|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online January 31, 2006
Journal of Experimental Biology 209, 731-747 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02032
A potassium channel (Kv4) cloned from the heart of the tunicate Ciona intestinalis and its modulation by a KChIP subunit
1 Department of Physiology, University of Alberta, Edmonton, AB, T6G 2H7,
Canada
2 Bamfield Marine Sciences Centre, Bamfield, BC, V0R 1B0, Canada
3 Department of Biological Sciences, University of Alberta, Edmonton, AB,
T6G 2E9, Canada
4 Department of Biology, Utah State University, Utah, 84322 USA
* Author for correspondence (e-mail: andy.spencer{at}shaw.ca)
Accepted 10 December 2005
 |
Summary |
|---|
 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Key words: Kv4, KChIP, potassium channel, Ciona intestinalis, tunicate, heart
|
Introduction |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
; Kv4.2:
Roberds and Tamkun, 1991; and
Kv4.3: Serôdio et al.,
1994). Kv4 channels mediate transient outward currents in cardiac
myocytes (Dixon et al., 1996),
smooth muscle (Amberg et al.,
2002) and neurons
(Serôdio et al., 1994).
The currents mediated by Kv4 channels play important roles in specifying the
firing properties of molluscan and lobster neurons
(Connor and Stevens, 1971;
Baro et al., 1996), suggesting
that Kv4 channels are conserved determinants of cellular excitability. The
three mammalian Kv4 channel isoforms differ in their biophysical properties.
For example, the fast component of inactivation of Kv4.1 channels contributes
significantly less to inactivation than the fast inactivating component of
Kv4.2 and Kv4.3 channels (Pak et al.,
1991; Jerng and Covarrubias,
1997). The full significance of these differences in properties
between the three Kv4 channel isoforms is not yet understood. However, since
expression of Kv4.2 and Kv4.3 isoforms is complementary in brain and heart
(Serôdio et al., 1996),
it seems likely that the specific properties of each of these isoforms
contribute to determining the function of the particular tissue area where it
is expressed. These differences between the three Kv4 channel isoforms likely
represent adaptive features of these channels. To identify adaptive and
conserved features of mammalian Kv4 channels it is necessary to compare these
with invertebrate Kv4 channels such as jellyfish Shal
(Jegla and Salkoff, 1997),
arthropod Shal (Baro et al.,
1996) and a tunicate Shal
(Nakajo et al., 2003).
KChIPs, a family of Ca2+ binding proteins
(Burgoyne and Weiss, 2001),
are integral components of Kv4 channel supra-molecular complexes
(An et al., 2000). KChIP
isoforms 1-3, but not KChIP4, increase the density of Kv4 currents
(Shibata et al., 2003). All
four KChIP isoforms modify the kinetics and gating properties of Kv4 channels
(An et al., 2000;
Holmqvist et al., 2002).
Modulation of Kv4 channels by KChIP subunits is likely a conserved mechanism
to modulate tissue excitability, because the effects of human KChIP1 on the
lobster and mammalian Kv4 channels are similar
(Zhang et al., 2003). Thus,
KChIP1 increased current amplitude, slowed the rate of inactivation, and
shifted activation and inactivation midpoints of lobster Kv4 channels
(Zhang et al., 2003). In the
present study, we address the question of how tunicate Shal is modulated by
endogenous KChIP subunits.
The vertebrate multi-gene Kv4 and KChIP families are
represented by single genes in the tunicate genome. Scaffolds 168 and 457 of
the genome database for Ciona intestinalis
(http://genome.jgi-psf.org/ciona4/ciona4.home.html,
release version 1.0) contain the Kv4 and the KChIP gene,
respectively. We cloned the transcripts for a Kv4 channel (CionaKv4)
and a KChIP subunit (CionaKChIP) from myocardial tissue of Ciona
intestinalis. Repolarization of the action potential in the tunicate
heart is complex with several repolarization phases, which are presumably an
adaptation for pumping blood by means of peristaltic contractions that can
propagate in both directions through the tubular heart
(Kriebel, 1967). Since Kv4
channels play a crucial role in determining the excitability properties of
cardiac tissues of vertebrates, we suspected that these channels were playing
a similar role in the tunicate heart.
We characterized the potassium currents produced by CionaKv4
channels heterologously expressed in Xenopus oocytes, in the presence
and absence of CionaKChIP. Our biophysical data for CionaKv4
reinforces and complements what is known for tunicate Shal
(Nakajo et al., 2003). Because
the N terminus of Kv4 channels appears to be essential for modulation by KChIP
(Bähring et al., 2001b),
an N-terminal deletion mutant of CionaKv4, lacking amino acids 2-32,
was constructed (ntCionaKv4), and the effects of CionaKChIP
on this mutant CionaKv4 channel were evaluated. In this paper we
describe the modulation of kinetic and gating parameters of a tunicate Shal
channel (CionaKv4) by a tunicate KChIP subunit (CionaKChIP),
the first KChIP subunit cloned from either an invertebrate or a non-vertebrate
chordate. Our data and those provided by Zhang et al.
(2003) on modulation of
lobster Shal by KChIP1, are the first indications that modulation of Kv4
channels by KChIP subunits might be an ancient mechanism to modulate the
excitability of electrically active tissues.
|
Materials and methods |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Rapid amplification of cDNA 3'end (3'-RACE) for Kv4 and KChIP homologues
The GeneRacer Kit (Invitrogen, Carlsbad, CA, USA) was used to perform a
3'-RACE assay. Briefly, cDNA was synthesized from total RNA using an
oligo-dT primer, with a unique sequence at the 3' end, provided with the
kit. Each 50 µl 3'-RACE assay contained: 1x Opti-Prime Buffer
(Stratagene, La Jolla, CA, USA) (10 mmol l-1 Tris-HCl, 1.5 mmol
l-1 MgCl2,25 mmol l-1 KCl, pH 8.3), 0.5 unit
of Taq polymerase, 0.25 mmol l-1 of each dNTP, 1 µmol
l-1 each of forward primer and reverse primer and 2 µl cDNA. The
sense primers (5'-GACGGATCTACAGCCAAAATCAAAGACA-3' for Kv4 and
5'-CAGTTGTTGGGATCGCATGT-3' for KChIP) were bound to an internal
sequence of the Kv channel or the KChIP subunit transcript;
the anti-sense primer, supplied with the kit, bound to the specific sequence
of the oligo-dT primer. The temperature conditions were: 30 s at 94°C,
followed by 5x (30 s at 94°C, 30 s at 65°C, 3 min at 72°C),
25x (30 s at 94°C, 30 s at 60°C, 3 min at 72°C) followed by
5 min at 72°C. 3'-RACE products were sequenced with the forward
primers used in these assays. Next, new forward primers were designed to bind
downstream within these sequences in nested 3'-RACE assays. Successive
nested RACE assays were done until the first 3'-RACE assay product was
fully sequenced.
