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J. Biol. Chem., Vol. 275, Issue 22, 16969-16978, June 2, 2000
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From the Section of Developmental Biology and Biophysics,
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, March 9, 2000, and in revised form, March 27, 2000
Potassium leak conductances were recently
revealed to exist as independent molecular entities. Here, the genomic
structure, cardiac localization, and biophysical properties of a murine
example are considered. Kcnk3 subunits have two pore-forming P domains and unique functional attributes. At steady state, Kcnk3 channels behave like open, potassium-selective, transmembrane holes that are
inhibited by physiological levels of proton. With voltage steps, Kcnk3
channels open and close in two phases, one appears to be immediate and
one is time-dependent ( Leak currents are considered essential to normal electrical
function in sympathetic ganglia (1, 2), myelinated axons (3-6),
carotid body type 1 cells (7), and cardiac myocytes (8-12).
Nonetheless, their existence as independent transport entities, rather
than residual flux through other pathways, was controversial until the
cloning of KCNKØ from Drosophila melanogaster (13). KCNKØ
(previously ORK1), encodes a potassium channel subunit with two P
domains and four predicted transmembrane segments
(2P/4TM)1 (13). KCNKØ
channels are open across the physiological voltage range, show no delay
in current development with voltage steps, and "openly rectify,"
that is, they operate like potassium-selective holes in an electric
field (13).2 KCNKØ channels
are tightly regulated; activation yields an open probability
(Po) close to 1, and inhibition produces
channels that are almost always closed
(63).3 Mammalian genes
homologous to KCNKØ, now enumerated KCNK1-9, are emerging
rapidly. Like KCNKØ, those that show function are potassium-selective
leak conductances (16-24).
Based on homology to KCNKØ we isolated Kcnk3 from a murine
cardiac cDNA library (16, 24), localized it to chromosome 5B in
mouse and 2p23.3-p24.1 in human (25), and called the predicted protein
product OAT1. Two other groups cloned Kcnk3 concurrently and
called the encoded subunit TASK1 (26) and TBAK1 (27). For clarity, we
will now employ the Human Genome Organization nomenclature:
KCNK3 gene and KCNK3 channel for human isolates and
Kcnk3 and Kcnk3 for mice. Significant discrepancies exist between the findings of the three groups. Although all agree that Kcnk3 predicts 2P/4TM subunits that form pH-sensitive,
openly rectifying potassium channels, there is no consensus as to
tissue distribution (atria or ventricle), function (instantaneous
or time-dependent, low or high open probability), or the
predicted protein sequence.
In this report, five points are highlighted. First, the genomic
sequence for murine Kcnk3 is determined to confirm the
accuracy of the cDNA under study; this reveals an intron in the
midst of the coding sequence for the signature motif (G YG) of the
first P loop (an arrangement seen to be conserved in the family from nematodes to humans). Second, Kcnk3 messenger RNA is
localized to murine cardiac ventricle and, at lower levels, in the
atria; Kcnk3 protein is then confirmed to have the same cardiac
distribution. Third, half-maximal blockade of Kcnk3 channels by
external protons is confirmed to be near physiological pH and shown to
be sensitive to external potassium. Fourth, Kcnk3 currents are seen to
develop with voltage changes in two phases; one appears to be immediate and one is time-dependent; the fraction of current in each
phase is responsive to external potassium. Fifth, single Kcnk3 channels are shown to open only briefly (to one of two conductance levels) and
rarely; although open probability increases with depolarization, it is
not significantly augmented by a wide array of stimuli including activation or inhibition of protein kinase A or C, application of
volatile anesthetics or metabolic poisons, changes in osmotic strength,
or exposure to low oxygen tension.
Based on its location and similar functional attributes, we hypothesize
Kcnk3 to be the correlate of a native cardiac current that remains
active throughout the action potential plateau but whose molecular
basis has been unclear, IKp or
IKsus (8-12, 28). The findings support the idea
that Kcnk3 channels link cardiac excitability to changes in acid-base status.
Cloning of Kcnk3
cDNA--
A homology search of NCBI data base using the
BLAST program suite (29) and the coding sequence of the ORK1 channel
(KCNKØ) as query (accession number U55321) identified expressed
sequence tag W09160. Northern blot analysis using the 801-bp cDNA
fragment in W09160 detected an abundant single message at ~3.8 kb in murine heart (24). The 801-bp cDNA fragment was used to screen a
random primed and oligo(dT)-primed murine heart cDNA library in
Genomic--
A murine genomic DNA library in bacteriophage P1
was screened with two oligonucleotide primers corresponding to the 3'
end of the coding region of Kcnk3 cDNA as described
(31). These primers, 5'-GCAGACGCAGCCGCAGTATG-3' and
5'-GCCTGGCCGTTGTGCGTGAGCAGGG-3', amplify a 168-bp fragment from
murine genomic DNA. Polymerase chain reaction-positive clones were
purified and subcloned into pGEM-7Z plasmid vectors (Promega Corp.,
Madison, WI). Subcloned fragments were analyzed by restriction
endonuclease digestion, Southern blotting, and nucleotide sequencing.
Northern Blot and in Situ Hybridization Analyses
32P[ATP]-labeled probes used for Northern blots
were the 801-bp fragment (W09160) and a Western Blot Analyses
Rabbit antibodies recognizing residues 252-269 of the human
KCNK3 subunit, EDEKRDAEHRALLTRNGQ, were purchased from Alamone (APC024,
Jerusalem, Israel). Frozen mouse heart ventricle and atria were
purchased (Pel-Freez Biologicals, Rogers, AZ), and crude membrane
fractions of each tissue were prepared by a modified method (34).
Proteins were extracted with 1% Triton X-100 and analyzed by
SDS-polyacrylamide gel electrophoresis and Western blot with the rabbit
antibody, followed by goat anti-rabbit horseradish peroxidase-conjugated antibody and visualization by enhanced
chemiluminescent substrate.
Electrophysiology
Oocytes were isolated from Xenopus laevis frogs
(Nasco, Atkinson, WI), subjected to collagenase treatment to ease
removal of the follicle, and injected with 46 nl of sterile water
containing 2-4 ng of Kcnk3 cRNA.
