ACCELERATED PUBLICATION
Block of Kcnk3 by Protons
EVIDENCE THAT 2-P-DOMAIN POTASSIUM CHANNEL SUBUNITS
FUNCTION AS HOMODIMERS*
Coeli M. B.
Lopes,
Noam
Zilberberg, and
Steve A. N.
Goldstein
From the Departments of Pediatrics and Cellular and Molecular
Physiology, Boyer Center for Molecular Medicine, Yale University School
of Medicine, New Haven, Connecticut 06536
Received for publication, April 11, 2001, and in revised form, May 16, 2001
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ABSTRACT |
KCNK subunits have two pore-forming P
domains and four predicted transmembrane segments. To assess the number
of subunits in each pore, we studied external proton block of Kcnk3, a
subunit prominent in rodent heart and brain. Consistent with a
pore-blocking mechanism, inhibition was dependent on voltage, potassium
concentration, and a histidine in the first P domain (P1H). Thus, at pH
6.8 with 20 mM potassium half the current passed by
P1H channels was blocked (apparently via two sites ~10% into the
electrical field) whereas channels with an asparagine substitution
(P1N) were fully active. Furthermore, pore blockade by barium was
sensitive to pH in P1H but not P1N channels. Although linking two Kcnk3
subunits in tandem to produce P1H-P1H and P1N-P1N channels bearing four
P domains did not alter these attributes, the mixed tandems P1H-P1N and P1N-P1H were half-blocked at pH ~6.4, apparently via a single site.
This implicates a dimeric structure for Kcnk3 channels with two (and
only two) P1 domains in each pore and argues that P2 domains also
contribute to pore formation.
 |
INTRODUCTION |
Leak currents are key to normal electrical activity of sympathetic
ganglia, myelinated axons, carotid body type 1 cells, and cardiac
myocytes (1). 50 years after their description (2, 3) leak currents
have been revealed to pass via potassium channels formed by subunits
with two P domains (1). Preceded by TOK1, a non-voltage-gated
outward rectifier from Saccharomyces cerevisiae with eight
predicted membrane-spanning segments (a 2P/8TM1
topology) (4), the first canonical leak channel, KCNK0,
was isolated from Drosophila
melanogaster and has a predicted 2P/4TM topology (5-7). To date,
15 mammalian genes (KCNK1-15) have been identified to
encode subunits with a 2P/4TM topology (1). Family members that show
function (KCNK0, -2, -3, -4, -5, -6, -9, -10, -13, and -15) are
potassium-selective leak channels; open at rest, they pass potassium
without apparent delay in response to changes in membrane potential
across the physiological voltage range. The functional repertoire of
KCNK channels is, however, more varied, as phosphorylation can
reversibly transform cloned and hippocampal KCNK2 between an open and
voltage-dependent phenotype (8). Other KCNK subunits have
yet to reveal their biophysical attributes (KCNK1, -7/8, and -12).
Previously, we studied the genomic structure, cardiac localization, and
biophysical properties of murine Kcnk3 (9). Thus, at steady-state,
Kcnk3 channels behave like potassium-selective, transmembrane holes
inhibited by physiological levels of protons. With voltage steps, Kcnk3
currents activate (or decay) in two phases; one phase appears to be
instantaneous, and another appears to be time-dependent
(
~ 5 ms). Both proton block and
time-dependent gating are potassium-sensitive, producing an
anomalous increase in outward flux as external potassium levels rise
because of decreased proton block. At physiological levels of
potassium, proton inhibition at
120 mV is half-maximal at ~pH 7.6, indicating that Kcnk3 is poised to sense and respond to changes in pH
value. Moreover, Kcnk3-like channels have been recorded in rat central
nervous system nuclei, where they were shown to influence spike
frequency in response to volatile anesthetics and neurotransmitters
(10-14), and in murine cardiac myocytes, where they appear to
contribute to the action potential plateau current called IKp (9, 15). These findings suggest a potential role for Kcnk3 in the physiology of
cerebral and cardiac acidosis.
