Calmodulin Mediates Calcium-dependent Activation of
the Intermediate Conductance KCa Channel,
IKCa1*
Christopher M.
Fanger
§,
Sanjiv
Ghanshani
§,
Naomi J.
Logsdon¶§,
Heiko
Rauer
,
Katalin
Kalman
,
Jianming
Zhou¶,
Kathy
Beckingham
,
K. George
Chandy
**,
Michael D.
Cahalan
, and
Jayashree
Aiyar¶
From the
Department of Physiology and Biophysics,
University of California, Irvine, California 92697, the ¶ Target
Discovery Department, Zeneca Pharmaceuticals,
Wilmington, Delaware 19850, and the
Department of
Biochemistry and Cell Biology, Rice University,
Houston, Texas 77251
 |
ABSTRACT |
Small and intermediate conductance
Ca2+-activated K+ channels play a crucial
role in hyperpolarizing the membrane potential of excitable and
nonexcitable cells. These channels are exquisitely sensitive to
cytoplasmic Ca2+, yet their protein-coding regions do not
contain consensus Ca2+-binding motifs. We investigated the
involvement of an accessory protein in the
Ca2+-dependent gating of hIKCa1, a
human intermediate conductance channel expressed in peripheral
tissues. Cal- modulin was found to interact strongly with the
cytoplasmic carboxyl (C)-tail of hIKCa1 in a yeast
two-hybrid system. Deletion analyses defined a requirement for the
first 62 amino acids of the C-tail, and the binding of calmodulin to
this region did not require Ca2+. The C-tail of
hSKCa3, a human neuronal small conductance channel, also
bound calmodulin, whereas that of a voltage-gated K+
channel, mKv1.3, did not. Calmodulin co-precipitated with
the channel in cell lines transfected with hIKCa1, but not
with mKv1.3-transfected lines. A mutant calmodulin,
defective in Ca2+ sensing but retaining binding to the
channel, dramatically reduced current amplitudes when co-expressed with
hIKCa1 in mammalian cells. Co-expression with varying
amounts of wild-type and mutant calmodulin resulted in a
dominant-negative suppression of current, consistent with four
calmodulin molecules being associated with the channel. Taken together,
our results suggest that Ca2+-calmodulin-induced
conformational changes in all four subunits are necessary for the
channel to open.
 |
INTRODUCTION |
Ca2+-mediated signaling events are central to the
physiological activity of diverse cell types. Opening in response to
changes in intracellular Ca2+
([Ca2+]i), Ca2+-activated
K+
(KCa)1 channels
play an important role in modulating the Ca2+ signaling
cascade by regulating the membrane potential in both excitable and
nonexcitable cells. Historically, these channels have been classified
as large (BKCa), intermediate (IKCa), and small
(SKCa) conductance channels based on their single-channel conductance in symmetrical K+ solutions (1).
BKCa channels have a single channel conductance of 100-250
pS, are opened by elevated [Ca2+]i as well as by
depolarization, and are blocked by the scorpion peptides charybdotoxin
(ChTX) and iberiotoxin (2). These channels are abundant in smooth
muscle and in neurons and are also present in other cells (2).
BKCa channels are composed of an
- and a
-subunit.
The
-subunit, encoded by the Slo gene (3-5), is a
seven-transmembrane region protein with an extracellular N terminus
(6). The
-subunit is a two-transmembrane region protein that, when
associated with the channel, enhances the Ca2+ sensing and
toxin binding properties of the channel (7, 8).
SKCa channels have unitary conductances of 4-14 pS; are
highly sensitive to [Ca2+]i, with activation in
the 200-500 nM range; and are voltage-independent (9, 10).
SKCa channels are highly expressed in the central nervous
system, where they modulate the firing pattern of neurons via the
generation of slow membrane after-hyperpolarizations (10).
SKCa channels have also been described in skeletal muscle (11) and in human Jurkat T-cells (12). These channels are blocked by
apamin, a peptide from bee venom, and by the scorpion peptide
scyllatoxin (12-14). Three genes (SKCa1-3) within a novel subfamily encode SKCa channels (13). SKCa1-3
gene products bear 70-80% amino acid sequence identity to each other,
and hydrophilicity analysis predicts that these proteins have six
transmembrane helices with intracellular N and C termini (13, 15). The
hSKCa3 gene has recently been implicated in schizophrenia
(15, 16).
IKCa channels, unlike SKCa channels, are
predominantly expressed in peripheral tissues, including those of the
hematopoietic system, colon, lung, placenta, and pancreas (17-23).
These channels have intermediate single channel conductance values of
11-40 pS and can be pharmacologically distinguished from
SKCa channels by their sensitivity to block by ChTX and
clotrimazole and by their insensitivity to apamin (20, 22). Both
SKCa and IKCa channels are voltage-independent
and steeply sensitive to a rise in [Ca2+]i. At
least one gene encoding an IKCa channel has been cloned
from human and mouse tissues. Called IKCa1 (also called KCa4, SK4, and KCNN4), this gene has
been shown to encode the native IKCa channel in human
T-lymphocytes (22, 23) and erythrocytes (24-27); some
patients with Diamond-Blackfan anemia lack one allele of this gene
(23). hIKCa1 shares little sequence identity with the
Slo proteins, and only about 40% identity with the
SKCa1-3 gene products. Thus, hIKCa1 constitutes
a distinct subfamily within the extended K+ channel
supergene family.
The Ca2+ sensor for BKCa channels resides in a
negatively charged Ca2+ bowl domain in the C-tail of the
-subunit (28, 29). The
-subunit also contributes to the gating of
these proteins (7). In marked contrast, the protein-coding regions of
SKCa1-3 and hIKCa1 do not contain any EF-hand or
Ca2+ bowl motifs in their primary amino acid sequence,
despite their exquisite Ca2+ sensitivity. This observation
led us to speculate that the Ca2+ sensor for these channels
either resides in a novel motif intrinsic to the channel or is provided
by an accessory subunit that is tightly linked to channel activity. We
investigated the latter possibility in a yeast two-hybrid system using
hIKCa1 as our prototype. The Ca2+-binding
protein calmodulin (CAM) was identified as a strong interacting partner
of the C-tail of hIKCa1. Recently, CAM was shown to confer Ca2+ sensitivity to SKCa channel subfamily
members (30). Here, we report that CAM binds to and is required for
Ca2+-dependent activation of hIKCa1.