Whole Cell--
Macroscopic currents were measured 1-4 days
after cRNA injection by two-electrode voltage clamp using a Geneclamp
500 amplifier (Axon Instruments, Foster City, CA). Data were sampled at
4-20 kHz and filtered at 1-5 kHz. Data acquisition and analysis
were performed using Pulse (Instrutech, Great Neck, NY) and Sigmaplot (Jandel Scientific, San Rafael, CA) software. Electrodes were made from
1.5-mm borosilicate glass tubes (Garner Glass Co., Claremont, CA),
contained 3 M KCl, and had resistances between 0.3 and 1 M Membrane Patch--
Voltage clamp recordings were made in both
on-cell and outside-out configuration using an Axopatch 200A amplifier
(Axon Instruments). The vitelline layer was removed prior to recording
with a pair of fine forceps after a 1-2-min incubation in hypertonic
solution 200 mM potassium aspartate, 20 mM KCl,
1 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.4, with NaOH. Pipettes were fabricated from 7052 glass (Garner Glass Co., Claremont, CA) coated with Q-Dope (GC
Electronics, Rockford, IL) and fire-polished. The electrode solution
for outside-out patches was 100 mM KCl, 5 mM
EGTA, 1 mM MgCl2, 5 mM HEPES, pH
7.4, with KOH. Bath solution contained 100 mM KCl, 0.3 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.4. External potassium concentration was
varied by substitution of NaCl for KCl. Pipette resistance ranged from
3-5 M Calculations--
Equilibrium reversal potentials were
determined in the indicated solutions by linear regression.
Current-voltage relations were studied in various potassium solutions
and fit to the Goldman (35) and Hodgkin and Katz (36) current
relationships.
Kcnk3 Encodes a Two P Domain Subunit with Four Predicted
Transmembrane Segments--
Three independent cDNA clones for
Kcnk3 isolated from a murine cardiac library were found to
be identical (accession number AF065162) (24). Relative to the
predicted initiator methionine, the cDNA contains an A in position
Alignments of the P domains for subunits predicted to have two P
domains reveals that Kcnk3 is most similar to the open and outward
rectifiers (KCNKØ, KCNK2, Kcnk4, and KCNK5), more distantly related to
clones with as yet undefined function (KCNK1, Kcnk6, Kcnk7, and KCNK8)
and most distinct from the outward rectifier of yeast cells, TOK1 (Fig.
1B). Homology among the genes is insignificant except for
the P domain segments where pairs can achieve ~30% identity.
We verified the predicted Kcnk3 cDNA sequence by
comparison to the genomic DNA sequence (accession numbers AF241798 and AF242508). Three Kcnk3 genomic clones were identified by
polymerase chain reaction screening, and one was studied in detail by
restriction enzyme analysis, Southern blotting, and limited nucleotide
sequencing. The region of this clone containing the Kcnk3
gene, including 5'and 3'-untranslated sequences and the coding region,
were sequenced on both strands. Comparison of cDNA and genomic
sequences showed that Kcnk3 is a two exon gene spread over
~21 kb (Fig. 1C). Evaluation of the exon/intron boundaries
revealed the AG:GT rule was not violated and that no AG nucleotide
pairs were present in the 15 bp upstream of the 3' (acceptor) splice
junction. The single exon/intron boundary is located at a functionally
critical position in the channel, in the midst of the selectivity
filter "signature sequence" (G YG) of the first P domain.
Kcnk3 mRNA Is Abundant in Murine Cardiac
Ventricle--
Northern blot analysis using an 801-bp Kcnk3
cDNA fragment as probe detected a strong signal in heart with less
abundant message in lung and brain (Fig.
2A). Only a single band at
~3.8 kb was detected. Faint signals were visualized after exposure
for extended periods in skeletal muscle and kidney (not shown). When
the distribution of Kcnk3 message in mouse heart was
examined by in situ hybridization, a strong, specific signal
was apparent throughout both ventricles with the antisense probe (Fig.
2C). A weak antisense signal was also visualized in the
atria, indicating a lower level of transcript in those cells. Because
this localization was at odds with prior reports (26), we evaluated the
cardiac expression pattern of Kcnk3 protein.
Kcnk3 Protein Is Prominent in Ventricle--
Anti-peptide
antibodies were used to visualize Kcnk3 protein in homogenates of
murine atrial and ventricular tissue (Fig. 2D). A strong
signal near the predicted mass for Kcnk3 was apparent in ventricular
samples (Fig. 2D, lane 1); a weaker signal was found in atrial preparations despite the presence of similar amount of
total protein in the lane (Fig. 2D, lane 2). The
signal was demonstrated to be specific for Kcnk3 protein because it was
competitively depleted by co-incubation with the peptide fragment
recognized by the antiserum (Fig. 2D, lanes 3 and
4).
Kcnk3 Protein Forms Potassium-selective, Openly Rectifying Ion
Channels--
When Kcnk3 cRNA was injected into X. laevis oocytes, a new current was observed by two-electrode
voltage clamp (Fig. 3A) that was not present in control cells. In response to changes in voltage, the current rose to a new steady state level. Once activated, inactivation was not observed (10 s pulses; not shown). At
physiological levels of bath potassium (5 mM) and pH (7.4),
the channel passed large outward currents with depolarizing voltage
steps but only small inward currents at hyperpolarized potentials (Fig.
3A, left panel). Increasing external potassium
concentration produced a shift in reversal potential and large inward
currents (Fig. 3, A and B). The change in
reversal potential indicated that the channel was selective for
potassium over sodium and chloride. Thus, increasing external potassium
levels from 5 to 100 mM (by isotonic substitution of NaCl
with KCl) produced a shift in reversal potential of 56 ± 3 mV/10-fold change in potassium (Fig. 3C) in good agreement
with the Nernst relation, which predicts an ~58 mV change for a
perfectly selective channel under these conditions.
Changes in current-voltage relationships with altered external
potassium indicated that Kcnk3 channels were openly rectifying. Thus,
inward currents were smaller than outward currents when external
potassium was low and increased to equal magnitude when potassium
levels were approximately the same across the membrane (Fig. 3,
A and B). This behavior was reasonably well
approximated by the Goldman-Hodgkin-Katz relation (Equation 1) for
current through across an ion-selective partition at differing
transmembrane gradients of permeant ion (Fig. 3B). It was
notable that Equation 1 failed to approximate the experimental data at
5 mM bath potassium because outward currents were smaller
than predicted. This was subsequently explained by
potassium-dependent proton inhibition of Kcnk3 channel
currents (see below).
Kcnk3 channels exhibit an Eisenman type III permeability series (Fig.