Four KCNK channels are notably sensitive to extracellular pH in the
physiological range, (KCNK3, -5, -9, and -15) (1, 16). KCNK3 and -9 appear to be closely related sharing 54% amino acid identity and a
histidine at position 98 in the first P domain (P1) in the midst of
signature sequence residues that form the potassium selectivity filter
(GYGH); KCNK5 and -15 do not possess this histidine. Kim et
al. (17) have demonstrated that histidine 98 in KCNK9 confers
sensitivity to external pH. In this study, we demonstrate a similar
role for histidine 98 in Kcnk3 and employ this finding to evaluate
subunit stoichiometry. Our findings support the conclusions that there
are two P1 domains present in the channel that contribute equivalently
to pore formation and, thus, that two Kcnk3 subunits assemble to form
each channel.
 |
MATERIALS AND METHODS |
Molecular Biology--
Cloning of Kcnk3
(GenBankTM accession number AB008537) and the pBF2 vector
have been described (9). Point mutants were produced by Pfu
mutagenesis with a QuickchangeTM kit (Stratagene, Inc., La
Jolla, CA) followed by insertion of mutant gene fragments into
translationally silent restriction sites. Sequences were confirmed by
DNA sequencing. To produce dimer constructs, a cDNA fragment
encoding all but the first 6 amino acids of Kcnk3 was excised (with
FspI and EcoRI) and ligated behind another copy
of the gene replacing the last 71 amino acids (from ScaI to
EcoRI). The result was two linked but truncated Kcnk3
subunits. the first lacks 71 C-terminal residues, and the second lacks
6 N-terminal residues. Wild type (P1H) and H98N (P1N) subunits were
combined in this fashion to form P1H-P1H, P1H-P1N, P1N-P1H, and
P1N-P1N. cRNA was produced with T3 RNA polymerase and a kit (Ambion,
Austin, TX).
Electrophysiology--
Oocytes were isolated from Xenopus
laevis frogs (Nasco, Atkinson, WI), treated with collagenase to
ease follicle removal, and injected with 0.1-0.4 ng of Kcnk3 cRNA in
46 nl of sterile water. Currents were measured 1-4 days after
injection by two-electrode voltage clamp using a Geneclamp 500 amplifier (Axon Intruments, Foster City, CA). Data were sampled at 4 kHz, filtered at 1 kHz, and analyzed using Pulse (Instrutech, Great
Neck, NY) and Sigmaplot (Jandel Scientific, San Rafael, CA) software.
Electrodes of 1.5-mm borosilicate glass tubes (Garner Glass Co.,
Claremount, CA) contained 3 M KCl and had resistances of
0.3 to 1 megohms. Oocytes were studied at room temperature with
perfusion at 0.4-1 ml/min using 5 mM potassium solution as
follows (in mM): 93 NaCl, 5 KCl, 1 MgCl2, 0.3 CaCl2, 5 HEPES, pH 7.4, with NaOH. As indicated, KCl was
isotonically substituted for NaCl. At pH 6.0, MES was substituted for HEPES.
Calculations--
Dose response curves were fit to Equation 1,
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(Eq. 1)
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where [B] is proton or barium concentration,
K1/2 is the concentration of B
required to achieve 50% inhibition, and n is the Hill
coefficient. Voltage dependence was modeled for data accumulated in 20 mM potassium solution using a simplification of the
Woodhull approach (18), represented by Equation 2,
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(Eq. 2)
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where [B] is proton or barium concentration,
n is the Hill coefficient, z is the electronic
charge of the blocker, and
is the apparent electrical distance
traversed by the blocker to reach its site.
 |
RESULTS |
Native Kcnk3 and a Mutant--
Native Kcnk3 channels expressed in
oocytes are notable for at least four reasons. First, the currents
activate and deactivate in response to voltage changes in two phases,
one instantaneous, another time-dependent (Fig.
1A). A non-zero holding
current at voltages from
120 to 60 mV (Fig. 1, A and
B) indicates that the channels are open across the
physiological voltage range, as has been observed directly at the
single channel level (9). Second, Kcnk3 currents are non-inactivating,
remaining at a stable level until voltage is again altered. Third,
Kcnk3 currents show open rectification; as expected for an open,
potassium-selective portal, the relationship of current and voltage
becomes linear as potassium is raised to nearly equal levels across the
membrane (Fig. 1B). Conversely, at physiological levels of
bath potassium (~5 mM), Kcnk3 passes large outward
currents and small inward currents, because permeant ions flow more
readily from a side of high concentration. Fourth, Kcnk3 channels are
inhibited by protons in the physiological pH range. Currents are close
to maximal at pH 8.0, partially suppressed at pH 7.0, and essentially
absent at pH 6.0 (Fig. 1C).