Cloning of full-length cDNA of CionaKv4 and CionaKChIP
The full-length open reading frames for CionaKv4 and
CionaKChIP were amplified with sense primers containing the start
codon and a Kozak consensus site for the 5' end, and anti-sense primers
designed to bind downstream of the translational termination codon
(CionaKv4-sense:
5'-GGCTCGAGGCCGCCACCATGGCAACAGCAGTAGC-3';
CionaKv4-antisense:
5'-GGCCTAGGCAAAGTCCCGCCGCTACAGTGAG-3'; CionaKChIP-sense:
5'-GGCTCGAGGCCGCCACCATGTCTCTCGCTATCTTAACCATGGTGAC-3';
CionaKChIP-antisense:
5'-GGACTAGTAACGCCAGAAACGCCGCCTTGATGGAGCTATAACGC-3'). Primers also
contained restriction sites to allow directional insertion of the PCR products
into pXT7 (Dominguez et al.,
1995). Each 50 µl PCR reaction contained: 1x Opti-Prime
Buffer (Stratagene, La Jolla, CA, USA; 10 mmol l-1 Tris-HCl, 1.5
mmol l-1 MgCl2, 25 mmol l-1 KCl, pH 8.3), 0.5
unit of Taq polymerase, 1 unit of Pfu, 0.2 mmol l-1 of each dNTP, 1
µmol l-1 each of forward primer and reverse primer, and 2 µl
cDNA. Temperature conditions were: 30 s at 94°C, 25x (30 s at
94°C, 30 s at 60°C, 3 min at 72°C) followed by 5 min at 72°C.
The resulting PCR products were then digested with appropriate restriction
enzymes, ligated with appropriately digested pXT7 plasmid and transformed into
E. coli. Clones were sequenced to verify that no mutation had been
introduced by PCR. This construct was linearized with SalI and cRNA
was synthesized using the T7 mMessage mMachine kit (Ambion, Austin, TX,
USA).
Construction of the N-truncation mutant (ntCionaKv4) of CionaKv4
To generate a mutant channel lacking amino acids 2-32 of the N terminus
domain, a diluted sample of the CionaKv4 clone was amplified by PCR.
The composition of the PCR reactions and the temperature conditions were as
described in the previous section. The forward primer
(5'-GGCTCGAGGCCGCCACCATGAACCGACGTAAAACAAAAGAC-3') was designed to
bind to the CionaKv4 clone, starting from nucleotide 97. A start
codon, Kozak consensus sequence and an XhoI site were added to this
sequence. The reverse primer was the same as used for the full-length clone.
These PCR products were inserted into pXT7 and transformed into E.
coli. Clones were sequenced to verify the deletion and to check that the
undeleted sequence had no mutations introduced by PCR.
Alignment and phylogenetic tree
Amino acid sequences of a set of phylogenetically representative Kv4
channels (18) and KChIP subunits (17) were aligned with T-Coffee software
(Notredame et al., 2000). Kv4
channel regions that contained extensive gaps were edited out, leaving a data
matrix that included the T1 domain and the membrane-spanning core
(trans-membrane domains S1 to S6), giving a total of 405 characters. MrBayes
v3.0b (Huelsenbeck and Ronquist,
2001) was used to determine the phylogenetic relationships of the
channel sequences. The default parameters for protein sequences were used. A
total of 200 000 cycles were run; the topology and branch length of every
100th tree was saved. The Bayesian maximum likelihood tree was obtained by
taking a consensus of the collected trees after a 101 tree burn-in. The nodes
on the tree are labelled with the posterior probability for each node; nodes
with a probability of one were left unlabelled. The tree was visualized using
Treeview (Page, 1996).
Oocyte preparation
Oocytes from Xenopus laevis were surgically removed and
dissociated using 2 mg ml-1 collagenase 1A (Sigma Aldrich, St
Louis, MO, USA) in a solution containing (in mmol l-1): NaCl (96),
KCl (4), MgCl2 (20) and Hepes (5), pH 7.4. Oocytes were incubated
at 18°C in culture medium containing (in mmol l-1): NaCl (96),
KCl (2), MgCl2 (1), CaCl2 (1.8), Hepes (5), sodium
pyruvate (2.5), pH 7.4 with 100 mg l-1 gentamycin and 3% horse
serum (Gibco BRL, Carlsbad, CA, USA). 24 or 48 h following oocyte isolation,
40 nl of CionaKv4 cRNA (
16 ng) or ntCionaKv4 (8
ng) were injected into each Xenopus oocyte. For co-expression
experiments, 20 nl (8 ng) of CionaKv4 or 20 nl (4 ng) of
ntCionaKv4 cRNA solution was mixed with 20 nl (7 ng) of
CionaKChIP cRNA solution, to give a final volume of 40 nl that was
injected into each oocyte. The approximate molar ratio was [1
CionaKv4:3 CionaKChIP] or [1 ntCionaKv4:6
CionaKChIP]. Immediately prior to each experiment the vitelline
membrane was manually removed with forceps after treatment in a hyperosmotic
solution containing (in mmol l-1): NaCl (96), KCl2 (2),
MgCl2 (20), Hepes (5) and mannitol (400), pH 7.4.
Voltage-clamp recordings
All recordings were made from cell-attached macro-patches. The pipettes
contained ND96 solution, with the following composition (in mmol
l-1): NaCl (96), KCl (2), MgCl2 (1), CaCl2
(1.8) and Hepes (5), pH 7.4. During the recordings, oocytes were bathed in a
grounded, isopotential solution, thereby ensuring that the potential applied
by the recording pipette would be the true membrane potential for the
macro-patch. This bath solution contained (in mmol l-1): NaCl
(9.6), KCl (88), EGTA (11), Hepes (5), pH 7.4. Patch pipettes were pulled from
aluminosilicate glass, coated with dental wax, and fire-polished before each
experiment. Only pipettes that had resistances between 0.7 and 1.4 M
were used.
Membrane seals were obtained by applying negative pressure. Voltage-clamp and data acquisition were carried out using an EPC-9 patch-clamp amplifier (HEKA, Lambrecht, Germany) controlled with PULSE software (HEKA) running on a Power MacIntosh G4 computer. Data were acquired at sampling intervals of 50 µs and low-pass filtered at 5 kHz during acquisition. Bath temperature was maintained at 12±0.2°C using a Peltier device controlled by an HCC-100A temperature controller (Dagan, Minneapolis, MN, USA). The holding potential was set at -100 mV and leak subtraction was performed with a P/4 protocol. PulseFit (HEKA), Igor Pro (Wavemetrics, Lake Oswego, OR, USA) and Instat (GraphPad Software, San Diego, CA, USA) software were used for analyses and graphing.
