus. In our model (Fig. 1C) MaxiK channels have a
unique transmembrane segment at the N terminus (S0) and
regions S1-S6 similar to voltage-gated ion channels. These
S1-S6 regions were first assigned based on sequence homology
(7, 16); we now show experimental evidence supporting this
view. We will be referring to S0-S6 as shown in our model (Fig.
Several lines of evidence indicate that the S1-S6 region of
MaxiK has the same membrane topology (Figs. 1 A and C) of
six membrane spanning regions and a pore loop between S5
and S6 as voltage-gated ion channels: (i) The accessibility of a
tag introduced between regions S1 and S2 (HF3), under
nonpermeabilizing conditions, shows that this region is extra-
cellular. (ii) The inaccessibility of an epitope tag between
transmembrane regions S0 and S1 (HF2) to antibody binding
under nonpermeabilizing conditions is consistent with this
region being intracellular. (iii) Transferring the linker between
S5 and S6, carrying a signature sequence for a K?selective
pore, from Dslo into Hslo leads to a loss of IbTx blockade
indicating that this region indeed forms the pore. (iv) MaxiK
channels carry an intrinsic voltage sensor (29–31), which
with the conservation of charged residues in segment S4 (15)
involved in voltage sensing (33, 34). Mutating positive charges
in the MaxiK S4 region results in changes in their voltage-
dependent gating (P.M. and L.T., unpublished results). All
these findings are consistent with our suggested model in Fig.
1C, and epitope tagging experiments exclude the N-terminal
folding of the alternative model shown in Fig. 1B.
Hydrophobicity analysis of the C-terminal regions (S7-S10)
with S0, S1, or S6. This argues against, but does not exclude,
the possibility that these regions are membrane spanning. For
example, a low hydrophobicity is also observed for transmem-
brane regions S2, S3, and especially S4. In these cases, it seems
that the charged residues in one membrane spanning segment
are neutralized by oppositely charged residues in other mem-
brane spanning regions (32). These charged residues, embed-
ded in a hydrophobic environment, considerably diminish the
overall hydrophobicity. Although unlikely, the same could be
the case for these C-terminal regions.
Despite a low sequence conservation, the hydrophobicity
patterns of Shaker channels and other voltage-dependent ion
channels (including S1 to S6 as assigned in Fig. 1C of MaxiK
channels) look very much alike (not shown). In contrast, for
regions S8 and S9, which show high sequence conservation
translation of the tail region containing S9 and S10. Hslo-tail and
Dslo-tail cRNAs were in vitro translated (see Materials and Methods).
The calculated molecular weights are: Hslo-tail, 48.2 kDa; Dslo-tail,
Coexpression of core and tail in the same oocytes produce functional
channels (17). Currents in cell-attached mode with test pulses from
?50 mV to ?136 mV every 6 mV (Vh ? 0 mV). (C) Assembly of
functional MaxiK channels by cross-cramming: (Left) Cell attached
patch in a core injected oocyte does not show any current. (Right)
Functional channels assemble within few minutes when the cell-
attached patch was excised and crammed into a tail injected oocyte.
Currents were elicited using the same protocol as in B. The larger K?
currents in the cross-cramming experiments compared with those in B,
may be due to diffusion of external Ca2?into the oocyte during
Cytosolic C terminus tail (containing S9-S10). (A) In vitro
14070Neurobiology: Meera et al.Proc. Natl. Acad. Sci. USA 94 (1997)
between Hslo and Dslo, the hydrophobicity is not preserved
and is considerably less pronounced in Dslo. Because of their
similar function, it is highly unlikely that Dslo has a different
membrane topology than Hslo. Therefore, this argues against
the possibility that S7 and S8 are membrane spanning in both
Dslo and Hslo.
Our experimental finding that the epitope tag (HF4) intro-
duced between S7 and S8 cannot be labeled from the external
side by an antibody is consistent with the idea that this region
is intracellular. However, the results obtained by in vitro
translation of a protein containing S7 and S8 suggests that one
or both of these regions could be membrane spanning as 50%
of the protein remains membrane associated even after high
pH treatment. Therefore, the membrane topology of hydro-
phobic regions S7 and S8, remains an open question. Argu-
ments in favor of a cytosolic nature of S7-S8 regions are: (i)
their low hydrophobicity; (ii) the lack of antibody binding to
intact cells expressing HF4; and (iii) that alternative splicing in
this region results in profound changes in the ‘‘intracellular’’
Ca2?sensitivity of Dslo channels (45). Therefore, in our model
we have shown S7 and S8 hydrophobic regions as intracellular.
The cross-cramming experiments show that the tail region
forms and behaves as a functional domain. This feature
strengthens the conclusion, drawn from in vitro translation
experiments, that the tail region is a soluble protein. Both
cross-cramming and in vitro translation indicate that the tail
region (containing S9-S10) is indeed cytosolic. The possibility
that the tail region inserts into the membrane during cross-
cramming experiments is remote because it does not show
tendency to be incorporated into membranes during in vitro
translation with microsomal membranes. In addition, a puta-
tive calcium binding site (Ca2?bowl) (15) is located between
S9 and S10 and should be cytosolic.
All voltage-dependent ion channels have two major discern-
ible structural and functional motifs in common; the voltage
sensor (roughly transmembrane segments S1-S4) and the
conduction pathway (S5 to S6). MaxiK channels share these
two motifs but have unique flanking parts appended to
accommodate the modulatory effects of Ca2?(C terminus)
and the ? subunit (N terminus). We have shown that the tail
region folds and functions as an independent unit, presumably
binding and mediating the facilitating effects of Ca2?. The N
terminus mediates the facilitating effects of the regulatory
?-subunit (16). Therefore, MaxiK channels constitute a re-
markable example of a modular protein design.
We thank Drs. John Adelman and Armado Lagrutta for the
generous gift of Dslo. This work was supported by National institutes
of Health Grant HL54970 (L.T.). L.T. is an Established Investigator
of the American Heart Association.
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