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Fig. 1.
Kcnk3 channels are open rectifiers, show
time-dependent gating, and are blocked by lowered pH values
in wild type but not mutant form. A, Kcnk3 currents in
whole-cell mode for a representative oocyte bathed in 5 and then 100 mM potassium (Ko) held at 80 mV and pulsed
from 120 to 45 mV in 15-mV voltages steps lasting 50 ms, followed by
a 20-ms step to 120 mV with a 1-s interpulse interval. The
dashed line indicates zero current level. The
time-dependent phase of current development is partially
obscured in whole cell mode by the capacitance transient. Scale
bars, 2 µA, 20 ms. B, steady-state
current-voltage relationships for groups of five oocytes studied as in
panel A in 5 (open circles) and then 100 mM potassium (filled circles); mean ± S.E.
C, inhibition of Kcnk3 channels by lowered external pH
values. Kcnk3 currents in whole-cell mode for a representative oocyte
bathed in 5 mM potassium at pH 8.0, 7.0, and 6.0; protocol
was as in A. D, P1N channels show little
inhibition with lowered external pH. Whole-cell mode for an oocyte
bathed in 5 mM potassium at pH 8.0 and 7.0; protocol was as
in A. E, steady-state current-voltage
relationships for six oocytes as in panel A in 5 mM (open circles) and then 100 mM
(filled circles) potassium solution; mean ± S.E.
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Mutation of Kcnk3 histidine 98 (P1H) to asparagine (P1N) markedly
diminishes pH sensitivity of Kcnk3 so that little block is now seen at
neutral pH (Fig. 1D). Moreover, wild type channels show
anomalously low outward current in 5 mM potassium compared with 100 mM at pH 7.4 because of increased proton blockade
(Fig. 1B) (9) whereas this effect is absent in the mutant
(Fig. 1E). Otherwise, the P1N mutation did not appear to
significantly change channel-gating kinetics, rectification properties,
or ion selectivity (Fig. 1, D and E, and data not
shown). These findings suggested that pH inhibition of Kcnk3 resulted
from protonation of histidine 98 in the selectivity filter.
Potassium and Voltage Affect Proton Block--
The influence of
external potassium concentration on inhibition of Kcnk3 by proton was
next studied (Fig. 2). At
120 mV, with
5 mM potassium, Kcnk3 current was half-maximal at pH
7.6 ± 0.6 (n = 16 cells) whereas with 20 mM and 100 mM potassium, half-block required
lowering the pH to 6.83 ± 0.01 and 6.35 ± 0.02, respectively (n = 10 and 20 cells). Moreover, the
current-pH relationship with 5 mM potassium showed a Hill
coefficient of 0.96 ± 0.06 whereas with 20 and 100 mM
potassium, Hill coefficients of 2.02 ± 0.14 and 1.86 ± 0.21 were determined. This suggested that protonation of a single pore site
was sufficient to inhibit at low bath potassium whereas protonation of
more than one site was required at higher potassium levels.

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Fig. 2.
Kcnk3 channels are inhibited by protons in an
external potassium-dependent manner. The dependence of
Kcnk3 current at 120 mV on bath pH (mean ± S.E.) for groups of
10-20 oocytes in 5, 20, and 100 mM potassium solution. The
solid lines represent fits of the data in Equation 1.
K1/2 was determined to be 7.6 ± 0.6, 6.83 ± 0.01, and 6.35 ± 0.02, and Hill coefficients were
0.96 ± 0.06, 2.02 ± 0.14, and 1.86 ± 0.21 for 5, 20, and 100 mM potassium (Ko), respectively.