Conductances were calculated using the equation GK=Ipeak/(V-EK), where GK is the potassium conductance, Ipeak is the peak measured amplitude of the K+ current to a test pulse, V is the voltage at which the current was measured, and EK is the measured potassium equilibrium potential, in this case -90 mV. Conductance-voltage relationships and steady-state inactivation curves were fitted in Igor Pro by a sigmoid (Boltzmann) distribution of the form: f(V)=Gmax/{1+exp[(V0.5-V)/K]}, where Gmax is the maximal conductance, V is the voltage of the depolarizing pulse (for activation) or the prepulse voltage (for inactivation), and K is the slope factor. For a first order Boltzmann, used to fit conductance-voltage relationships and steady-state inactivation curves, V0.5 is the voltage at which activation or inactivation is half maximal.
Time constants of activation were obtained from a fit of the rising phase
of the currents to the Hodgkin-Huxley model of the form:
I(t)=Imax[1-exp(-t/
)]n
where Imax is the maximum current obtained in the absence
of inactivation, is the activation time constant, t is time and
n=4. To study the decay of the current as a function of time, the
decaying phases of the outward currents evoked by test pulses between +30 mV
and +80 mV were fitted to the equation:
I(t)=I0+I1exp(-t/1)+I2exp(-t/2),
where 1 and 2 represent the fast and the slow
time constants of inactivation, respectively, I1 and
I2 represent the relative contribution of each component
to inactivation, and I0 is the offset.
|
The voltage dependence of steady-state inactivation was determined by measuring the peak current evoked with a depolarizing pulse to +50 mV as a function of the voltage of a preceding 10 s prepulse test (between -130 and -30 mV). A double-pulse protocol was used to assess the rate of recovery from inactivation at -100 mV. A test pulse to +50 mV of 1 s duration was separated by a recovery period (at -100 mV) of increasing duration (50-2000 ms) from a second test pulse to +50 mV. The currents evoked by the second pulse of a double-pulse protocol were normalized to the currents produced by the first pulse and plotted against the duration of the interpulse interval.
Statistical analyses
Statistical comparisons were carried out using the Student t-
test, or the alternative Welch t-test when there were significant
differences between the standard deviations of the two groups. Data are
presented as the mean ± s.e.m., and N is the number of
macro-patches or oocytes, because recordings from one macro-patch per oocyte
were included in the analyses.
|
Results |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
17.8 kb, of
which 2.4 kb encode the ORF. The first exon (exon 0) encoded the first amino
acids of the N terminus. The second exon (exon 1), 1070 bp in length, coded
most of the channel protein, including the T1 domain, trans-membrane domains
S1-S5, and the first part of the pore domain. The second intron interrupted
the sequence of the K+ selectivity filter motif (GYG), since the
last base pair of the second exon and the first two base pairs of the third
exon encoded the first G of this motif. The second part of the pore region and
the S6 trans-membrane domain were encoded by exon 2. Exons 3-5 encoded parts
of the C-terminal cytoplasmic domain. The last exon (exon 6), encoded the rest
of the C terminus of CionaKv4, and contained the stop codon (TAG) and
the 3'-UTR sequence.
Fig. 2 shows an alignment
between the predicted amino acid sequences of CionaKv4 and selected
vertebrate and invertebrate Kv4 channels. The six trans-membrane domains
(S1-S6), the pore region, and the first 23 amino acids of the N terminus
of all Kv4 channels showed high sequence similarity. The C-terminal motif
PTPP, which is involved in the interaction of Kv4 channels with filamin and is
located 30 amino acids upstream of the C termini of mammalian Kv4
channels (Petrecca et al.,
2000), was not found in CionaKv4, although
CionaKv4 shares the first residue of this motif (P) and the preceding
residue (I) with all mammalian Kv4 channels (residues 651-652 in
CionaKv4, Fig. 2). The
conserved di-leucine motif, consisting of 16 amino acids that span positions
474-489 and is involved in the targetting of membrane proteins
(Rivera et al., 2003), was
also found in CionaKv4 (Fig.
2). The C-terminal domain of CionaKv4 and the N terminus
contained, respectively, two and one putative sites for phosphorylation by
cAMP-dependent protein kinase A (PKA). Additionally, the N terminus of
CionaKv4 contained four sites for phosphorylation by protein kinase C
(PKC); another four putative sites for PKC phosphorylation were found in the C
terminus; and another such site was found in the intracellular loop between
membrane-spanning domains S4 and S5.
|
7 kb of genomic DNA, of which the
ORF occupied 0.7 kb, distributed in seven exons with sizes between 69 and
162 bp (Fig. 1D).
Fig. 3 shows an alignment
between the deduced amino acid sequences of CionaKChIP and
representatives of the four major isoforms of mammalian KChIPs (KChIP1-4). As
with other KChIPs, the sequence of the N terminus of CionaKChIP was
the most variable region of the protein (An
et al., 2000) whereas the sequence corresponding to the C terminus
was conserved, containing four EF-hand Ca2+-binding motifs
(Kretsinger, 1987). The N
terminus of CionaKChIP was encoded by exons 1 and 2. The coding
region for the first EF-hand was partitioned between exons 2 and 3. Similarly,
the codons for the second and the third EF-hands were partitioned between
exons 4 and 5 (second EF-hand), and between exons 5 and 6 (third EF-hand). The
fourth EF-hand was encoded by exon 6 and the last section of the C-terminus
was coded by exon 7. A Prosite scan of CionaKChIP suggested the
location of six putative sites for PKC phosphorylation but no sites for PKA
phosphorylation (Fig. 3).
|
) formed a single clade, as would be expected. This
tunicate clade is the sister group to the clade containing the Kv4.1, Kv4.2
and Kv4.3 paralogues from vertebrates, indicating that the duplication and
divergence that gave rise to the three vertebrate Kv4 channels occurred after
the divergence of the vertebrate lineage from the tunicate lineage. In the
phylogenetic tree shown in Fig.
4B, the single CionaKChIP branched basal to the four
mammalian KChIP isoforms. This tree indicated that there is a 99% likelihood
that the KChIP1 isoform is the sister group of the other three isoforms.
However, the phylogenetic relationships between the KChIP isoforms 2, 3 and 4
were unclear, with posterior probabilities of approximately 50-60%. There was
strong support for the presence of all four isoforms in the common ancestor of
modern fish (represented by zebrafish) and the tetrapods (represented by
human, mouse, rat and chicken). All of the vertebrate KChIP sequences
partitioned robustly into one of the four paralogous families, indicating that
the paralogues all originated prior to the origin of the vertebrate clade.
|
|
|
In all cases, with or without N-terminal truncation and with or without co-expression of KChIP, current levels reached a plateau at stimulation voltages above +50 mV, indicating that conductance decreases above this voltage. The mechanism for this decrease in conductance is unknown. We speculate that the outward current is inhibited at higher depolarization voltages by blocking of the internal face of the ion pore by a positively charged component of the cytoplasm. If the block is similar to Mg2+ or polyamine blocking of Kir channels then the affinity of the cytoplasmic blocker must be lower than that seen for polyamine block of Kir channels since blockage of CionaKv4 does not appear until + 50 mV.