Biochemical studies demonstrate that both hIKCa1 and
hSKCa3 are prebound tightly to CAM in a
Ca2+-independent fashion. Finally, we show by expression
and patch-clamp recording that four CAMs are required to mediate the
Ca2+-dependent channel activity of the
hIKCa1 tetramer.
 |
EXPERIMENTAL PROCEDURES |
Clones, Mutants, and Vectors--
We have previously reported
the cloning of hIKCa1 (22, 23), hSKCa3 (15), and
mKv1.3 (31). Drosophila wild-type (WT) and mutant
(B1234Q) CAMs with differing Ca2+ sensitivities have been
reported previously (32, 33). The B1234Q mutant has all four EF-hands
mutated; glutamates 31, 67, 104, and 140 are replaced by glutamine
(33). PAGA2 vector was a kind gift of Lutz Birnbaumer (University of
California, Los Angeles, CA). This vector is a pGEM3-based version of
the pAGA vector, both of which contain the 5'-untranslated region of
alfalfa virus RNA 4 and a 92-base pair poly(A) tail to increase
stability of message and for efficient in vitro translation.
The segments of DNA encoding the C-terminal tails of hIKCa1
(nucleotides 1252-1678; GenBankTM accession AF022797),
hSKCa3 (nucleotides 1632-2193; GenBankTM
accession number AF031815) and mKv1.3 (nucleotides
1736-2112; GenBankTM accession number M30441) were
subcloned into the PAGA2 vector using the polymerase chain reaction
with engineered restriction sites. Both CAM clones were also subcloned
into the PAGA2 vector. For co-precipitation and electrophysiology
experiments (see below), the full-length hIKCa1
(GenBankTM accession number AF033021) and mKv1.3
coding regions were fused in-frame with a N-terminal
His6 tag in the pcDNA3.1-His-C vector
(Invitrogen, Carlsbad, CA). All clones were verified by sequencing.
Yeast Two-hybrid Screening--
A 426-base pair fragment of
hIKCa1 coding for residues 286-427 in the cytoplasmic
C-terminal tail of the channel was subcloned into the GAL4 DNA-binding
vector (pAS2-1, CLONTECH, Palo Alto, CA) using
polymerase chain reaction and engineered restriction sites. This
construct was used as bait to screen an activated human leukocyte
cDNA library (HL4021AB, CLONTECH). Screening
procedures were performed according to the manufacturer's
recommendations (CLONTECH PT3061-1). Several
thousand putative positives were identified after first-round selection
in growth medium; they were then subjected to the colony-lift
lacZ assay. Positive blue colonies were sequenced using
vector-specific primers.
Calmodulin Binding--
Two methods were used to test for CAM
binding to the channel proteins. The initial deletion constructs of
hIKCa1 were generated by polymerase chain reaction as
glutathione S-transferase (GST) fusions in the pGEX-6P-1
vector (Amersham Pharmacia Biotech), expressed in the Escherichia
coli strain BL21-De3, and synthesis of the fusion
proteins was induced with 0.1 mM isopropyl
-D-thiogalactoside in a liquid culture grown to
A600 of ~1.0. After 2.5 h at 37 °C, cells were collected by centrifugation, resuspended in NETN lysis buffer (0.5% Nonidet P-40, 1 mM EDTA, 20 mM
Tris-HCl (pH 8.0), 100 mM NaCl; 1.0 ml per 20 ml of
culture) containing protease inhibitor mixture (complete protease
inhibitor mixture tablets, Boehringer Mannheim), and lysed by
sonication. The lysate was cleared by centrifugation at 10,000 × g for 10 min at 4 °C. GST fusion proteins in the
supernatant were adsorbed for 30 min at room temperature to
glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) in NETN (1 volume of lysate:0.4 volume of 50% (v/v) slurry of Sepharose-GSH beads
(Amersham Pharmacia Biotech) in NETN), which were then washed with
binding buffer containing 1% (v/v) polyoxyethylene-9-lauryl ether, 100 mM NaCl, 20 mM Tris-HCl, pH 8.0. For
experiments investigating Ca2+ dependence, the above buffer
contained, in addition, 1 mM CaCl2 or 2 mM EDTA. Slurries (50% (v/v)) of the bound Sepharose-GSH beads (Sepharose-GSH:GST fusions) were then incubated for 30 min at
room temperature in 50 µl of binding buffer containing
[35S]methionine-labeled hCAM, synthesized by
coupled transcription-translation (TnT, Promega, Madison, WI) as
described (34). The bound beads were washed three times with binding
buffer and resuspended in 15 µl (three volumes) of 2× Laemmli's
sample buffer. Proteins released from the beads by boiling in the
presence of reducing reagent were analyzed by 4-20% gradient SDS-PAGE
followed by autoradiography to detect retention of hCAM by
the channel-GST fusion proteins. To ensure equivalent protein loading,
gels were stained with colloidal blue (Novex, San Diego, CA) to
visualize the major protein band in each lane prior to autoradiography.
Binding of WT- and B1234Q-CAMs to the C-tail of hIKCa1 was
also determined using the GST pull-down method as above.
For all other experiments, channel constructs in the pAGA2 vector were
radiolabeled with [35S]methionine during coupled
transcription-translation using reagents from Promega. These constructs
were incubated with CAM-Sepharose 4B beads (Amersham Pharmacia
Biotech). Briefly, slurries of CAM beads (50% (v/v)) in binding buffer
(as described above) were incubated with radiolabeled channel proteins
that had been normalized for radioactive incorporation using a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Equal cpms of each
specific protein were added to 50 µl of binding buffer including
either 2 mM EDTA or 1 mM Ca2+.
Binding and washing conditions were the same as for the GST-Sepharose experiments above. Proteins released from the beads by boiling in the
presence of reducing reagent were analyzed by 18% SDS-PAGE followed by autoradiography.