3D). To assess relative permeability compared with potassium (the predominant internal permeant ion), a test cation replaced the
sodium and potassium in the bath solution to achieve a pseudo bi-ionic
condition in whole cell mode and Equation 2 was used. Permeability was
highest for rubidium (1.1 ± 0.1, n = 12) and potassium (= 1), intermediate for cesium and ammonium (0.30 ± 0.02 and 0.23 ± 0.03, n = 8, respectively), and
lowest for sodium and lithium (less than 0.031 ± 0.003 and
0.031 ± 0.002, n = 8, respectively). Although
rubidium had a greater relative permeability than potassium its
relative conductance was over 2-fold lower (Fig. 3D).
Proton Inhibition of Kcnk3 Channels at Physiological pH Is
Potassium-sensitive--
With 5 mM potassium in the bath,
Kcnk3 currents were maximal at pH 8.0, significantly blocked at pH 7.4, and completely inhibited at pH 6.0 (Fig.
4A). Proton block was well fit
to Equation 3 with a half-maximal blocking concentration
(pKa) of 7.24 ± 0.03 and a Hill coefficient of
1.02 ± 0.06, suggesting that one proton was required to block
(Fig. 4B). As external potassium levels rose, block by
protons was diminished (Fig. 4C); at pH 7.0, the fraction of
unblocked current at 30 mV in a potassium-free bath solution was
0.32 ± 0.02 and increased to 0.54 ± 0.02, 0.73 ± 0.02 and 0.94 ± 0.02 with 5, 20, and 100 mM bath
potassium, respectively. Increasing proton levels inhibited Kcnk3
channels despite elevated potassium levels (not shown). The effect of
potassium on proton inhibition explained the anomalous increase in
outward current seen with elevation of external potassium (Fig.
3A); although increasing bath potassium decreased the
outward driving force for potassium flux, it also diminished proton
inhibition (Fig. 4C) leading to an overall increase in
outward current.
External Potassium Alters the Fraction of Kcnk3 Current That Is
Time-dependent--
The rise and fall of Kcnk3 currents
showed a phase that appeared immediate and another that was delayed. In
whole cell mode, ~40% of activation was judged to be
time-dependent with steps from Voltage Alters the Fraction of Time-dependent Kcnk3
Currents--
Kcnk3 currents in on-cell patches showed changes with
voltage consistent with altered open probability (Fig.
6). Thus, the fraction of
time-dependent current decreased with more positive holding
potential (from Voltage Influences the Kinetics of Time-dependent Kcnk3
Currents--
Activation and deactivation were well approximated by
single exponential relationships, and both rates showed a weak
dependence on voltage. The rate of activation was greater at more
positive test potentials (Fig. 6B), although deactivation
was faster at more negative voltages (Fig. 6D). With 5 mM external potassium at pH 7.4, the time constant (
The time constant for current decay was 5.0 ± 0.2 ms
(n = 5) at
A rough upper limit for the time course of "immediate" current
development with voltage steps was determined in patch mode from the
inflection point between the fall of the capacitance transient and the
rise of the time-dependent current (as the immediate phase
was judged to be complete by this time). By this strategy, the rise of
the immediate current (with a step from Single Kcnk3 Channels and Unitary Conductance--
A new potassium
channel was observed at the single channel level in oocytes expressing
Kcnk3. Open infrequently, its slope conductance was
estimated to be 11 ± 1 pS in on-cell patches with approximately
symmetric 100 mM potassium (Fig.
7, A and B,
left panel). Although ~70% of openings were to this
level, ~30% were to a conductance level ~1.6-fold larger (based on
11,000 single channel transitions). This suggested our estimate of
unitary conductance was for a dominant substate (Fig. 7B,
middle panel). Unitary conductance estimated by noise
variance analysis was 10 ± 1 pS (Figs. 7C).
Single Kcnk3 channels studied in outside-out patch mode recapitulated
observations made macroscopically; single channels were selective for
potassium, openly rectifying, and sensitive to external pH (Fig.
8A). Thus, Kcnk3 channels were
observed at both depolarized and hyperpolarized voltages in
approximately symmetric 100 mM potassium with a reversal
potential of ~0 mV (Fig. 8A, left panel). When
external potassium was lowered to 5 mM, no inward currents were observed, and the reversal potential was ~ Latency to First Opening and Open Probability of Single Kcnk3
Channels--
The response of single channels to voltage was assessed
by holding outside-out patches at the reversal potential for potassium (
In on-cell patches at
Important sources of error in these estimates were uncertainty about
whether more than one channel was present in a patch (in which case
open probability estimates were too large) and missed brief openings as
the minimal detectable event was 90 µs at the filter frequency
employed (2 kHz) and mean open time was ~300 µs (tending to depress
estimates of open probability). Errors because of unappreciated
channels with open probability ~1 were ruled out because lowering pH
had no effect on current base line. Finally, one of seven patches
thought to have a single Kcnk3 channel showed an
Po of ~0.14; this patch was excluded from
analyses but may have represented Kcnk3 channels in a different
functional mode.
Modulation of Kcnk3 Currents--
The presence of multiple
regulatory consensus sites in the carboxyl-terminal segment of Kcnk3
and its low open probability suggested that the channel might be
down-regulated in oocytes. Moreover, whole cell Kcnk3 currents tended
to slowly "run down," ~15% over 30 min (Table
I). Therefore, a variety of common
potassium channel inhibitors and activators were evaluated in the
presence of 20 mM external potassium to permit ready
measurement of both inward and outward currents (Table I). Only
depolarizing voltage steps were found to increase open probability
significantly. Kcnk3 channels were found to be relatively insensitive
to common blockers added to the bath (tetraethylammonium,
4-aminopyridine, and glibencamide and sensitive to amiodorone and
barium). PMA (50 nM), an activator of protein kinase C, had
no rapid effect (10 min) but decreased current by ~ The superfamily of genes encoding potassium channel subunits with
two P domains has emerged with remarkable speed since the cloning of
TOK1 (with a predicted 2P/8TM topology) (40) and KCNKØ (with a
predicted 2P/4TM topology) (13). At present, more than 70 genes are
listed in public data bases. Mammalian genes, now identified as
KCNK1-9 by the Human Genome Organization, all have a
predicted 2P/4TM topology. Thus far, KCNK channels that function are
"leak" conductances; they have a non-zero open probability across
the physiological voltage range. KCNK2 and KCNK5 are outward rectifiers, a phenotype first observed for TOK1 (40-42); under symmetric ionic conditions these pass large outward potassium currents
and small inward potassium currents (16, 18, 43). KCNKØ, Kcnk3, and
Kcnk4 are open rectifiers, showing linear current-voltage relationships
under symmetric conditions and Goldman rectification (35, 36) when
potassium levels are unequal across the membrane (13, 17, 24, 26, 27,
44).2 Kcnk9 is an inwardly rectifying clone (23), whereas
KCNK1, Kcnk6, Kcnk7, and KCNK8 do not show reproducible function (16, 19-22). Previously accessible only in native cells, cloned leak channels are now available for detailed evaluation under controlled conditions.