The average time constant of activation of currents produced by
CionaKv4 channels during a pulse to +50 mV (3.4±0.2 ms) was
not affected by the presence of CionaKChIP (3.3±0.2 ms), as
shown in Fig. 6Bi
(P>0.05, Student t-test). The voltage-dependence of the
time constant for activation was similar for channels expressed alone or with
CionaKChIP, increasing by e-fold every 29 or 33 mV, respectively.
Deletion of amino acids 2-32 from CionaKv4 channels was also without
effect on the rate of activation, since the average time constant of
activation of currents produced by this N-terminally deleted CionaKv4
channel was 3.2±0.3 ms (P>0.05, Student t-test).
The voltage-dependence of activation kinetics was similar for
CionaKv4 and ntCionaKv4 channels, increasing by a factor of
e for every 26 or 29 mV of voltage change, respectively
(Fig. 6Bii). Activation
kinetics of ntCionaKv4 in the presence of CionaKChIP was
3.5±0.3 ms, which was not significantly different from the value for
ntCionaKv4 alone (P>0.05, Student t-test).
Interestingly, the voltage-dependence of activation kinetics was different for
ntCionaKv4 channels alone (e-fold increase every 26 mV) than when
co-expressed with CionaKChIP (e-fold increase every 37 mV),
which suggests that CionaKChIP slightly decreased the voltage
sensitivity of activation kinetics of ntCionaKv4 channels
(Fig. 6Biii).
CionaKChIP enhanced activation of CionaKv4 by decreasing the voltage necessary to activate half the channels (Fig. 6Ci), since first order Boltzmann fits to the relationships between normalized peak conductance vs voltage had midpoints (V0.5) of -0.5±1.7 mV for CionaKv4 and -29.2±1.1 mV for CionaKv4/KChIP (P<0.0001, Student t-test). The slope values of the Boltzmann curves were 15.5±0.8 mV/e-fold (CionaKv4) and 13.4±0.3 mV/e-fold (CionaKv4/KChIP) (P<0.05, Student t-test).
Deletion of the N terminus of CionaKv4 also shifted the midpoint of activation of CionaKv4 in the hyperpolarizing direction (Fig. 6Cii). First order Boltzmann fits to normalized peak conductance-voltage relationships had significantly different midpoints of activation (V0.5) of -0.5±1.7 mV for
CionaKv4 and -12.3±1.6 mV for ntCionaKv4 (P<0.0001, Student t-test). The slopes for these curves were 15.5±0.8 mV/e-fold and 14.3±0.4 mV/e-fold, respectively (P>0.05, Welch t-test).
Deletion of the N terminus eliminated the effects of CionaKChIP on activation midpoint (Fig. 6Ciii). The V0.5 of first order Boltzmann fits to the conductance-voltage relationships were -15.2±1.3 mV for ntCionaKv4 in the presence of CionaKChIP (-12.3±1.6 mV for ntCionaKv4 alone, P>0.05, Student t-test). The slope factors of these fits were 14.3±0.4 mV/e-fold for ntCionaKv4 channels expressed alone, and 13.4±0.2 mV/e-fold for ntCionaKv4 channels co-expressed with CionaKChIP subunits (P>0.05, Student t-test).
All the biophysical parameters determined in this study are summarized in Table 1.
|
Inactivation and recovery from inactivation properties of CionaKv4 and an N-deletion mutant of CionaKv4 (ntCionaKv4) in the presence/absence of CionaKChIP
The decaying phase of CionaKv4 currents obtained with test pulses
between +20 mV and +80 mV was well fitted by a double exponential function
with fast (1) and slow (2) time constants of
inactivation. Currents produced by CionaKv4/KChIP complexes
inactivated more slowly than currents produced by CionaKv4 channels
(Fig. 5B). The decay phases of
the currents produced by CionaKv4/KChIP complexes were well fitted
with single exponential functions, whose single inactivation time constants
were larger than the slow inactivation time constants of currents produced by
CionaKv4 channels (Fig.
7Ai and Table 1).
For instance, the double exponential fit to the decay of currents produced by
CionaKv4 in response to a pulse to +50 mV had a fast time constant of
26±2 ms, which contributed to 82% of total current decay, and a
slow time constant of 147±26 ms, which contributed to 18% of the
current decay. However, a single exponential fit to the inactivation phase of
currents conducted by CionaKv4/KChIP complexes at the same potential
had a time constant of 291±42 ms.
|
The decaying phase of currents produced by N-terminally deleted CionaKv4 channels was also well fitted by a double exponential function, but the inactivation time constants were slower than for wild-type channels (Fig. 7Aii) and they contributed equally to total inactivation (Table 1). For instance, the decay phase of ntCionaKv4 currents during a test pulse to +50 mV was best described by a double exponential function with a fast time constant of 56±6 ms and a slow time constant of 381±50 ms. Interestingly, co-expression of ntCionaKv4 channels with CionaKChIP subunits slowed inactivation kinetics relative to those for ntCionaKv4 channels alone (Fig. 5D). The decaying phases of these currents were best fitted with single exponential functions, whose time constant was similar to that from untrucated CionaKv4 channels expressed with CionaKChIP (Fig. 7Aiii and Table 1). These results demonstrate that CionaKChIP still forms a functional complex with CionaKv4 channels that lack the N terminus.
Since Kv4 channels inactivate mostly from the closed state (Bahring et al., 2001a), we hypothesized that CionaKChIP slowed inactivation kinetics of CionaKv4 channels by interfering with channel closure. To address this question, we measured and compared the kinetics of tail currents produced by CionaKv4 channels and CionaKv4/KChIP complexes in response to re-polarizing pulses from -160 to -110 mVdelivered after a brief (10-15 ms) depolarizing pulse to +50 mV, which would activate a maximum number of channels. Our results suggest that channel closing of CionaKv4 channels is indeed inhibited by CionaKChIP subunits (Fig. 7Bi). For example, during a re-polarizing pulse to -130 mV, CionaKv4 tail currents deactivated with a time constant of 4.1±0.3 ms, whereas CionaKv4/KChIP tail currents deactivated with a time constant of 8.3±0.4 ms. These values were significantly different (P<0.0001, Welch t-test). Deletion of the N terminus of CionaKv4 also slowed closure kinetics (Fig. 7Bii), indicating that this domain of CionaKv4 has a role in accelerating channel closure. For example, during a pulse to -130 mV, currents produced by ntCionaKv4 deactivated with a time constant of 5.7±0.6 ms, which was significantly slower (P<0.05, Welch t-test) than for wild-type channels (4.1±0.3 ms). The N terminus of CionaKv4 seems to be required for modulation of closing kinetics by CionaKChIP because deactivation time constants of tail currents produced by ntCionaKv4 channels and ntCionaKv4/KChIP complexes were not significantly different (Fig. 7Biii). For example, at the re-polarizing voltage of -130 mV, the deactivation time constant for ntCionaKv4 in the presence of CionaKChIP was 6.5±0.6 ms, which was not significantly different (P>0.05, Student t-test) from the average deactivation time constant for ntCionaKv4 channels expressed alone (5.7±0.6 ms).