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Consistent with the idea that proton enters the superficial portion of
the Kcnk3 pore to block, inhibition was sensitive to transmembrane
voltage. In the presence of 20 mM potassium the apparent
fraction of the electric field traversed by proton to reach its site
(
) was estimated to be 0.08 ± 0.01 (n = 6; see "Materials and Methods").
pH Alters Pore Block by Barium--
To assess the effect of proton
blockade on ions entering and leaving the Kcnk3 pore, blockade by
external barium was studied. Inward Kcnk3 currents are inhibited by
barium in a voltage-dependent manner (z
= 0.45 ± 0.01; n = 5 cells) (9), apparently via occupancy of
a potassium site in the pore as in 1-P-domain (19, 20) and another
2-P-domain channel (7). Thus, with 20 mM potassium at pH
8.0, Kcnk3 was half-blocked by 0.40 ± 0.02 mM barium
with a Hill coefficient of 1.1 ± 0.1 (
120 mV; n = 7 cells). When the pH value was lowered from 8.0 to 6.5, inhibition
by 1 mM barium decreased from 66 ± 3% to 37 ± 4% (n = 8 cells; see Fig.
3A). Although the onset of
barium block was not resolved from the capacitance transient associated
with a voltage step to
150 mV, release from steady-state barium
inhibition at
150 mV could be assessed by a step to 60 mV;
off was slowed ~2-fold (from 53 ± 8 and 100 ± 10 ms) when the pH value was lowered from 8.0 to 6.5 (n = 7 cells) (Fig. 3B).

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Fig. 3.
Barium block is pH-sensitive in wild type but
not mutant Kcnk3 channels. A, Kcnk3 channels are
inhibited by barium in a pH-dependent manner for wild type
(P1H) but not for mutant (P1N) channels. Data is shown for
inhibition by 1 mM barium at 120 mV in 20 mM
potassium (n = 7 cells). B, left,
barium off rate ( off) is pH-dependent for
wild type (P1H) but not for mutant (P1N) channels. Oocytes were held at
150 mV for 1 s and then pulsed to 60 mV. Data is shown for
inhibition by 1 mM barium with 20 mM potassium
(n = 7 cells) as in B. The relaxation was
fit with a single exponential. Right, representative trace
at pH 6.5 for cells expressing wild type (P1H) or mutant (P1N)
channels. Current was normalized to peak. Current relaxation in the
absence of barium for P1H was complete in less than 5 ms.
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Studies with the proton-insensitive Kcnk3 channel mutant (P1N)
supported the conclusion that the proton(s) that modify barium blockade
reside on histidine 98. Thus, P1N channels were inhibited by barium
like wild type (K1/2 was 0.42 ± 0.06 with a Hill coefficient of 0.9 ± 0.1; n = 8 cells). However, blockade of P1N channels by 1 mM barium
was insensitive to pH, showing 77 ± 2% and 75 ± 3%
inhibition at pH 8.0 and 6.5, respectively (n = 5 cells; see Fig. 3A). Moreover, unblock kinetics for P1N
channels were less sensitive to pH than wild type channels, showing
off of 59 ± 3 and 67 ± 5 ms at pH 8.0 and
6.5, respectively (n = 5 cells; see Fig.
3B).
Proton Block of Tandem Dimers--
When two subunits were linked
in tandem to produce constructs with four P domains, those with two
native subunits (P1H-P1H) or two mutant subunits (P1N-P1N) showed the
same sensitivity to proton as channels formed by the related monomer
subunits, P1H or P1N (Fig.
4A). Thus, with 20 mM potassium at
120 mV the K1/2 and Hill coefficient for wild type tandems was 6.75 ± 0.04 and 1.84 ± 0.11, respectively, similar to wild type monomer,
suggesting protonation at more than one site. In contrast, tandem
constructs with a single histidine in the first or second P1 position
(P1H-P1N and P1N-P1H, respectively) showed decreased pH sensitivity and evidence for a single proton blocking site. Values for P1H-P1N channels
were 6.28 ± 0.05 and 1.10 ± 0.14 whereas those for P1N-P1H channels were 6.34 ± 0.04 and 1.18 ± 0.21. These results
support the conclusion that two P1H sites are protonated in channels
formed by wild type subunits, and one P1H is protonated in mixed
tandems whether it resides in the first or downstream position.

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Fig. 4.
Two P1 domains are present in the Kcnk3
pore. A, number of histidine residues present at the
pore determines pH dependence of Kcnk3 channels. The dependence of
Kcnk3 currents at 120 mV on bath pH (mean ± S.E.) for groups of
at least five oocytes in 20 mM potassium solution is shown.