His Tag Pull-down Assays--
mKv1.3 and
hIKCa1 expression constructs in pcDNA-3.1His(C) were
transfected into COS-7 cells using Fugene6 (Boehringer Manheim) according to the supplied protocol. About 40 h after transfection, 5 × 106 cells were lysed in 10 mM HEPES
(pH 7.4), 40 mM KCl, 0.75 mM EDTA (free
Ca2+ concentration, <1 nM), 1% Triton X-100,
10 mM
-mercaptoethanol, 0.25% deoxycholate, and
protease inhibitors. After 20 min on ice, cells were Dounce-homogenized
and centrifuged at 2900 × g for 15 min to remove
insoluble material. The soluble lysate was transferred to a clean tube
and mixed with an equal volume of 2× binding buffer (20 mM
HEPES (pH 7.4), 200 mM KCl, 20% glycerol, 60 mM imidazole, 20 mM
-mercaptoethanol, and
protease inhibitors). The diluted lysate containing the membrane
fraction was incubated with Ni+-NTA resin (Qiagen,
Valencia, CA) for ~2 h at 4 °C in order to immobilize the
His-tagged channel protein. After extensively washing the resin with
wash buffer (10 mM HEPES (pH 7.4), 100 mM KCl, 10% glycerol, 0.25% Triton X-100, 30 mM imidazole, 0.2 mM EDTA, 10 mM
-mercaptoethanol, and
protease inhibitors), the channel protein was eluted with elution
buffer (same as wash buffer but containing 400 mM
imidazole). Proteins from the elution fraction, as well as from the
flow-through, were separated by SDS-PAGE and transferred to
polyvinylidene fluoride membranes. To determine whether CAM was
preassociated with hIKCa1 or mKv1.3, a Western blot analysis was performed using an anti-CAM monoclonal antibody (Upstate Biotechnology, Lake Placid, NY).
Preparation of cRNA, Microinjection, and Whole Cell
Recording--
Rat basophilic leukemia (RBL) cells were maintained in
a culture medium of Eagle's minimum essential medium (BIO-Whittaker, San Diego, CA) supplemented with 1 mM
L-glutamine (Sigma) and 10% heat-inactivated fetal calf
serum (Summit Biotechnology, Fort Collins, CO) and grown in a
humidified, 5% CO2 incubator at 37 °C. Cells were
plated to grow nonconfluently on glass 1 day prior to use for cRNA
injection and electrophysiological experiments. T-lymphocytes were
isolated from human peripheral blood and activated with
phytohemagglutinin (DIFCO, Detroit, MI) as described previously (20).
Prior to experimentation, cells were plated for 15 min on glass
coverslips coated with poly-L-lysine (Sigma). For other experiments, we stably transfected the COS-7 cell line with
hIKCa1; the biophysical properties of the hIKCa1
channels in these cells are indistinguishable from those of
IKCa channels in T-cells (data not shown). Plasmids
containing the entire coding sequence of the hIKCa1 gene,
WT-CAM, and B1234Q-CAM were linearized with NotI and
in vitro transcribed with the T7 mMessage mMachine system (Ambion, Austin TX). Plasmids containing the mKv1.3 coding
sequence were linearized with EcoRI and in vitro
transcribed with the Sp6 version of the same kit. The resulting cRNA
was phenol/chloroform-purified and stored at
75 °C. RNA
concentrations were determined to an accuracy of 25%, based on
intensity of bands in agarose gel electrophoresis. The cRNA was diluted
with fluorescein isothiocyanate-dextran (Sigma) (average
Mr, 10,000; 0.1% in 100 mM KCl).
RBL cells were injected with an Eppendorf (Hamburg, Germany)
microinjection system (Micro-manipulator 5171 and Transjector 5246)
using injection capillaries (Femtotips®, Eppendorf) filled with the
cRNA/fluorescein isothiocyanate solution, as described previously (35).
Cells were visualized by fluorescence, and hIKCa1-specific
currents were measured 4-8 h after injection. Cells measured in the
whole cell configuration were normally bathed in normal Ringer solution
containing 160 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2,
10 mM HEPES, 10 mM glucose; adjusted to pH 7.4 with NaOH, with an osmolarity of 290-320 mosM. In
K+ Ringer solution, Na+ was replaced by
K+. A simple syringe-driven perfusion system was used to
exchange the bath solutions in the recording chamber. The internal
pipette solution with 1 µM free Ca2+
contained 145 mM K+ aspartate, 8.5 mM CaCl2, 2 mM MgCl2,
10 mM HEPES, 10 mM K2 EGTA; adjusted to
pH 7.2 with KOH, with an osmolarity of 290-310 mosM. EGTA
was omitted in the high Ca2+ internal solution containing 1 mM CaCl2. Pipettes were pulled from glass
capillaries, coated with Sylgard (Dow-Corning, Midland, MI), and
fire-polished to resistances measured in the bath of 2-5 M
.
Membrane currents were recorded with an EPC-9 patch-clamp amplifier
(HEKA elektronik, Lambrecht, Germany) interfaced to a computer running
acquisition and analysis software (Pulse and PulseFit; HEKA
elektronik). Data were filtered at 1.5 kHz, and all voltages were
corrected for a liquid junction potential offset of
13 mV for
aspartate-based solutions. The holding potential in all experiments was
80 mV. For characterization of the hIKCa1 current, voltage
ramp stimuli were used to assess channel activation by elevated
[Ca2+]i. RBL cells express an endogenous inwardly
rectifying K+ channel that did not interfere with
KCa currents seen at depolarized potentials. Experiments
were performed at room temperature (21-25 °C). The CAM antagonists
W7, trifluoperazine (TFP), and calmidazolium were purchased from
Calbiochem (La Jolla, CA). ChTX was obtained either from Peptides
International, (Louisville, KY) or from BACHEM Biosciences (King of
Prussia, PA). Clotrimazole was purchased from Sigma. All slope
conductances reported in the text are written as mean ± S.E.
(number of cells or experiments).
 |
RESULTS |
CAM Binds the C-tail of hIKCa1 in a Ca2+-independent
Manner--
We searched for accessory molecules that bind to
hIKCa1 using the yeast two-hybrid system. We reasoned that
if such a molecule is involved in Ca2+ sensing, it must be
common to IKCa and SKCa channels, because their
Ca2+ sensitivities and gating behavior are remarkably
similar (10, 13, 20, 22, 24). An amino acid alignment of
hIKCa1 with hSKCa1, rSKCa2, and hSKCa3
revealed that except for the pore and the transmembrane regions, the
proximal half of the cytoplasmic C-tail was the most highly conserved
(Fig. 1); the C-tail of hIKCa1 was therefore employed as the bait. We chose an activated human leukocyte cDNA library to screen for interaction partners because hIKCa1 has been previously shown to be highly up-regulated
in activated lymphocytes (20, 22). A primary screen using the triple
nutrient selection (Trp+Leu+His+)
resulted in the identification of several thousand positive clones. A
subsequent subscreen of 500 colonies yielded nine clones that were
positive for
-galactosidase activity. Seven of these clones encoded
CAM.