Addition of CionaKChIP shifted the midpoint of steady-state inactivation of CionaKv4 to the left (Fig. 7Ci). Midpoints of single order Boltzmann fits to steady-state inactivation curves of CionaKv4 and CionaKv4/KChIP were -72±2 mV for CionaKv4 and -88±2 mV for CionaKv4/KChIP (P<0.001, Student t-test). The slope factors of these Boltzmann fits to steady-state inactivation were also significantly different: 3.6±0.2 mV/e-fold for CionaKv4 and 4.3±0.2 mV/e-fold for CionaKv4/KChIP (P<0.001, Student t-test). The N terminus of CionaKv4 does not appear to contribute to steady state inactivation (Fig. 7Cii) since midpoints of inactivation of N-terminally deleted channels (-73±2 mV) were not significantly different (P>0.05, Student t-test) from inactivation midpoints of wild-type channels (-72±2 mV). The slopes of the steady-state inactivation curves were not significantly different (P>0.05, Student t-test): 3.6±0.2 mV/e-fold for CionaKv4 and 3.7±0.2 mV/e-fold for ntCionaKv4. However, the effect of CionaKChIP on steady-state inactivation parameters was dependent on the N terminus of CionaKv4, since deletion of this domain abolished these effects (Fig. 7Ciii). Midpoints of inactivation were -73±2 mV for ntCionaKv4 and -74±2 mV for ntCionaKv4/KChIP. Slope factors were 3.7±0.2 for ntCionaKv4 and 4.1±0.3 for ntCionaKv4/KChIP (P>0.05, Student t-test, for both midpoint and slope values).
The rate of recovery from inactivation of CionaKv4 channels at
-100 mV was also affected by CionaKChIP
(Fig. 7Di). Recovery from
inactivation was measured as the fractional recovery of current as a function
of time at -100 mV. This relationship was well fitted by a single exponential
function with recovery time constants from inactivation (rec)
of 387.4±46.6 ms for CionaKv4 and 330.6±49.5 ms for
ntCionaKv4 (P>0.05, Student t-test), indicating
that the N-terminal peptide sequence alone is not a significant factor in
recovery (Fig. 7Dii). The
recovery of unmutated CionaKv4 channels was significantly slowed by
the presence of CionaKChIP, with a time constant of recovery of
927±47 ms (Fig. 7Di and
Table 1). The recovery of the
ntCionaKv4 channel was also slowed significantly by the presence of
CionaKChIP, with a time constant of recovery of 587±76 ms
(Fig. 7Diii and
Table 1), although the effect
was less than on the unmutated CionaKv4.
|
Discussion |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
), early chordates (Komuro
and Izumo, 1993; Holland et
al., 2003) and vertebrates
(Davidson and Levine, 2003),
it is reasonable to suppose that hearts are homologous across chordate
phylogeny (Romer and Parsons,
1986) and also within certain invertebrate lineages. Therefore, it
was not surprising that the message for a Kv4 channel was present in RNA
extracted from the heart of the tunicate Ciona intestinalis, since
these channels are known to mediate a transient current responsible for the
`notch' in cardiac action potentials from fish to mammals
(Dixon et al., 1996;
Brahmajothi et al., 1999; Nurmi and
Vornanen, 2002; Roden et al.,
2002). Similarly modulation of Kv4 mediated currents by KChIP and
analogous calcium-binding accessory subunits is common across the Metazoa
(Rosati et al., 2001;
Zhang et al., 2003). Although
genes for KChIP subunits have been identified by BLAST searches in the genomes
of Drosophila and Anopheles, CionaKChIP is the first KChIP
subunit from an invertebrate or non-vertebrate chordate that has been cloned
and characterized.
Comparing the genomic structure of CionaKv4 and CionaKChIP with their mammalian counterparts
The genomic structure of CionaKv4
(Fig. 1B) was similar to the
genomic structures of the three mammalian Kv4 channel genes, which have been
previously characterized (Isbrandt et al.,
2000). Interestingly, the CionaKv4 gene contains an
additional intron that partitions the codons for the first 17 amino acids onto
an additional exon. For comparison, exons 1-7 of CionaKv4 were
numbered 0-6 because the boundaries and relative sizes of exons 2-7 correspond
to exons 1-6 of mammalian Kv4 channels. The exon that encodes the first 17
amino acids of CionaKv4 is referred to as `exon 0' and is located
8 kb upstream of exon 1 (Fig.
1B). As in mammalian Kv4 channel genes, exon 1 of
CionaKv4 encodes the transmembrane domains S1-S5 and the first part
of the pore domain of CionaKv4, exon 2 encodes the second part of the
pore and transmembrane domain S6, and exons 3-6 encode the C terminus. The
intron between exons 1 and 2 in CionaKv4 interrupts the coding
sequence of the K+ selectivity sequence GYG at exactly the same
position as the equivalent intron of mammalian Kv4 genes. The sixth exon of
CionaKv4 is larger in this gene than in its mammalian counterparts,
encoding a comparatively longer C terminus.
Comparison between the genomic structures of CionaKChIP
(Fig. 1D) and the KChIPs cloned
from several mammalian species revealed that most exon/intron boundaries of
KChIPs are highly conserved. For example, the last exon of
CionaKChIP, KChIP2 and KChIP3 are identical in length
(Decher et al., 2004;
Spreafico et al., 2001). Each
of the EF-hands 1 to 3 of CionaKChIP is encoded by two exons, as in
KChIP3, and the partition of these three EF-hands into two exons occurs in the
same positions in CionaKChIP and KChIP3. The fourth EF-hand is
similarly encoded in a single exon in CionaKChIP, KChIP2 and KChIP3
(Decher et al., 2001;
Spreafico et al., 2001). The
number of exons that encode the different mammalian KChIPs is variable, since
numerous splice variants from a single KChIP gene are possible; for example,
the gene encoding KChIP2 subunits can be spliced differently to produce at
least eight isoforms that differ in their N terminus: KChIP2a
(An et al., 2000), KChIP2b
(Bähring et al., 2001b),
KChIP2c (Decher et al., 2001),
KChIP2d (Patel et al., 2002),
KChIPs 2e-g (Decher et al.,
2004), and KChIP2t
(Deschênes et al.,
2002). The fact that CionaKChIP is encoded by several
exons, in a similar fashion to the mammalian KChIP isoforms
(Fig. 1D), suggests that
several splice variants of CionaKChIP are possible, although only one
variant was detected in our experiments. Although most splice variants of
KChIPs differ in their N termini, variants that differ in their C termini have
been also cloned (Decher et al.,
2004). Our results suggest that splice variants of
CionaKChIP that differ in their C termini are not expressed, or are
expressed at very low levels in the heart of Ciona intestinalis,
because otherwise these would have been detected with the 3'-RACE assay.