Genomic Organization--
Kcnk3 has an intron in the
first P domain. This boundary is located at the same location in
KCNKØ, KCNK2, Kcnk4, and Kcnk5 based
on their genomic sequences as reported in the GenBankTM
data base of NCBI4 and
Kcnk75 as well as
20 predicted two P domain channel genes in the nematode Caenorhabditis elegans (45). Maintenance of this
organization suggests either that this noncoding region serves an
important regulatory role or that its natural deletion is infrequently
propagated because random excision risks damage to the signature
sequence residues that are required for function. That the genomic
organization of Kcnk7,5 which is nonfunctional
in experimental cells, is like that of Kcnk3 indicates the
intron/exon boundary does not designate which KCNK isolates will show function.
The functional attributes we determine for Kcnk3 differ in a number of
respects from those reported by others; most notable, they did not
observe time-dependent currents (26, 27, 44). Sequence
differences, although minor, may explain functional discrepancies between the clones under study. Based on combined cDNA and genomic sequencing, it appears that the Kcnk3 variant we study is
derived from the same gene as TASK1 (26) and TBAK1 (27). Alternative splicing cannot explain variations among the three cDNA sequences. Thus, these differences must result from RNA editing (46), or they are
due to sequence errors introduced during reverse transcription or
polymerase chain reaction.
Kcnk3 Channel Protein: a Potential Correlate of Cardiac
IKp--
Whereas we visualize Kcnk3 transcripts
throughout the heart with a predominance in ventricle by in
situ hybridization (Fig. 2B), others find message only
in cardiac atria (26). Here we support ventricular localization by
direct demonstration that Kcnk3 protein is also in ventricular tissue
and at lower levels in atrial samples (Fig. 2C). The
conclusion that Kcnk3 channels function in cardiac myocytes (rather
than nonmuscular cells) is supported by a report that Kcnk3
message can be amplified from single cardiac myocytes by polymerase
chain reaction (27), rare observation of single channels in rat
ventricular myocytes with the same unitary conductance as murine Kcnk3
(47), and suppression of mouse ventricular currents by Kcnk3
antisense
oligonucleotides.6
Currents in native cells with attributes similar to those we delineate
for Kcnk3 channels have been reported (8-12, 28). Like Kcnk3,
IKp in guinea pig cardiac myocytes is prominent
in ventricle, activates rapidly, and does not inactivate with sustained depolarization (8, 9). Moreover, IKp is
insensitive to external tetraethylammonium and 4-aminopyridine and
sensitive to external barium (8); the effect of low pH on guinea pig
IKp has not been reported. Although differences
between IKp and Kcnk3 in open probability and
barium block argue against identity, species differences in homologous
potassium channels is well recognized (48).
Leak channels remain active at all potentials in contrast to inwardly
rectifying cardiac potassium channels (49, 50). Thus, Kcnk3 channels
are expected to contribute not only to establishing resting membrane
potential but to the height and length of action potentials and,
therefore, the duration of myocardial contraction. Indeed, the function
of pH-sensitive leak potassium conductance has been recognized in
native cardiac cells (51-53).
Kcnk3 Shows Potassium-dependent Proton Block--
A
key property of Kcnk3 channels is their sensitivity to changes in
external pH in the physiological range. Here we show that proton block
is more effective at lower external potassium (Fig. 4). This served to
explain the anomalous increase in outward current observed as external
potassium level is elevated. The mechanism of
potassium-dependent, proton block is the subject of another report.7
Time-dependent Changes in Kcnk3 Open Probability with
Voltage Steps--
Classical voltage-gated potassium channels sense
changes in membrane potential via a transmembrane segment that has
multiple basic residues (54-57). Shaker channels show a half-maximal
activation voltage of ~0 mV and an e-fold change in open
probability per ~25 mV (these parameters are insensitive to potassium
reversal potential or absolute level). Voltage-dependent
changes in Shaker current magnitude take time because they result from
changes in channel conformation. Immediate increases in current (seen
when channels open before a voltage step) are rarely observed with voltage-gated channels in native cells for two reasons: resting membrane potential is usually below the voltage required for opening and voltage steps that open channels also favor their entry into a
nonconducting conformation, the inactivate state.
Kcnk3 manifests both immediate (
Kcnk3 channels appear to reside in at least four states (at all
voltages) and to undergo a reversible voltage-dependent
gating process. A simple model must include two open states of
different conductance (O1 and O2) and two
closed states that feed into the open states with time constants of 5 and 65 ms (C1 and C2). Single-channel recordings do not reveal whether channels traverse O1 to
enter O2. That Kcnk3 enters a closed conformation is
suggested by observation of a latency to first opening for single
channels (Fig. 8), time-dependent development of current
(Fig. 3), and the absence of evidence for blockade as a cause for entry
into the nonconducting mode (Table I). The closed to open transition is
seen to be voltage-sensitive by the effect of potential on the fraction
and kinetics of time-dependent current (Figs. 5 and 6); a
single rate-limiting voltage-gated step is suggested by good
approximation of current relaxation with a single exponential.
Although KCNKØ was used to isolate Kcnk3 and is also a 2P/4TM open
rectifier, it shows only instantaneous changes in current magnitude
with voltage steps at both the single-channel and macroscopic level.2 Conversely, TOK1 (the 2P/8TM outward rectifier)
shows weak voltage dependence and sensitivity to potassium (40, 42,
58). Recently, Loukin and Saimi (59) showed that TOK1, like Kcnk3,
visits two kinetically distinct closed states, a nearly open state
(whose dwell time depends on membrane potential and potassium reversal potential), and a deeply closed state (responsive on voltage and external potassium). They observed that temperature had a significant effect on activation from the deep closed state but little effect on
nearly open state. They concluded that the deep closed state reflected
function of a channel gate, whereas the nearly open state was an effect
of ions in the pore (59). A role for permeant ion occupancy of the pore
in voltage-dependent gating is well recognized in chloride
(60) and potassium channels (54, 61, 62). Our results also suggest a
role for occupancy of the pore by permeant ion in gating.