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Fig. 1.
Amino acid alignment of the C-terminal tails
of hIKCa1, rSKCa2, hSKCa3, and hSKCa1.
Dashes represent identical residues, and
asterisks correspond to conserved residues. Shaded
areas represent identical or conserved residues. Numbering shown
above the sequence alignment is based on the first amino
acid in the C terminus (post-S6), as predicted by Kyte-Doolitle
hydropathy plots. The numbers shown at the right of each
sequence correspond to the actual positions of residues in the channel
sequence. The C-tail of hIKCa1 corresponds to residues
286-427 in the protein; the C-tail of hSKCa1
corresponds to residues 383-561 (GenBankTM accession
U69883); the C-tail of rSKCa2 corresponds to residues
395-580 (GenBankTM accession number U69882); and the
C-tail of hSKCa3 corresponds to residues 543-731.
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We next examined the ability of 35S-labeled in
vitro translated hIKCa1-C-tail to bind CAM-Sepharose as
assessed by running the product on an SDS-PAGE gel followed by
autoradiography. As shown in Fig.
2A, a radiolabeled band of
~28 kDa is visible (hIKCa1) consistent with the size of
the hIKCa1 C-tail (Fig. 2B), indicating that
hIKCa1 and CAM interact. CAM also binds to the C-tail of the
SKCa channel, hSKCa3 (Fig. 2A,
~22-kDa band (hSKCa3)), but not the C-tail of the
voltage-gated K+ channel, mKv1.3 (Fig
2A, mKv1.3). As an additional specificity control, we performed GST pull-down experiments; CAM did not bind GST
alone but interacted with the GST-hIKCa1 C-tail (Fig.
3A). Thus, CAM interacts
specifically with members of the IKCa and SKCa
family.

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Fig. 2.
CAM binds to the C-tail of hIKCa1
and hSKCa3, but not mKv1.3. A,
35S-labeled C-tails of hIKCa1,
hSKCa3, and mKv1.3 were incubated with
CAM-Sepharose beads in the presence (right panel) and in the
absence (left panel) of 1 mM Ca2+,
washed, boiled, and loaded on SDS-PAGE gels. Bands were visualized by
autoradiography. A faint nonspecific band seen in the mKv1.3
lane in the presence of Ca2+ was also detected when the
fragment was incubated with GST-Sepharose beads alone (data not shown).
B, an autoradiogram of 35S-labeled products,
synthesized by coupled transcription translation (TnT) and loaded prior
to incubation with CAM produced equivalent band intensities in all
three lanes.
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Fig. 3.
Deletion analysis of hIKCa1
C-tail. A, glutathione-Sepharose beads containing
GST fusion constructs of either the entire C-tail or deletion fragments
of hIKCa1 C-tail were incubated with 35S-labeled
CAM in the presence of 1 mM Ca2+ (top
panel) or in the presence of 2 mM EDTA and no added
Ca2+ (bottom panel). Coomassie gels ensured
equivalent loading of protein in all lanes (data not shown).
B, deletion constructs 1-82, 1-72, and 1-62 were
35S-labeled by transcription-translation and incubated with
CAM-Sepharose beads in the presence of 1 mM
Ca2+ (left panel) or in buffers containing 2 mM EDTA and no added Ca2+ (right
panel).
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Surprisingly, the C-tail fragments of hIKCa1 and
hSKCa3 bound CAM efficiently in buffers containing 2 mM EDTA and no added Ca2+ (Fig. 2A,
hIKCa1 and mKv1.3, right panel). Deletion analysis revealed that the shorter 1-98 and 1-62 fragments of the
hIKCa1 C-tail also bound CAM efficiently in
Ca2+-free conditions (Fig. 3A, bottom panel;
Fig. 3B, right panel). Four additional deletion fragments
(45-98, 37-77, 1-72, and 1-82) bound CAM, but only in the presence
of 1 mM Ca2+ (Fig. 3A, top panel;
Fig. 3B, left panel), whereas two others (1-50 and 93-142)
did not bind CAM at all (Fig. 3). Thus, CAM interacts with the C-tails
of hIKCa1 and hSKCa3 in the absence of
Ca2+, and this property resides in a domain within the
first 62 residues of the hIKCa1 C-tail. The segment between
residues 62 and 82 appears to mask the Ca2+-independent
interaction of CAM with hIKCa1, because the 1-72 and 1-82
fragments bind CAM only in the presence of Ca2+, whereas
residues 82-98 appear to reverse the negative effect of 62-82.
Removal of as yet unidentified motifs between residues 1 and 37 appears
to unmask a Ca2+-dependent interaction with
CAM. Interestingly, the 1-98 segment of the hIKCa1-C-tail,
which contains the Ca2+-independent and
Ca2+-dependent modulatory domains, shares a
high degree of sequence similarity with the three members of the
SKCa family (Fig. 1).
CAM Co-precipitates with Full-length hIKCa1 in Transfected
Cells--
The binding data described above suggest that CAM is
preassociated with the channel in cells with resting low
[Ca2+]i. If this were the case, it should be
possible to co-precipitate CAM from cells expressing hIKCa1.
To test this hypothesis, we expressed an N-terminal His-tagged fusion
protein of hIKCa1 in COS-7 cells, prepared a crude membrane
lysate in a Ca2+-free solution, and passed the lysate
through a nickel chelate column to allow the hIKCa1 channel
to bind to the column via a His-nickel interaction. The column was
washed extensively, and the unbound fraction was collected in the
flow-through. The His-tagged hIKCa1 channel (along with any
prebound accessory proteins) was then eluted with 400 mM
imidazole. We examined the flow-through and the
hIKCa1-containing eluate fraction for CAM using a anti-CAM monoclonal antibody. As negative controls, we used membrane lysates from untransfected cells and lysates from cells expressing a His-tagged version of the mKv1.3 channel that does not bind CAM (Fig.