Since we did not perform a 5'-RACE assay it is possible that
alternatively spliced variants of CionaKChIP with one or more
alternative N-terminal exons, variants containing the information encoded by
possible additional exons, or splice variants without an N terminus, similar
to the minimal isoform KChIP2d (Patel et
al., 2002), are expressed in the heart of C.
intestinalis. However, we can suggest that splice variants that do not
contain the information of one or more of exons 2-5 are not expressed, or
expressed at relatively low levels, otherwise it is likely that these would
have been detected when amplifying the ORF of CionaKChIP.
Comparing the function of CionaKv4 and other Kv4 channels
CionaKv4 produced A-type currents that activated and inactivated
with relatively rapid kinetics. The relative contribution of the fast
inactivation component (82%) was comparable to that of lobster
Shal (Baro et al.,
1996), Kv4.2 (Bähring et
al., 2001a) and Kv4.3 (Wang et
al., 2002), but was significantly greater than the <20%
recorded for Kv4.1 channels (Jerng and
Covarrubias, 1997). The fast inactivation time constant of
CionaKv4 at 12°C was about 26 ms, similar to lobster
Shal (31 ms at 16°C; Baro
et al., 1996) and mammalian Shal (23-23°C;
Pak et al., 1991). The fact
that this inactivating component has a similar time constant at very different
temperatures suggests that inactivation kinetics of Kv4 channels are not
constrained by temperature. Low Q10 values are common for marine
animals in shallow temperate waters.
|
0 mV
(Fig. 6Ci and
Table 1), is considerably
depolarized with respect to that of the Kv4 channel cloned from another
tunicate (Halocynthia), around -20 mV, as measured from
Fig. 2 of Nakajo et al.
(2003) and with respect to
most mammalian Kv4 channels, approximately -10 to -15 mV (see records for Kv4
family in
http://vkcdb.biology.ualberta.ca/).
The first N-terminal amino acids of CionaKv4 are likely involved in
regulating membrane expression, since deletion of these residues resulted in
increased current amplitude relative to wild-type channels when expressed in
Xenopus oocytes (Fig.
6Ai). This result mimicked the effect of deleting a similar
fragment from mammalian Kv4 channels
(Bähring et al., 2001a;
Shibata et al., 2003). It has
been shown that the hydrophobicity of most of the first 30 amino acids of
Kv4 channels impedes the trafficking of these channels to the membrane
(Shibata et al., 2003).
Deletion of amino acids 2-32 of CionaKv4 slowed the fast component
of inactivation approximately twofold (Fig.
7Aii) and slowed deactivation kinetics
(Fig. 7Bii). However, the N
terminus of CionaKv4 does not play a role in steady-state
inactivation (Fig. 7Cii). These
data suggest that the role of the N terminus of CionaKv4 in
inactivation is comparable to the role of the N terminus of Kv4.2 channels,
because deleting the first 40 amino acids of Kv4.2 channels had similar
effects on the rate of inactivation and steady-state inactivation
(Bähring et al., 2001a).
The role of the N terminus of Kv4.1 channels is divergent, as its deletion
resulted in loss of the fast component of inactivation of this isoform
(Pak et al., 1991;
Jerng and Covarrubias, 1997).
Interestingly, the N terminus of CionaKv4 also contributes to the
voltage-dependence of activation, since deletion of this domain resulted in a
significant shift of the activation midpoint by about 12 mV in the
hyperpolarizing direction relative to wild-type channels
(Fig. 6Cii and
Table 1).
Like the N terminus of mammalian Kv4 channels (Bähring et al., 2001), the N terminus of CionaKv4 is essential for modulation of some channel properties by KChIP subunits, since in the absence of this domain, CionaKChIP did not increase current amplitude, shift midpoint values of activation or inactivation or affect the kinetics of deactivation of CionaKv4 channels (see below). However, the macroscopic decay of ntCionaKv4/KChIP currents was slower than the decay of ntCionaKv4 channels alone. It is likely that CionaKChIP, in addition to interacting with the proximal amino acids of the N terminus of CionaKv4, also binds to a second domain of the N terminus that was not affected by the deletion performed on CionaKv4 channels. Mammalian KChIPs interact with at least two domains of the N terminus of Kv4 channels, one within residues 7-11 and another comprising residues 71-90 (Scannevin et al., 2004), and this second binding site is sufficient for binding to KChIPs.
KChIP modulation of current amplitude and activation parameters
As do mammalian KChIP isoforms 1-3 (An
et al., 2000), CionaKChIP increased the amplitude of
CionaKv4 currents (Figs
5B,
6Ai). The mammalian KChIP
isoforms do not increase the Kv4 current amplitude by a direct effect on
single channel conductance (Beck et al.,
2002), but by preventing aggregation of their hydrophobic N
terminus in the ER that would interfere with folding. Proper folding favours
the insertion of Kv4 channels into the plasma membrane and increases their
half-life in the membrane (Shibata et al.,
2003). This study did not determine whether CionaKChIP
increases current amplitude by facilitating proper insertion of these channels
in the plasma membrane or by increasing the conductance of CionaKv4
channels. CionaKChIP shifted the midpoint of activation of
CionaKv4 in the hyperpolarizing direction
(Fig. 6Ci), which is consistent
with the effect of most mammalian KChIPs
(An et al., 2000;
Van Hoorick et al., 2003).
KChIP modulation of inactivation
CionaKChIP slowed inactivation of the transient currents produced
by CionaKv4. This was reminiscent of the effects of the mammalian
isoform KChIP4a on mammalian Kv4 channels
(Holmqvist et al., 2002). The
N terminus of KChIP4a [K inactivation suppressor domain (KIS)] is required for
its effect on inactivation kinetics
(Holmqvist et al., 2002). To
determine whether the N terminus of CionaKChIP also has the KIS
domain, we aligned the first 40 amino acids of CionaKChIP with the
first 40 amino acids of KChIP4a using T-Coffee software
(Notredame et al., 2000). The
results of this alignment are shown in Fig.
8A. For comparison, we also aligned the first 40 amino acids of
CionaKChIP with the first 40 amino acids of representatives of the
other vertebrate KChIP subfamilies (KChIP1-3). Interestingly, the N terminus
of CionaKChIP was more similar to the N terminus of the KChIP4a
isoform than to the N termini of other KChIP isoforms. The alignment between
CionaKChIP and KChIP4a revealed that two leucines, at positions 3 and
6 in both subunits, and a methionine, at position 8 also in both subunits, are
conserved between KChIP4a and CionaKChIP
(Fig. 8A). Other residues that
are conserved between the N terminus of KChIP4a and CionaKChIP are a glutamic
acid residue and a glycine residue at positions 19 and 22, in
CionaKChIP (positions 23 and 26 in KChIP4a), an alanine-glycine motif
located at position 28 in CionaKChIP (position 30 in KChIP4a), and a
valine residue at position 34 in CionaKChIP (position 32 of KChIP4a;
Fig. 8A). These conserved
residues may be crucial to the modulation by KChIP subunits on inactivation
kinetics. The alignments between CionaKChIP and KChIP2a or KChIP3a
had extensive gaps (Fig. 8C,D).