The solid line represents a fit of the data in Equation 1.
IC50 and the native (P1H-P2-P1H-P2) and mutant
(P1N-P2-P1N-P2) tandems of the Hill coefficient were 6.75 ± 0.04 and 1.84 ± 0.11 and 5.70 ± 0.15 and 0.95 ± 0.40, respectively, whereas the values for mixed tandems were 6.28 ± 0.05 and 1.10 ± 0.14 and 6.34 ± 0.04 and 1.18 ± 0.21 with P1H in the first (P1H-P2-P1N-P2) and downstream (P1N-P2-P1H-P2)
position, respectively. B F, possible arrangement of Kcnk3
subunits assuming the pore is formed by four P domains. B,
upper, two P1H subunits; lower, one P1H-P1H
tandem in an equivalent configuration. C, upper,
two P1N subunits; lower, one P1N-P1N tandem in an equivalent
configuration. D, upper, P1H-P1N tandem as in
B and C; lower, P1N-P1H tandem.
E, left, four P1H subunits arranged as if only P2
domains are pore-forming; right, two P1H-P1N tandem subunits
arranged so some P1 domains are not pore-associated. F,
left, four P1H subunits arranged as if only P1 domains are
pore-forming; right, two P1N-P1H tandem subunits arranged as
if only P1 domains are pore-forming.
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 |
DISCUSSION |
The First P Domain of Kcnk3 Contributes to the Pore--
Here, we
demonstrate that Kcnk3 senses pH value changes in the physiological
range via protonation of histidine 98 leading to channel blockade. We
employ proton blockade to demonstrate that the first P domain in Kcnk3
subunits contributes to pore function and then provide evidence that
the pore contains two P1 domains. Four experiments support the
conclusions that inhibition results from protonation of sites in the
pore and that P1 domains are pore-forming. First, block is mediated by
histidine 98, which is located in P1 adjacent to residues that
coordinate potassium ions in the ion-conduction pathway (21). Second,
block depends on voltage as if the protons enter the transmembrane
electric field to bind. Third, protons alter steady-state affinity of
barium on its pore site (as well as barium block kinetics). Fourth,
increasing external potassium decreases the sensitivity of the channel
to proton (perhaps because of increased occupancy of the pore by potassium). Finally, evaluation of linked subunits bearing four P
domains (P1-P2-P1-P2) demonstrate that P1 mediates proton blockade in
the first or third position arguing for their functional equivalence.
Kcnk3 Subunit Number--
Because four P domains are necessary and
sufficient to form the pore in voltage-gated and inwardly rectifying
potassium channels we expect the same number of P domains in each Kcnk3
pore. The arrangement and number of Kcnk3 subunits should account for
the following four findings in this report: first, wild type channels are expected to have more than one proton binding site (based on a Hill
coefficient of ~2 in 20 mM potassium); second, occupancy of one of the sites is sufficient to block (based on a Hill coefficient of ~1 in 5 mM potassium); third, the site is formed with
histidine residues on the external side of the pore (as the P1N mutant
is insensitive); and fourth, only one site is available in channels formed by mixed tandem constructs with a single P1H domain (based on a
Hill coefficient of ~1 in 20 mM potassium).
The most likely model for pore formation is a dimer with two P1 and two
P2 domain as this provides two histidine residues to bind protons in
P1H (Fig. 4B, upper) and P1H-P1H channels (Fig. 4B, lower), no histidine sites in P1N (Fig.
4C, upper) and P1N-P1N channels (Fig.
4C, lower), and one histidine in P1H-P1N (Fig. 4D, upper) and P1N-P1H channels (Fig.
4D, lower). It is appropriate to rule out both
channels formed by four P2 domains, as P1 domains contribute to pore
function (Fig. 4E, left), and channels formed only by the central P domains in linked tandems, because P1H-P1N channels (Fig. 4E, right) show proton blockade.