2A). As expected for a ubiquitous protein expressed at high
levels in mammalian cells, CAM was detected in the flow-though
fractions from untransfected cells (Fig.
4), mKv1.3-transfected cells,
and hIKCa4-transfected cells. In contrast, CAM was detected
only in the hIKCa1-containing eluate, but not in the eluates
from untransfected or mKv1.3-transfected cells (Fig. 4).
Thus, CAM specifically co-precipitates with full-length
hIKCa1, but not mKv1.3, suggesting that the
IKCa channel is tightly bound to CAM under basal conditions
in mammalian cells.

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Fig. 4.
CAM co-precipitates with hIKCa1
in transfected cells. Lysates from untransfected COS-7 cells
or lysates from COS-7 cells transfected with either N-terminal
His-tagged mKv1.3 or N-terminal His-tagged
hIKCa1, as indicated, were passed through a nickel-chelate
column. The flow-though (unbound fraction) and eluate (channel + prebound accessory proteins) fractions were analyzed for CAM by Western
blotting using anti-CAM antibodies.
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CAM Antagonists Do Not Alter KCa Channel
Function--
In T lymphocytes or in mammalian cells expressing
hIKCa1, elevating [Ca2+]i rapidly
opens IKCa channels, revealing a voltage-independent K+ current with a reversal potential near
80 mV (20, 22).
To investigate the role of CAM in the function of IKCa
channels encoded by hIKCa1, we tested whether CAM
antagonists might disrupt KCa currents activated by
dialysis of human T cells with a pipette solution containing 1 µM [Ca2+]i. Whole cell recordings
revealed two components of K+ current, an immediately
active voltage-gated K+ current encoded by
hKv1.3, along with a rapidly activating IKCa current (Fig. 5A, traces
1 and 2), as reported previously (20). The time course
of the slope conductance of this IKCa current is shown in
Fig. 5B. Treatment with the CAM antagonist W7 (10 µM) had no effect on the IKCa current at
physiological potentials. Although it blocked both currents at
depolarized potentials (Fig. 5A), this suppression is
voltage-dependent and is thought to be mediated by a direct
effect on the channel, rather than via CAM modulation (36). Another CAM
antagonist, TFP (10 µM), also had no effect on
IKCa currents when applied acutely; the slope conductance ratio, pre-TFP/post-TFP was 1.3 ± 0.2 in six cells. Intact cells were also pretreated with TFP, W7, or 2 µM calmidazolium
for 15-30 min prior to recording, with no effect (not shown).
Inclusion of 10 µM W7 inside the patch pipette in
combination with such pretreatment also had no effect; the slope
conductance ratio in seven drug-treated cells relative to untreated
cells was 1.3 ± 0.3. Similar results were observed in COS-7 cells
stably transfected with hIKCa1. We conclude that CAM
antagonists do not interfere with the activation of hIKCa1
channels.

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Fig. 5.
CAM antagonists do not inhibit
hIKCa1. A, currents in activated T-cells were
elicited by voltage ramps from 120 to 30 mV over 200 ms. At
hyperpolarizing potentials, where only IKCa currents are
observed, acute application of 10 µM W7 to the bath
solution had no effect, whereas at depolarized potentials, the current
was inhibited. Numbers on traces correspond to time of ramp
(shown in B), where ramp 1 was taken immediately after
break-in, ramp 2 at steady-state hIKCa1 current, and ramp 3 after ~6 min of exposure to 10 µM W7. B,
time course of hIKCa1 slope conductance activated by
dialysis with 1 µM free Ca2+, determined at
80 mV. After complete and stable activation of hIKCa1
currents, 10 µM W7 was applied to the bath solution
(indicated by the bar), but no effect on the KCa
current was observed. Ratio of mean slope conductance of
KCa 2 min after treatment relative to the same cell prior
to treatment, 0.9 ± 0.2 (six cells); same ratio for
KV + KCa current at +25 mV, 0.76 ± 0.02 (six cells)
|
|
IKCa Channel Function Requires WT CAM--
The
association of hIKCa1 C-tail with CAM in very low
[Ca2+] (Figs. 2 and 4), as well as the inability of CAM
antagonists to alter current through these channels (Fig. 5), supports
the idea of a stable, nonconventional association between CAM and
hIKCa1. Therefore, to study the interaction of these
proteins, simultaneous new synthesis of each might be required. We
injected combinations of cRNA encoding channel proteins plus cRNA
encoding WT or mutant CAM into RBL cells, enabling us to investigate
the effects and interactions of the resulting gene products using
electrophysiological techniques. First, we characterized the
physiological and pharmacological properties of hIKCa1
expressed after injection of the encoding cRNA alone. Robust currents
exhibiting all the hallmarks of IKCa channels were seen
4-7 h postinjection (Fig. 6). The
currents reversed near
80 mV in normal Ringer solution, and switching the bath solution to K+-Ringer (164.5 mM
K+) shifted the reversal potential to ~0 mV, as expected
from the Nernst equation for a K+-selective channel (not
shown). ChTX or clotrimazole reduced the current in a
dose-dependent manner with the expected potency for IKCa channels in native tissue (20, 22, 24).

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Fig. 6.
Microinjection of hIKCa1
into RBL cells. Characterization of currents after injection
of FITC only (dashed line) or FITC + hIKCa1 cRNA
into RBL cells; typical hIKCa1 currents were detected 4-7 h
postinjection. Currents were elicited by 200-ms voltage ramps, using an
internal solution with 1 µM free Ca2+.
Application of 5 nM ChTX and 100 nM
clotrimazole blocked the IKCa conductance.
|
|
To determine whether CAM was responsible for the
Ca2+-mediated gating of these channels, we exploited the
availability of a Drosophila mutant (B1234Q) CAM.
Drosophila and human CAM are identical at the amino acid
level except at five positions. In B1234Q, glutamates (Glu31, Glu67, Glu107, and
Glu140) at the
Z coordination positions in each of the
four Ca2+-binding sites have been replaced by glutamine,
resulting in a dramatically lower affinity for Ca2+ (33).