Kcnk3 Channels in Oocytes Have a Low Open Probability--
Kcnk3
channels had a low open probability despite numerous experimental
manipulations known to alter the opening and closing of other potassium
channels (Table I). The paucity of openings does not result from block
by protons because channels show low Po above pH 8.0 where they are not
inhibited (Fig. 5B); nor is there evidence found for block
by magnesium, calcium, or heavy metal contaminants (Table I). Although
activation of PMA increases the open probability of KCNKØ dramatically
and staurosporine closes that channel (63), these agents were only
mildly inhibitory on Kcnk3. Arachidonic acid, reported to activate
Kcnk4 (17, 64), also inhibited Kcnk3 (Table I). The volatile anesthetic halothane was reported to activate Kcnk3 by ~50% at doses lethal to
humans (1 mM, ~5 MAC, where 1 MAC is the minimum alveolar
concentration required to anesthetize half of the human population)
(14, 15), and our results support the agents weak effect. The striking
regulation of KCNKØ (63)3 and the failure of KCNK1, Kcnk6,
Kcnk7, and KCNK8 to show function in experimental cells suggests that
an as yet unidentified regulator or channel subunit may act to increase
the open probability of Kcnk3 channels.
We are grateful to C. Wong and R. Goldstein
for expert technical assistance.
*
This work was supported by a grant 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, March 27, 2000, DOI 10.1074/jbc.M001948200
2
N. Ilan and S. A. N. Goldstein,
submitted for publication.
3
N. Zilberberg, N. Ilan, R. Gonzalez-Colaso, and
S. A. N. Goldstein, submitted for publication.
4
N. Zilberberg and S. A. N. Goldstein,
unpublished observation.
5
D. Bockenhauer, M. A. Nimmakayalu, D. C. Ward, S. A. N. Goldstein, and P. G. Gallagher,
submitted for publication.
6
C. M. B. Lopes, M. Apkon, and S. A. N. Goldstein, unpublished observation.
7
C. M. B. Lopes, F. Sesti, N. Zilberberg, M. E. Buck, and S. A. N. Goldstein,
unpublished observation.
The abbreviations used are:
2P/4TM, two P
domains and four predicted transmembrane segments;
bp, base pair(s);
kb, kilobase(s);
MES, 4-morpholineethanesulfonic acid;
PMA, phorbol
12-myristate 13- acetate.
Proton Block and Voltage Gating Are
Potassium-dependent in the Cardiac Leak Channel
Kcnk3*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
= ~5 ms). Both proton block and gating are potassium-sensitive; this produces an anomalous increase in outward flux as external potassium levels rise because of
decreased proton block. Single Kcnk3 channels open across the physiological voltage range; hence they are "leak" conductances; however, they open only briefly and rarely even after exposure to
agents that activate other potassium channels.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
t11 (CLONTECH, Palo Alto, CA). Of 28 clones
that hybridized to the probe, eight were purified and subcloned, and
their ends subjected to automated DNA sequencing; three clones were
sequenced in their entirety. This yielded a 5'-untranslated sequence,
an open reading frame, and a 3'-untranslated sequence. An additional 160 bp of the upstream 5'-untranslated sequence was obtained by 5'
rapid amplification of cDNA ends using 1 µg of total RNA prepared from murine cardiac muscle, as described (30) and primer A
(5'-CACCAGCAGGTAGGTGAAG-3'). Single-stranded ligation and amplification
were carried out with primers D
(5'-GCCAAGCTTGCGGTGGCCCTCAGGTCCAGCTC-3') with B
(5'-GCCGCCGCTGCTGCCCCGGA-3') and D with C
(5'-CTCCACGCCGGGCACCAGCTCCGCAC-3'), respectively. Analyses of
nucleotide and predicted amino acid sequences were performed utilizing
GCG software from the University of Wisconsin (Madison, WI). The
cDNA sequence is listed (accession number AB008537). The coding
sequence was placed between the 5'- and 3'-untranslated regions the of
Xenopus 
-globin gene in pBF2 (a gift of Bernd Fakler,
Tuebingen, DE) and cRNA produced using T3 RNA polymerase and a kit
(Ambion, Austin, TX). Transcripts were quantified by spectrophotometer
and compared with control samples separated by agarose gel
electrophoresis. Our cDNA sequence (accession number AF065162) was
verified by comparison with the genomic sequence (accession numbers
AF241798 and AF242508) and varies from the partial sequence reported
for murine TASK1 at amino acid residues 4 (Gln replaces Glu), 123 (Val
replaces Ile), and 286 (where an additional Gly is added) (26) and
murine TBAK1, which includes a 9-residue amino-terminal extension and a
single residue difference at position 101 (Pro replaces Ala) (27).
These differences do not coincide with known consensus sites in the
Kcnk3 genomic clone (see below) for splice junctions or
editing and are judged to be errors in the sequences reported by others.
-actin cDNA (Amersham
Pharmacia Biotech) (32). In situ hybridization was performed
with adult C57BL6 mice (Jackson Labs, Bar Harbor, ME) using sense and
antisense probes from the 801-bp Kcnk3 fragment, as
described (33). [
-35S]UTP (Amersham Pharmacia Biotech)
incorporation into the 801-bp Kcnk3 fragment was 70-85%.
Heart sections (8 µm) were hybridized overnight, treated with
ribonuclease-A, and dehydrated by soaking in 100% EtOH in 0.6 M ammonium acetate. Emulsion radiographs were generated by
dipping slides in photographic emulsion with development and fixation 2 days later. Slides were placed on Kodak SB5 film to generate images.
. Oocytes were studied while perfused at 0.5-1 ml/min with 5 mM KCl bath solution 93 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.3 mM CaCl2, 5 mM HEPES, pH 7.4, with
NaOH. In indicated cases, KCl was substituted for NaCl. For solutions
at pH 6.0, MES replaced HEPES. Studies were performed at room temperature.
for single channel recordings and 0.3-0.6 M for
macropatches; seal resistance was 4-15 G. Data were sampled at
10-40 kHz, filtered at 0.5-5 kHz with ACQUIRE software (Instrutech
Corp.), and analyzed off-line by TAC (Instrutech Corp.) and IGOR
(Wavemetrics, Lake Oswego, OR) software.
where PK is the permeability of
potassium, z, V, F, R, and
T have their usual meanings, and an internal K+
concentration of 90 mM is assumed, as reported previously
(37). Permeability ratios were calculated according to the following equation.