Finally, it seems extremely unlikely but remains possible that four P1
domains form the pore (Fig. 4F) if KCNK P2 domains are
non-functional (more on this below). Indeed, four glutamate residues
(on four 1P/6TM subunits) form only two proton binding sites in a
cyclic nucleotide-gated channel pore (22), and a single proton site is
formed by a cluster of four glutamate residues in an L-type
cardiac voltage-dependent calcium channel (23). However, to
account for our results with mixed tandem constructs, P1 domains would
either need to function non-equivalently (which is not what we
observed), and/or single protons must block via imidazole/imidazolate
pairs (an arrangement that has not been described in other proteins),
and/or single protons must block across the mouth of the pore (Fig.
4E, right). Thus, it is reasonable to argue that
Kcnk3 channels are homodimeric (Fig. 4B,
upper).
Functional evidence that 2-P-domain channels are dimeric has been
expected since the isolation of the first channel clones (1).
Indeed, an early report argued that a cysteine in the first external
loop of KCNK1 was required for both dimer formation and function (24).
However, that channel (TWIK1) has subsequently been demonstrated to be
non-functional in oocyte plasma membranes, and the cysteine residue
revealed to be absent from other KCNK channels (1).
How Protons Block--
Protonation of Kcnk3 histidine 98 is likely
to alter channel gating rather than permeation. Thus, Kim et
al. (15, 17) found that proton blockade decreased open probability
of Kcnk3 and KCNK9 whereas single channel conductance was not
significantly altered. Moreover, increasing external permeant ion
concentration destabilizes the long-lasting closed state of single
KCNK0 channels2 whereas
changing the external permeant ion from potassium to rubidium increases
the open probability of single KCNK2 channels (8). These findings
suggest that protonation favors Kcnk3 channel closure whereas permeant
ions in the pore favor the open state.
The position homologous to P1H in Kcnk3 is also a histidine in KCNK1,
-3, -7/8, and -9 but is occupied by aspartate (GYGD) in TOK1 and
asparagine (GYGN) in KCNK0, -2, -4, -5, -6, -12, -13, and -15. This
indicates that blockade of KCNK5 and -15 channels by external proton
proceeds by a different mechanism than for Kcnk3 and KCNK9. The
analogous position in the second P domain of all KCNK channels is
aspartate (GYGD) as found in voltage-gated potassium channels; when
this P2D was mutated to histidine there was no evidence for Kcnk3
channel function whether a histidine, asparagine, or aspartate was in
position 98 (not shown) demonstrating that homologous P1 and P2
residues are not functionally equivalent.
Kir2.1 inward rectifier potassium channels are formed by 1P/2TM
subunits and have an arginine residue in the P domain (GYGFR) that has
been argued to form an electrostatic barrier to divalent cation
permeation, because a histidine at the site (GYGFH) yields channels
with increased sensitivity to magnesium until proton levels increase
and the site is thought to bear a proton (26); a similar
mechanism may explain the effects of pH on barium blockade via P1H in
Kcnk3 (Fig. 3).
Little Is Known about P2 Domains--
Remarkably, a P2 domain has
yet to be shown to contribute to pore formation. Thus far, it has been
demonstrated that mutations in the TOK1 P2 domain (and flanking
transmembrane segments) alter potassium-sensitive gating kinetics
without affecting its single channel current (25, 27, 28). Thus,
external potassium concentration influences both the magnitude and rate
of TOK1 current development, and mutations in some P2 sites modify this
effect. As homologous sites in Shaker channels are known to affect the
potassium sensitivity of C-type inactivation, these P2 residues have
been suggested to contribute to channel regulation. A homodimeric
stoichiometry for Kcnk3 demands that P2 domains are, indeed,
pore-forming.
 |
ACKNOWLEDGEMENTS |
We are grateful to F. Sesti, M. Buck, and
R. Goldstein for generous advice and technical support.
 |
FOOTNOTES |
*
This work was supported by grants from the National
Institutes of Health (to S. A. N. G.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: 295 Congress Ave., New
Haven, CT 06536. Tel.: 203-737-2214; Fax: 203-737-2290; E-mail:
steve.goldstein@yale.edu.
Published, JBC Papers in Press, May 17, 2001, DOI 10.1074/jbc.C100184200
2
N. Zilberberg, N. Ilan, and
S. A. N. Goldstein, submitted for publication.
 |
ABBREVIATIONS |
The abbreviations used are:
TM, transmembrane;
MES, 4-morpholineethanesulfonic acid.
 |
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