We reasoned that co-expressing hIKCa1 along with B1234Q would result in a significant reduction of current amplitudes. In order
for this hypothesis to be tested by co-expression, it was first
important to show that B1234Q bound hIKCa1 normally. The apo
form of B1234Q is structurally similar to WT-CAM, the UV CD signal at
222 nm for the B1234Q-apo form being approximately 80% of the
wild-type value (33). The apo form of B1234Q would therefore be
expected to bind the hIKCa1 C-tail in a
Ca2+-independent fashion. Consistent with this prediction,
35S-labeled Drosophila WT- and B1234Q-CAM bound
to GST-hIKCa1 C-tail both in the presence (Fig.
7, lanes 4 and 5)
and absence (lanes 9 and 10) of Ca2+,
as did 35S-labeled hCAM (lanes 3 and
8). These CAMs did not bind GST alone in the presence or
absence of Ca2+ (Fig. 7, lanes 1, 2, 6, and
7).

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Fig. 7.
Wild-type Drosophila CAM and
a mutant CAM, B1234Q, interact with the C-tail of hIKCa1.
Glutathione-Sepharose beads containing the GST fusion construct of the
hIKCa1 C-tail were incubated with 35S-labeled
mammalian WT-CAM, Drosophila WT-CAM, or B1234Q-CAM in the
presence of 1 mM Ca2+ (left) or in
the presence of 2 mM EDTA and no added Ca2+
(right).
|
|
Co-injection into RBL cells of WT-CAM cRNA together with cRNA encoding
the hIKCa1 channel produced robust hIKCa1
currents in the whole cell mode with 1 µM free
Ca2+ in the pipette. In marked contrast, co-injection of
B1234Q cRNA with hIKCa1 cRNA resulted in an average 17-fold
reduction in current amplitude. Fig.
8A shows the ramp currents
obtained from individual cells. Aside from the endogenous inwardly
rectifying K+ current, almost no additional current was
seen in the B1234Q-containing cells, as compared with a large outward
current observed in the cells with WT-CAM. From experiment to
experiment, great variation was noted in the magnitude of currents seen
in WT CAM-microinjected cells. However, on a given day, the currents
observed in mutant or WT CAM-microinjected cells co-varied in a
consistent manner, preserving a statistically significant difference in
current ratio. Table I summarizes these
results. Overall, injection of mutant CAM reduced currents to 6% of WT
CAM-microinjected cells. A reduction in the whole cell KV
current was not observed when B1234Q was coinjected with
mKv1.3 cRNA (mean: 427 pA for WT and 460 pA for B1234Q),
ruling out the possibility that the effects on hIKCa1 currents were due to global inhibition of translation by B1234Q. The
results of a typical experiment are shown in Fig. 8B and
clearly demonstrate that B1234Q inhibits current through
hIKCa1. B1234Q is incapable of the normal high affinity
Ca2+ binding and concomitant conformational changes seen in
WT-CAM, but it does show a very limited conformational response that is completed upon attaining Ca2+ levels of 1 mM
(33). However, even with 1 mM Ca2+ in the patch
pipette, cells expressing B1234Q exhibited no appreciable current (data
not shown). Thus, the minimal conformational changes in B1234Q are not
adequate to gate the channel, and the normal conformational changes
associated with Ca2+ binding to CAM are essential for
channel opening.

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Fig. 8.
Co-injection of hIKCa1 with
mutant CAM inhibits IKCa currents. hIKCa1
cRNA was co-injected into RBL cells with either WT-CAM or B1234Q cRNA.
Slope conductance was determined at potentials between -20 and 40 mV
to avoid contamination with currents through the endogenous
inward-rectifier channel present at potentials below -70 mV.
A, ramp currents in RBL cells injected with
hIKCa1 in combination with WT-CAM (WT) or
hIKCa1 in combination with B1234Q (MUT).
B, comparison of the slope conductance of uninjected cells
and those co-injected with hIKCa1 or KV1.3 and either WT-CAM
(WT) or B1234Q (MUT). Each circle
represents the measurement of slope conductance of currents in a single
cell approximately 2 min after establishing the whole cell mode. The
bold lines illustrate the mean slope conductance for all
cells in each column. The difference between the mean slope conductance
of cells microinjected with B1234Q RNA and those
microinjected with WT-CAM RNA was statistically significant, as
illustrated by the one-tailed Student's t test
(p < 0.02). Cells co-injected with KV1.3
and MUT or WT-CAM showed no significant difference in current at +20
mV.
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Table I
B1234Q mutant CAM suppresses hIKCal currents
RBL cells were measured in whole cell patch-clamp experiments 4-7 h
after microinjection with hIKCal RNA + CAM RNA. The CAM
RNA consisted of wild-type (WT) or B1234Q mutant (MUT) RNA injected
separately or together in varying proportions, as indicated. All
experiments were performed as a paired comparison between WT and MUT
CAM RNA, or WT and the indicated ratio of WT:MUT CAM RNA, using a
constant amount of hIKCal RNA for each pair. Nine pairs of
experiments are shown in the table. Slope conductance values were
determined as described in the Fig. 8 legend and are reported in nS as
mean ± S.E. (number of cells), with data taken approximately 2 min after establishing the whole cell configuration. Similar ratios of
slope conductance were obtained for all experiments employing a given
proportion of WT:MUT CAM RNA (see Fig. 9). The day-to-day variations in
slope conductance resulted from injections of different amounts of
hIKCal RNA.
|
|
Dominant-negative Suppression of hIKCa1 by B1234Q--
Like most
K+ channels, hIKCa1 is anticipated to be
tetrameric, but although each subunit could bind a single CAM, the
Ca2+ binding requirement for channel activation is unknown.
It is conceivable that one functional CAM binding to Ca2+
is sufficient to activate an hIKCa1 channel, or perhaps
channel activity requires that all four subunits bind WT CAM and
undergo the Ca2+-induced conformational change. To explore
this question, we co-injected RBL cells with hIKCa1 cRNA and
a mixture of B1234Q mutant and WT CAM. The mutant and WT CAM
composition of hIKCa1 channels formed in such cells can be
predicted by the binomial distribution. The proportion,
P(r), of channels with r mutant subunits is given by the equation,
|
(Eq. 1)
|
where p is the fraction of mutant CAM in the CAM mix
and n is the total number of subunits, four in the case of a
K+ channel. The experimental results of microinjecting
mutant and WT CAM RNA mixtures can then be compared with these
predictions to determine the permitted number of CAM-binding subunits
that most accurately represents the observed conductance. Because the mutant CAM appears totally unable to activate hIKCa1 (Fig.