The alignment between CionaKChIP and KChIP1a has fewer and smaller
gaps for other isoforms, perhaps revealing a closer relationship between the N
termini of these two subunits (Fig.
8B).
Most KChIP isoforms either do not affect the midpoint of inactivation (e.g.
KChIP1a; Nakamura et al.,
2001; Van Hoorick et al.,
2003) or shift the midpoint of inactivation in the depolarizing
direction (e.g. KChIP2b; Bähring et
al., 2001b). However, the splice variant KChIP2g, which has an
alternative exon 1 (Decher et al.,
2004) and KChIP 1b, which contains the information of an
additional exon in its N terminus (Van
Hoorick et al., 2003), shifts the midpoint of inactivation in the
hyperpolarizing direction as for CionaKChIP
(Fig. 7Ci). However, the N
termini of these mammalian KChIP splice variants and CionaKChIP are
not similar.
KChIP modulation of recovery from inactivation
CionaKv4 channels required 1 s to fully recover from
inactivation at -100 mV, with a recovery time constant of 387 ms
(Fig. 7D). Similar values have
been reported for jellyfish and fly Shal
(Jegla and Salkoff, 1997) and
Kv4.2 (Serôdio et al.,
1994) for the same membrane potential. Kv4.1 channels recover
fully from inactivation within 300-400 ms at -100 mV
(Serôdio et al., 1994),
while Kv4.3 channels recover from inactivation relatively rapidly, with
recovery time constants of less than 200 ms, also at -100 mV
(Dixon et al., 1996). In
conclusion, invertebrate and non-vertebrate chordate Kv4 channels, including
CionaKv4, exhibit lower rates of recovery from inactivation than the
mammalian Kv4.1 and KvV4.3 channel subtypes, suggesting that relatively slow
kinetics of recovery is a primitive feature of Kv4 channels.
The Kv4 channel and the KChIP subunit diversified during vertebrate evolution
Since the mammalian genome contains three Kv4 paralogues and four KChIP
paralogues, while the genome of Ciona intestinalis contains only one
gene for Kv4 channels and a single gene for KChIP subunits, it is probable
that both genes duplicated and diverged in the vertebrate lineage during
evolution. Current theory states that there were two major genome duplications
early in the evolution of the vertebrates
(Ohno, 1970). Most vertebrate
gene families (81%) are composed of two or three paralogues that derive from a
single ancestral gene (Escriva et al.,
2002), as is the case for the Kv4 channel gene family. Gene
families formed by three paralogues are believed to have lost a fourth
paralogue after the second large-scale genomic duplication
(Spring, 1997). Since the
KChIP gene family has four paralogues in vertebrates, it is unlikely that gene
loss occurred after the second large-scale duplication. The pattern of KChIP
clades in the vertebrate lineage (Fig.
4B), [A(B(CD))], is not consistent with two successive genome
duplications. The fact that this pattern is common within vertebrate gene
families composed of four paralogues has been used by Hugues
(1999) to argue against two
rounds of genomic duplication, as this duplication pattern would have rendered
a topology of the form: (AB)(CD). Sidow
(1996) has suggested that
duplications of single genes or fragments of the genome that were independent
of large-scale genomic duplications have played a role in the evolution of the
vertebrates and might explain these discrepancies. However, the support values
for the two internal branching events in the vertebrate KChIP clade are so low
that the relationships of these four paralogues are most reasonably described
by an unresolved polytomy rather than either of the specific evolutionary
models.
Conclusion
Until this study, invertebrate KChIPs had not been cloned. However it has
been shown that crustacean Kv4 channels can be modulated by a mammalian KChIP
(Zhang et al., 2003) in a
similar fashion to that shown by the effect of CionaKChIP on
CionaKv4 shown in this study. This is the first cloning of a
non-vertebrate KChIP, and since tunicates are the earliest diverging chordate
clade, these results show that modulation of Kv channels by calcium-binding
proteins in vertebrates is an ancient and conserved mechanism.
We conclude that tunicate KChIP subunits modulate expression, voltage sensitivity of gating, and kinetic behavior of tunicate Kv4 channels. Modulation by CionaKChIP requires an intact N terminus of CionaKv4, as in mammals. CionaKChIP dramatically slowed the rate of inactivation of CionaKv4 channels, similarly to the vertebrate KChIP4a isoform (Holmqvist et al., 2004) and our comparative analyses may have provided clues as to the N-terminal amino acids in KChIP that are responsible for this action. Our results are also the first report on the molecular nature of excitability in the tunicate myocardium, which may help further our understanding of its physiology and the evolution of hearts. We suggest that modulation of Kv4 channels by KChIP subunits is a conserved mechanism to modulate cardiac excitability.
rec
|
References |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Amberg, G. C., Koh, S. D., Hatton, W. J., Murray, K. J.,
Monaghan, K., Horowitz, B. and Sanders, K. M. (2002).
Contribution of Kv4 channels toward the A-type potassium current in murine
colonic myocytes. J. Physiol.
544,403
-415.
An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S. et al. (2000). Modulation of A-type potassium channels by a family of calcium sensors. Nature 403,553 -556.[CrossRef][Medline]
Bähring, R., Boland, L. M., Varghese, A., Gebauer, M. and
Pongs, O. (2001a). Kinetic analysis of open- and closed-state
inactivation transitions in human Kv4. 2 A-type potassium channels.
J. Physiol. 535,65
-81.
Bähring, R., Dannenberg, J., Peters, H. C., Leicher, T.,
Pongs, O. and Isbrandt, D. (2001b). Conserved Kv4
N-terminal domain critical for effects of Kv channel-interacting protein 2.2
on channel expression and gating. J. Biol. Chem.
276,23888
-23894.
Bairoch, A. and Cox, J. A. (1990). EF-hand motifs in inositol phospholipids-specifc phospholipase C. FEBS Lett. 269,454 -456.[CrossRef][Medline]
Baro, D. J., Coniglio, L. M., Cole, C. L., Rodriguez, H. E.,
Lubell, J. K., Kim, M. T. and Harris-Warrick, R. M.
(1996). Lobster shal: comparison with Drosophila
shal and native potassium currents in identified neurons. J.
Neurosci. 16,1689
-1701.
Beck, E. J., Bowlby, M., An, W. F., Rhodes, K. J. and
Covarrubias, M. (2002). Remodelling inactivation gating of
Kv4 channels by KChIP1, a small-molecular-weight calcium-binding protein.
J. Physiol. 538,691
-706.
Bodmer, R. (1993). The gene tinman is required for specification of the heart and visceral muscles in Drosophila. Development 118,719 -729.[Abstract]
Brahmajothi, M. V., Campbell, D. J., Rasmusson, R. L., Morales, M. J., Trimmer, J. S., Nerbonne, J. M. and Strauss, H. C. (2005). Distinct transient outward potassium current (Ito) phenotypes and distribution of fast-inactivating potassium channel alpha subunits in ferret left ventricular myocytes. J. Gen. Physiol. 113,581 -600.