![I<SUB><UP>S</UP></SUB>=P<SUB><UP>K</UP></SUB><UP>z</UP><SUP><UP>2</UP></SUP><SUB><UP>S</UP></SUB><FENCE><FR><NU>VF<SUP>2</SUP></NU><DE>RT</DE></FR></FENCE><FENCE><FR><NU>[K]<SUB>i</SUB>−[K]<SUB>o</SUB> <UP>exp</UP>(−z<SUB><UP>S</UP></SUB> VF/RT)</NU><DE>1−<UP>exp</UP>(−z<SUB><UP>S</UP></SUB> VF/RT)</DE></FR></FENCE>](/content/vol275/issue22/fulltext/16969/img001.gif)
(Eq. 1)
where PK and PX
are the permeability of potassium and the test cation, respectively; in
whole cell mode it is assumed that potassium is the only permeant ion
inside the cell. Dose response curves were fit to the following
function.
(Eq. 2)
where [B] is the concentration of the blocker,
K1/2 is the concentration of the blocker
required to achieve 50% inhibition, and n is the Hill
coefficient. The voltage dependence of block was modeled by a
simplification of the approach of Woodhull (38) according to the
following equation.
![<FR><NU>1</NU><DE>1+<FENCE><FR><NU>[B]</NU><DE>K<SUB>1/2</SUB></DE></FR></FENCE><SUP>n</SUP></DE></FR>](/content/vol275/issue22/fulltext/16969/img003.gif)
(Eq. 3)
where z and
![<FR><NU>I</NU><DE>I<SUB><UP>max</UP></SUB></DE></FR>=<FENCE><FR><NU>[B]</NU><DE>K<SUB>1/2</SUB></DE></FR><UP>exp</UP><FR><NU><UP>−</UP>z&dgr;FV</NU><DE>RT</DE></FR>+1</FENCE><SUP><UP>−1</UP></SUP>](/content/vol275/issue22/fulltext/16969/img004.gif)
(Eq. 4)
represent charge on the blocker and the apparent
electrical distance traversed by the blocking particle to reach its
receptor site.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3 and a termination codon 209 bp upstream with no additional ATG
triplets in the intervening sequence. The open reading frame is 1227 bp, and secondary structure analyses predict a protein of 409 amino
acids with two classical P domains (2P) bounded by hydrophobic segments
that suggest the presence of four transmembrane segments (4TM) (Fig.
1A). A 2P/4TM topology is
consistent with the absence of a recognizable leader sequence, the
external disposition of one consensus site for N-linked glycosylation, and an internal location for two sites for protein kinase C, two for protein kinase A, three for calcium-calmodulin kinase
II, one for tyrosine kinase, and a carboxyl-terminal PDZ consensus
motif.
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Fig. 1.
Predicted topology, gene structure, and
relation to other KCNK channels. A, predicted membrane
topology of Kcnk3 (based on average free energy for transfer to water
with a window of 20 amino acids) and amino acids in each of the two P
domains. B, maximum likelihood tree for the relationship
among the second P domains of 10 predicted two P domain potassium
channel subunits. The scale bar indicates predicted genetic
distance. Each subtype has a bootstrap value of 100/100. cDNA
sequences are deposited under the following accession numbers: KCNKØ
(D. melanogaster, ORK1, U55321), KCNK1 (human OHO/TWIK,
U76996), KCNK2 (human TPKC1/TREK, AF004711), Kcnk3 (murine
OAT1/TASK1/TBAK, AF065162), Kcnk4 (murine TRAAK, AF056492), KCNK5
(human, AF084830), Kcnk6 (murine, AF158234), Kcnk7 (murine, AF110522),
KCNK8 (human, AF134149), and TOK1 (Saccharomyces cerevisiae,
U28005). Alignments were performed by ClustalW 1.6 with Blossum
algorithms with default gap opening and extension penalties and TOK1 as
outgroup. Genes names as assigned by the Genome Data Base Nomenclature
Committee and the Human Genome Organization. C, genomic
organization of Kcnk3 (accession numbers AF241798 and
AF242508). The cDNA sequence across the exon 1/2 boundary and
predicted P1 amino acids are indicated. NC,
noncoding.
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Fig. 2.
Kcnk3 message and protein are present in
murine cardiac ventricular cells. A, Northern blot
analysis. Samples of 2 µg of poly(A)+ mRNA from
various murine tissues were hybridized to a
[32P]dCTP-labeled 801-bp murine KCKN3 cDNA fragment.
Abundant message of ~3.8 kb was detected in heart, with lower amounts
detected in brain and lung. B, the blot used in A
was stripped and hybridized to a [32P]dCTP-labeled
-actin cDNA probe as a control for loading. Note that in
skeletal and cardiac muscle, both the expected 1.6-1.8- and 2.0-kb
signals are seen after hybridization with this probe. C,
in situ hybridization of mouse heart with a
35S-UTP-labeled 801-bp murine Kcnk3 cDNA
fragment demonstrates Kcnk3 message in murine ventricular
tissue. D, anti-KCNK3 antibodies and Western blot analysis
show that Kcnk3 protein is prominent in murine ventricular tissue.
Triton X-100 extracted ventricle or atrium homogenate (100 µg) was
loaded in each lane of a 10% SDS-polyacrylamide gel and probed with
anti-KCNK3 antibody (APC024). Lanes 1 and 3,
ventricular cell lysates; lanes 2 and 4, atrial
cell lysates; lanes 3 and 4 show loss of the
specific signal upon co-incubation of control antigen peptide with
KCNK3 antibody (1 µg peptide/1 µg antibody) prior to probing.
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Fig. 3.
Kcnk3 channels are openly rectifying and
potassium-selective. Kcnk3 whole cell currents were assessed in
oocytes by two-electrode voltage clamp with pulses from a holding
voltage of 80 mV with 50 ms steps from 120 to 45 mV in 15 mV
increments, followed by a 20 ms step to 120 mV. A 1-s interpulse
interval was used. A, Kcnk3 currents at 5, 20, and 100 mM external potassium solutions at pH 7.4. Scale
bars represent 2 µA and 20 ms. Arrows indicate the
zero current level. B, steady state current-voltage
relations in 5 (closed circle), 20 (open
triangle), 50 (closed triangle), and 100 mM
(open box) potassium solution for a single oocyte. The
solid lines are drawn according to Equation 2. C,
the reversal potential of Kcnk3 currents was determined with 5, 20, 50, and 100 mM potassium solutions (mean ± S.E.,
n = 6). Linear regression gave a shift of 56 ± 3 mV/10-fold change in potassium concentration. D, Kcnk3
steady state current-voltage relationships for groups of oocytes
studied with 100 mM rubidium, potassium (filled
circle), rubidium (filled square), cesium (open
circle), or lithium (open square) in the external
solution. The conclusion that permeability assessments were not
significantly altered by native conductances was supported by the
magnitude of Kcnk3 currents in cells expressing the channel and that
absence of less selective pathways (C).