8), channels with four B1234Q CAMs are presumed to be nonfunctional. Channels containing a mixture of mutant and WT CAM subunits would be
expected to conduct only if hIKCa1 can be activated by fewer than four functional CAM-bound subunits. Thus, the current in cells
microinjected with a mixture of mutant and WT CAM should reveal the
number of functional CAMs required for active channels. Fig.
9 shows the KCa current in
cells with mixed mutant and WT CAM normalized to control
KCa currents in cells microinjected with WT CAM in parallel
experiments performed on the same day. Table I summarizes the results
of all experiments. For the case in which even a single mutant subunit
will disrupt channel function, the binomial equation simplifies to the
equation,
|
(Eq. 2)
|
as represented by the solid line (curve 0) in Fig. 9, with
p plotted as the abscissa. If a single mutant subunit is
tolerated in a functional channel, the proportion of conducting
channels is shown by the equation,
|
(Eq. 3)
|
as depicted by Fig. 9, curve 1. Similarly expanded
equations can be written for cases in which two or three mutant CAM are allowed. The data are well fitted only by the equation in which a
single mutant subunit is sufficient to disrupt hIKCa1
function. Hence, the presence of a B1234Q CAM is dominant-negative for
the function of hIKCa1.

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Fig. 9.
Dominant-negative suppression of
hIKCa1 currents by B1234Q. RBL cells were
co-injected with hIKCa1 cRNA and either WT-CAM alone,
B1234Q-CAM alone, or a mixture of B1234Q- and WT-CAM cRNA.
For each experiment, the base-line conductance (the mean slope
conductance of uninjected cells) was subtracted and the resulting
number was divided by the mean slope conductance of cells from the same
experiment microinjected with WT-CAM. Each circle represents
the mean ratio ± S.E. of three independent experiments. The ratio
of mutant CAM RNA to total CAM RNA is shown on the x axis.
Each experiment consisted of 4-10 cells measured 4-7 h postinjection.
Error bars on the x axis indicate the maximal
anticipated error in RNA concentration. The solid line
(line 0) represents a fit to the binomial distribution for
the scenario in which no mutant subunits are allowed in a functional
channel. Dotted lines (lines 1-3) show the fits
to the same equation if one, two, or three mutant CAMs, respectively,
binding a single hIKCa1 could form a functional
channel.
|
|
 |
DISCUSSION |
In the present study, we have demonstrated that CAM is prebound to
the cytoplasmic C-tail of the intermediate conductance KCa
channel, hIKCa1, and mediates
Ca2+-dependent gating of these channels. The
first 98 amino acids in the C-tail of hIKCa1 contain
subdomains that are critical for both
Ca2+-dependent and Ca2+-independent
CAM binding. Although this region contains several positively charged
and hydrophobic residues reminiscent of CAM-binding sites (37, 38), its
lack of dependence on Ca2+ for binding is noteworthy. We
also show that known CAM antagonists W7 and TFP have no effect on
hIKCa1 current, indicating a novel binding surface for the
CAM-hIKCa1 interaction. Such Ca2+-independent
binding of CAM to its target protein, although uncommon, has been
reported for some molecules, including nitric oxide synthase, neurogranin, neuromodulin, phosphorylase kinase, and unconventional myosins (39).
Our results with an IKCa channel parallel and complement
those recently reported by Xia et al. (30) for
SKCa channels. In that study, CAM was shown to associate
tightly with the C-tails of SKCa channels in the absence of
Ca2+. Co-expression in Xenopus oocytes of
rSKCa2 and CAM mutants with lower Ca2+ binding
affinities resulted in a significant decrease in the Ca2+
sensitivity of the expressed channel, thus providing the first evidence
for a mechanism of Ca2+-gating by SKCa
channels. Our finding of an identical mechanism for hIKCa1
expressed in mammalian cells confirms a common mechanism of
Ca2+ gating for both SKCa and IKCa
channels, despite their ~40% overall sequence identity. Not
surprisingly, the region in the C-tail of hIKCa1 that we
identified as being critical for CAM binding (1-98) shows a high
degree of sequence similarity with the corresponding regions in the
three members of the SKCa subfamily of K+ channels.
In addition to demonstrating that CAM can bind to and mediate the
function of hIKCa1, we have demonstrated a strong
suppression of IKCa conductance when the channel is
co-expressed with a mutant CAM with four defective EF-hand motifs
(B1234Q CAM). The fact that the mutant CAM is so effective at competing
with the endogenous protein for binding to the channel subunits
suggests some mechanism for coassembly of newly synthesized CAM protein
with the cytoplasmic tails of new channel molecules as they are folded
on the endoplasmic reticulum membranes. Alternatively, it is possible
that there is, in essence, no pool of "free" CAM to compete with
the newly synthesized protein. At elevated
[Ca2+]i, this condition appears to apply, with
free (non-target bound) CAM representing about 0.1% of total CAM
protein (40). The situation at resting [Ca2+]i is
less well understood. However, studies in muscle cells suggest that the
levels of CAM and its target proteins are carefully co-regulated even
at resting levels of [Ca2+]i (41).
If each subunit of hIKCa1 channel tetramer binds one
molecule of Ca2+-free CAM, and if the concerted action of
all four molecules is necessary for gating, then perturbing one
interaction would impose a dominant-negative phenotype on the channel
currents. Consistent with this hypothesis, the currents observed in
cells microinjected with a 1:1 ratio of WT and mutant CAM cRNA along
with the channel cRNA exhibited about
th of the current
magnitude observed in cells microinjected with channel cRNA and WT CAM
alone. When the ratio of mutant to WT CAM was varied, we observed that the current relative to WT-CAM-microinjected cells agreed with the
equation for disruption of channel activity by binding of a single
mutant CAM (Fig. 9). Furthermore, even 1 mM
Ca2+ in the patch pipette, a concentration sufficient to
elicit the conformational changes seen in B1234Q, did not rescue
conductance when this mutant CAM was co-expressed with
hIKCa1. These results imply that Ca2+-induced
conformational changes must occur involving each prebound CAM in order
to open the channel (Fig. 10). The
requirement for four CAM molecules provides a structural basis for the
previously determined steeply cooperative Ca2+ dependence
for activation of the lymphocyte KCa channel encoded by
hIKCa1 (Hill coefficients of 3-4) (20, 42).