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Fig. 4.
Kcnk3 channels are inhibited by protons at
physiological pH. Kcnk3 currents were studied in whole cell mode
at the indicated pH and potassium solutions. Oocyte membrane potential
was held at 80 mV and pulsed from 120 to +45 mV in 15 mV voltage
steps for 50 ms, followed by a 20 ms step to 120 mV. A 1-s interpulse
interval was used. A, representative oocyte expressing Kcnk3
exposed to varying pH with 5 mM potassium bath solution.
Scale bars represent 1 µA and 20 ms. B,
dependence of Kcnk3 currents at 0 mV on bath pH (mean ± S.E.) for
groups of five cells in 5 mM potassium solution normalized
to pH 8.0. The solid line represents a fit of the data to
Equation 3. A pKa of 7.24 ± 0.03 and a Hill
coefficient of 1.02 ± 0.06 were obtained. C,
inhibition of Kcnk3 current by protons was
potassium-dependent. Steady state current-voltage
relationships for pH 8.0 (filled circle) and pH 7.0 (open circle) at indicated levels of bath potassium (in
mM).
80 to 60 mV at
physiological levels of pH (7.4) and potassium (5 mM) (Fig.
5A). Both raising external
potassium from 5 to 100 mM (Fig. 5B, left
panel) and decreasing protons from pH 7.0 to 8.0 (Fig.
5A, right panel) decreased the fraction of
current that was time-dependent
(ITD/I). Similarly, 55% of
deactivating current was judged to be time-dependent with a
step from 60 to 120 mV at pH 7.4 and 5 mM potassium (Fig.
5C), and raising external potassium (Fig. 5D,
left panel) and lowering proton level (Fig. 5D,
right panel) decreased the fraction of current that was
time-dependent. ITD/I
reflects the fraction of channels closed at rest; thus, higher
potassium and lower proton in the bath increased the fraction of
channels that were open before the test pulse. At physiological resting
potentials and ionic conditions, roughly half the Kcnk3 channels that
passed current upon depolarization were already open. The effects of
external potassium were consistent with the theory that increased
occupancy of the external pore by potassium (either by raising bath
levels or decreasing proton block) favored the open channel state.
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Fig. 5.
The fraction of time-dependent
Kcnk3 current is potassium and pH-dependent.
A, activation. Kcnk3 current traces measured in whole cell
mode in 5 and 100 mM potassium solution, pH 7.4. Oocyte
membrane potential was held at 80 mV and pulsed to 60 mV for 50 ms.
Currents are normalized to peak (8-10 µA).
ITD and INTD represent
the time-dependent and non-time-dependent
components of the current. Steady state and initial currents (see
dashed line) were estimated using a single exponential fit
of the data. B, activation. The fraction of
time-dependent current assessed as in A with 5, 20, and 100 mM potassium and pH 7.4 or 5 mM
potassium and pH 7.0 or 8.0. Mean ± S.E. for 3-6 cells.
C, deactivation. Kcnk3 current traces in whole cell mode in
5 and 100 mM potassium solution, pH 7.4. Oocyte membrane
potential was held at 60 mV and pulsed to 120 mV for 50 ms. Currents
are normalized to peak (2 to 13 µA). ITD
and INTD and fits are as in A. D, deactivation. The fraction of time-dependent
current assessed as in C with 5, 20, and 100 mM
potassium and pH 7.4 or 5 mM potassium and pH 7.0 or 8.0. Mean ± S.E. for 3-6 cells.
150 to 60 mV; Fig. 6A, middle
panel) or test pulse (from 0 to 60 mV; Fig. 6A,
right panel), indicating opening of channels by
depolarization. Deactivation showed a similar dependence on voltage;
activation at positive potentials (from 0 to 60 mV; Fig. 6B,
middle panel) opened more channels, increasing the fraction
of current that decayed upon subsequent hyperpolarization, and more
positive deactivation potential (from -150 to -60 mV; Fig.
6B, right panel) decreased
ITD/I, presumably by increasing the
number of channels that stayed open.
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Fig. 6.
Activation and deactivation of Kcnk3 channels
are weakly voltage-dependent. A,
activation. Left panel, Kcnk3 current traces in on-cell
patches pulsed for 50 ms from 80 mV to 60 mV (followed by a step to
120 mV) with 5 mM potassium, pH 7.4 in the pipette.
ITD and INTD represent
the time-dependent and non-time-dependent
components of the current. The dotted line indicates zero
current. Scale bars represent 100 pA and 10 ms. Middle
panel, holding voltage alters the fraction of
time-dependent Kcnk3 current measured at 40 mV for groups
of 5 cells (mean ± S.E.). Steady state and initial currents were
estimated using a single exponential fit of the data. Right
panel, activation voltage alters the fraction of
time-dependent Kcnk3 current for groups of 5 cells
(mean ± S.E.). The open symbols are the ratios by the
same protocol in whole cell mode. B, the command voltage
alters the time course of activation. The time constant () for
activation at various voltages for groups of five cells studied as in
A and fit with a single exponential of the form a + b*exp
(x/). C, deactivation. Left panel, Kcnk3
current traces in on-cell patches pulsed for 50 ms from a holding
potential of 60 mV to 120 mV with 100 mM potassium, pH
7.4, in the pipette. Otherwise conditions are as in A. Scale bars represent 100 pA and 20 ms. Middle
panel, prior activation voltage alters the fraction of
time-dependent Kcnk3 current at 120 mV for groups of 5 cells (mean ± S.E.). Steady state and initial currents were
estimated using a single exponential fit of the data. Right
panel, deactivation voltage alters the fraction of
time-dependent Kcnk3 current for groups of 5 cells
(mean ± S.E.). The open symbols are the ratios by the
same protocol in whole cell mode. D, the command voltage
alters the time course of deactivation. Time constant () for
deactivation at various voltages for groups of five cells were studied
as in the right panel in C and fit with a single
exponential as in B. E, deactivation. The time
course of recovery from prior activation was evaluated in on-cell mode
by increasing the time between two successive 50 ms activating test
pulses to 40 mV from 80 mV with 5 mM potassium, pH 7.4, solution in the pipette; the two pulses were separated by intervals of
2-100 ms, and traces are superimposed. Scale bars represent
100 pA and 20 ms.
)
for the rise in the current (with a step from 80 to 45 mV) was
4.4 ± 0.5 ms and showed an e-fold change per ~250 mV
over this voltage range (Fig. 6B). This rate of was largely
insensitive to both external potassium and pH. Thus, with 20 mM potassium at pH 7.4 was 4.1 ± 0.2 ms
(n = 3), whereas it was 4.4 ± 0.4 and 4.3 ± 0.6 ms at 5 mM potassium at pH 8.0 (n = 4)
and pH 7.0 (n = 4), respectively.
120 mV and changed e-fold per
~500 mV over this range of potentials (Fig. 6D). Another
measure of deactivation rate was the time required at 80 mV to
recover an initial ratio of immediate to time-dependent
current as measured by activation (Fig. 6E). The recovery
time constant at 80 mV was 5.7 ± 0.3 ms (n = 3), similar to the deactivation rate estimated directly at 80 mV, 5.5 ± 0.3 ms (n = 3).
80 to 45 mV) was less than
0.25 ms (Fig. 6A), consistent with the idea that these
channels were open before test pulse.
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Fig. 7.
Single Kcnk3 channel conductance. Single
channel currents were recorded in an on-cell patches with 100 mM potassium solution in the pipette. A,
representative single Kcnk3 channel at 120 mV. The solid
line indicates zero current, and the dotted line
indicates major open channel level. The scale bar represents
1 pA and 50 ms. Data were filtered at 2 kHz and sampled at 10 kHz.
B, left panel, the unitary current-voltage
relationship estimates a conductance of 11 ± 1 pS. Middle
panel, single channel data analyzed off-line allowed construction
of histograms using a 50% crossing method (21); ~70% of openings
were to a mean value of 1.2 pA, the other was to 1.6 pA, corresponding
to 10 and 13 pS. Right panel, representative open and closed
time histogram for single Kcnk3 channels at 120 mV in 100 mM potassium solution; the histograms represent 20 s
of data from six single channel patches (20,000 events) evaluated as in
A with maximum-likelihood fits to the data. C,
left panel, single channel conductance was estimated by
noise variance analysis in four patches pulsed 300 times to 80 mV for
15 ms from a holding voltage of 80 mV. Current variance and mean were
calculated. For a channel with low open probability the slope of the
current variance-mean relationship gives a valid approximation of
unitary current. Right panel, current-voltage relationship
for variance analyzed at different voltages offered an estimate of
single channel conductance of 10 ± 1 pS.
80 mV, close to the
equilibrium reversal potential for potassium (Fig. 8A,
middle panel). Lowering pH from 7.4 to 6.0 at 5 mM potassium completely inhibited single-channel activity
(Fig. 8A, right panel) in a reversible manner
(not shown).
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Fig. 8.
Single Kcnk3 channel first latency and open
probability. Single Kcnk3 channels were studied in outside-out
patches with 100 mM potassium solution in the pipette.
A, a patch with multiple Kcnk3 channels at various bath
conditions. Left panel, 100 mM potassium at pH
7.4. Middle panel, 5 mM potassium at pH 7.4. Right panel, 5 mM potassium at pH 6.0. Scale bars represent 2 pA and 100 ms. B,
representative single channel patch; traces elicited by depolarization
from 40 to 40 mV for 50 ms, followed by a 50-ms step to 120 mV
prior to returning to the holding potential with 20 mM
potassium, pH 7.4, in the bath. The driving force was similar for
inward and outward potassium currents at these test potentials.
Scale bars represent 1 pA and 50 ms. C, an
ensemble average of 300 pulses as in B. In 75% of traces no
openings or closings were observed (as in B, trace
5).
40 mV in this case) and repeatedly stepping to +40 and then 120
mV, test voltages with equal but opposite driving force for potassium.
Most traces were null with no openings or closings observed (Fig.
8B, trace 5); others showed evidence for brief openings with depolarization (Fig. 8B, traces 1 and 3) or hyperpolarization (Fig. 8B,
traces 2 and 4). The ratio of openings to the two
conductance levels was similar to that observed in steady state
recordings (~7:3, not shown).
120 mV with approximately symmetric 100 mM potassium (Fig. 7A) open probability was
~0.04 ± 0.01 at steady state. Dwell time for openings were well
approximated by a single time constant of 0.3 ± 0.1 ms,
suggesting that open times for the two conductance levels were similar
(Fig. 7B, right panel). Closed time distributions
were best fit by two time constants representing closed states with
mean dwell times of ~5 and 65 ms, with relative frequencies of 19 and
81%, respectively (Fig. 7B, right panel).
over 30 min (Table I). Bisindolylmaleimide I (5 µM), an inhibitor
of protein kinase C, slowly increased currents ~ over 30 min and suppressed PMA-induced down-regulation when the agents were
applied together (Table I). Activation of protein kinase A by 20 µM forskolin and 1 mM IBMX decreased current by ~40%, although PKA inhibitor H-89 at a standard dose (5 µM) had no effect (Table I). Small to moderate decreases
in Kcnk3 currents were also observed with manipulations known to
activate KATP channels (3 mM sodium azide)
(39), Kcnk4 channels (100 µM arachadonic acid) (17), and
a native leak channel (decreased oxygen tension achieved by purging the
bath solution with nitrogen) (7) (Table I). Minimal changes in current
were observed when bath osmotic and ionic strength were increased, bath
calcium and/or magnesium were increased or removed, calcium was
eliminated and 5 mM EGTA added, ultra-pure potassium was
employed as a sole source of external monovalent cations, or the
volatile anesthetics halothane or chloroform were applied at
supra-therapeutic levels (Table I). These manipulations did not
significantly alter the fraction of time-dependent current
at 60 or 120 mV (not shown).
Effects of various agents on Kenk3 current magnitude
80 to 30 mV for 50 ms with interpulse interval of
2 or 30 s (PMA, bisindolylmaleimide, forskolin, H-89, azide, and
hypoxia); all effects were reversible except for those of arachidonic
acid. z was calculated by Equation. The efficacy of the H-89
solution was confirmed by its effects on KCNKØ channels (not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
< 0.25 ms) and
time-dependent ( = ~5 ms) changes in current
magnitude. Observation of immediate currents supports the conclusion
that Kcnk3 channels open at all voltages. The fraction of open channels
is sensitive to external potassium and voltage (Figs. 5 and 6) and does
not appear to result from release from block. Thus, current magnitudes
were not significantly altered by either elevation or removal of bath
calcium and/or magnesium, addition of EGTA, or formulation of solutions
with 100 mM ultra-pure salts (Table I).
ACKNOWLEDGEMENTS
FOOTNOTES
Supported in-part by grants from the National Institutes of
Health, the Yale University Children's Health Research Center, and the
March of Dimes Birth Defects Foundation.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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