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Fig. 10.
Model depicting the site of interaction of
CAM with the C-tail. The inner helices of each channel subunit,
shown as black cylinders, are arranged in a bundle, as
suggested by the KcsA crystal structure (46). These helical rods cross
at the bottom cytoplasmic surface (bundle crossing) and
diverge at the extracellular surface to accommodate the P-regions. The
upper end of the helices correspond to the S6 (M2 in KcsA) segments
(black), and the region below the bundle crossing represents
residues 1-10 of the hIKCa1 C-tail (white).
Residues 11-62 of the critical C-tail CAM-binding region of each
subunit are shown (in scale) as a separate single helix based on both
Chou/Fasman and Robson algorithms for secondary structure predictions.
Two CAM molecules (actual structure in the absence of Ca
2+) are shown in apposition to the C-tail; the other two
subunits also associate with CAM. The spatial disposition of the CAMs
does not imply interaction with the C-tail in any particular
orientation.
|
|
Our results point naturally toward a kinetic model for gating of the
IKCa channel. In the following scheme,
C indicates closed channel conformations, and
asterisks represent activated subunit conformations.
Horizontal transitions represent Ca2+ binding to CAM on the
channel, and vertical transitions represent Ca2+-CAM-induced conformational changes in the channel
subunit, with the number of asterisks symbolizing the number of
activated subunits. In the above scheme, each subunit of a tetrameric
channel is associated with a single CAM, which can bind up to four
Ca2+ ions to induce a conformational change in the channel
subunit. For each of four independent subunits, we envision a
sequential two-step activation process: first Ca2+ binding
by preassociated CAM, and then a conformational change in the
IKCa channel subunit to an activated conformation. In this scheme, it is imagined that conformational changes in the absence of
Ca2+ binding to CAM are so energetically unfavorable that
the states with more activated subunits than Ca2+-CAM
moieties do not exist, consistent with the fact that no
IKCa conductance is seen at low
[Ca2+]i. Such states, if they existed, would fill
in the lower left-hand portion of the scheme. All four subunits must be
activated before the channel opens. Although speculative, the kinetic
diagram is similar to previous proposals for a variety of
KCa channels based upon single-channel data (43-45). These
schemes predict that the steep Hill coefficient determined in
functional measurements of the Ca2+ sensitivity of channel
opening arises from the requirement for Ca2+-induced
conformational changes by each of four subunits in BKCa, SKCa, and, as proposed here, IKCa channels.
How might a conformational change in the C-tail of hIKCa1
result in opening of the pore? A comparison of the sequence of this region with that of the structurally defined bacterial potassium channel, KcsA, from Streptomyces lividans (46)
suggests that the first 6-10 residues of the hIKCa1 C-tail
correspond to part of the inner helix that includes S6 (Fig. 10). More
specifically, these residues represent the stretch of the inner helix
lying below the "bundle crossing" (Fig. 10), and any
Ca2+-CAM-induced conformational change in this segment
could conceivably be transmitted along the helical rod, resulting in
channel opening. Interestingly, recent studies on the voltage-gated
K+ channel, Shaker, suggest that gating occurs
at the bundle crossing possibly due to conformational changes in this
region (47). Two different algorithms (Chou/Fasman and Robson) predict
that the remainder of the 1-62 segment has a high helical propensity, suggesting that the inner helix might extend further cytoplasmically (Fig. 10). Coupling of this segment with the inner helix might underlie
calcium gating of hIKCa1. This heuristic model requires direct structural verification.
Although second messenger cascades involving CAM are known to modulate
many ion channels (48), there is growing evidence of regulation by
Ca2+-CAM through direct binding (49). These phenomena have
been documented for the Paramecium
Ca2+-activated sodium channels (50), the
Drosophila Ca2+-permeable channels
trp and trpl (51, 52), the vertebrate photoreceptors and olfactory receptors involving cyclic nucleotide gated channels (53), the ryanodine receptor Ca2+-release
channels (54), and the N-methyl-D-aspartate
receptors (55). However, the region of the C-tail of hIKCa1
and hSKCa3 implicated in Ca2+-free CAM shows no
obvious similarity to sequences with a comparable function in
trpl (51) or the ryanodine receptor (56). In these examples,
channel modulation involves either activation or deactivation by CAM.
In contrast, the high affinity for Ca2+ and the rapid
activation kinetics of SKCa and IKCa channels
demands a fast gating mechanism (45). This "near-intrinsic"
requirement is provided by preassociated CAM molecules in a tight
multimeric complex with the channel tetramer, converting a modest
change in intracellular Ca2+ to a quick, robust
physiological response. Further biochemical, biophysical, and direct
structural studies will help elucidate the mechanisms by which
CAM-induced channel conformational changes in the C-tail translate into
opening of IKCa and SKCa channels, leading to
hyperpolarization and downstream signaling events.
 |
ACKNOWLEDGEMENTS |
We appreciate the expert technical assistance
of Luette Forrest and Annabelle Chia-ling Wu, as well as valuable
discussions with Drs. George Ehring and George A. Gutman.
 |
FOOTNOTES |
*
This study was supported by National Institutes of Health
Grants NS14609 and GM41514 (to M. D. C.), GMOD54872 and MH59222 (to
K. G. C.), 5T32CA09054 (from NCI) (to C. M. F.), GM49155 and Grant
C-1119 from the Welch Foundation (to K. B.), and by a fellowship from
the Alexander von Humboldt Foundation (to H. R.).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.
§
These authors contributed equally to this work.
**
To whom correspondence should be addressed: Joan Irvine Smith Hall,
Rm. 291, Medical School, University of California,
Irvine,CA 92697. Tel.: 949-824-2133; Fax: 949-824-3143; E-mail:
gchandy{at}uci.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
KCa, Ca2+-activated K+;
BKCa, large
conductance KCa;
SKCa small conductance
KCa, IKCa intermediate conductance
KCa;
C-tail, carboxyl-terminal tail;
CAM, calmodulin;
ChTX, charybdotoxin;
RBL, rat basophilic leukemia;
TFP, trifluoperazine;
WT, wild-type;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel electrophoresis.
 |
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