Burgoyne, R. D. and Weiss, J. L. (2001). The neuronal calcium sensor family of Ca2+-binding proteins. Biochem. J. 353,1 -12.[CrossRef][Medline]
Connor, J. A. and Stevens, C. F. (1971).
Prediction of repetitive firing behaviour from voltage clamp data on an
isolated neurone soma. J. Physiol.
213, 31-53.
Davidson, B. and Levine, M. (2003).
Evolutionary origins of the vertebrate heart: specification of the cardiac
lineage in Ciona intestinalis. Proc. Natl. Acad. Sci.
USA 100,11469
-11473.
Decher, N., Uyguner, O., Scherer, C. R., Karaman, B.,
Yüksel-Apak, M., Busch, A. E., Steinmeyer, K. and Wollnik, B.
(2001). hKChIP2 is a functional modifier of hKv4.3 potassium
channels: Cloning and expression of a short hKChIP2 splice variant.
Cardiovasc. Res. 52,255
-264.
Decher, N., Barth, A. S., Gonzalez, T., Steinmeyer, K. and
Sanguinetti, M. C. (2004). Novel KChIP2 isoforms
increase functional diversity of transient outward potassium currents.
J. Physiol. 557,761
-772.
Deschênes, I., DiSilvestre, D., Juang, G. J., Wu, R. C.,
An, W. F. and Tomaselli, G. F. (2002). Regulation of
Kv4.3 current by KChIP2 splice variants: a component of native cardiac Ito?
Circulation 106,423
-429.
Dixon, J. E., Shi, W., Wang, H. S., McDonald, C., Yu, H.,
Wymore, R. S., Cohen, I. S. and McKinnon, D. (1996).
The role of the Kv4.3 K+ channel in ventricular muscle: a molecular
correlate for the transient outward current. Circ.
Res. 79,659
-668.
Dominguez, I., Itoh, K. and Sokol, S. Y.
(1995). Role of glycogen synthase kinase 3-beta as a negative
regulator of dorsoventral axis formation in Xenopus embryos.
Proc. Natl. Acad. Sci. USA
92,8498
-8502.
Escriva, H., Manzon, L., Youson, J. and Laudet, V.
(2002). Analysis of lamprey and hagfish genes reveals a complex
history of gene duplications during early vertebrate evolution.
Mol. Biol. Evol. 19,1440
-1450.
Holland, N. D., Venkatesh, T. V., Holland, L. Z., Jacobs, D. K. and Bodmer, R. (2003). AmphiNk2-tin, an amphioxus homeobox gene expressed in myocardial progenitors: insights into evolution of the vertebrate heart. Dev. Biol. 255,128 -137.[CrossRef][Medline]
Holmqvist, M. H., Cao, J., Hernandez-Pineda, R., Jacobson, M.
D., Carroll, K. I., Sung, M. A., Betty, M., Ge, P., Gilbride, K. J.,
Brown, M. E. et al. (2002). Elimination of fast inactivation
in Kv4 A-type potassium channels by an auxiliary subunit domain.
Proc. Natl. Acad. Sci. USA
99,1035
-1040.
Huelsenbeck, J. P. and Ronquist, F. (2001). MRBAYES: Bayesian inference of phylogenetic trees. J. Bioinform. 17,754 -755.
Hugues, A. L. (1999). Phylogenies of developmentally important proteins do not support the hypothesis of two rounds of genome duplication early in vertebrate history. J. Mol. Evol. 48,565 -576.[CrossRef][Medline]
Isbrandt, D., Leicher, T., Waldschütz, R., Zhu, X., Luhmann, U., Michel, U., Sauter, K. and Pongs, O. (2000). Gene structures and expression profiles of three human KCND (Kv4) potassium channels mediating A-type currents ITO and ISA. Genomics 64,144 -154.[CrossRef][Medline]
Jegla, T. and Salkoff, L. (1997). A novel
subunit for Shal potassium channels radically alters activation and
inactivation. J. Neurosci.
17, 32-44.
Jerng, H. H. and Covarrubias, M. (1997). K+ channel inactivation mediated by the concerted action of the cytoplasmic N- and C-terminal domains. Biophys. J. 72,163 -174.[Medline]
Komuro, I. and Izumo, S. (1993). Csx: A murine
homeobox-containing gene specifically expressed in the developing heart.
Proc. Natl. Acad. Sci. USA
90,8145
-8149.
Kretsinger, R. H. (1987). Calcium coordination
and the calmodulin fold: divergent versus convergent evolution.
Cold Spring Harbor Symp. Quant. Biol.
52,499
-510.
Kriebel, M. E. (1967). Conduction velocity and
intracellular action potentials of the tunicate heart. J. Gen.
Physiol. 50,2097
-2107.
Nakajo, K., Katsuyama, Y., Ono, F., Ohtsuka, Y. and Okamura, Y. (2003). Primary structure, functional characterization and developmental expression of the ascidian Kv4-class potassium channel. Neurosci. Res. 45,59 -70.[CrossRef][Medline]
Nakamura, T. Y., Nandi, S., Pountney, D. J., Artman, M., Rudy, B. and Coetzee, W. A. (2001). Different effects of the Ca2+-binding protein, KChIP1, on two Kv4 subfamily members, Kv4.1 and Kv4.2. FEBS Lett. 499,205 -209.[CrossRef][Medline]
Notredame, C., Higgins, D. and Heringa, J. (2000). T-Coffee: a novel method for multiple sequence alignments. J. Mol. Biol. 302,205 -217.[CrossRef][Medline]
Nurmi, A. and Vornanen, M. (2002). Electrophysiological properties of rainbow trout cardiac myocytes in serum-free primary culture. Am. J. Physiol. Reg. Integr. Comp. Physiol. 228,R1200 -R1209.
Ohno, S. (1970). Evolution by Gene Duplication. Heidelberg: Springer-Verlag.
Page, R. D. (1996). TreeView: an application to display phylogenetic trees on personal computers. J. Comput. Appl. Biosci. 12,357 -358.
Pak, M. D., Baker, K., Covarrubias, M., Butler, A., Ratcliffe,
A. and Salkoff, L. (1991). mShal, a subfamily of
A-type K+ channel cloned from mammalian brain. Proc.
Natl. Acad. Sci. USA 88,4386
-4390.
Patel, S. P., Campbell, D. L. and Strauss, H. C.
(2002). Elucidating KChIP effects on Kv4.3 inactivation and
recovery kinetics with a minimal KChIP2 isoform. J.
Physiol. 545,5
-11.
Petrecca, K., Miller, D. M. and Shrier, A.
(2000). Localization and enhanced current density of the Kv4.2
potassium channel by interaction with the actin-binding protein filamin.
J. Neurosci. 20,8736
-8744.
Rivera, J. F., Ahmad, S., Quick, M. W., Liman, E. R. and Arnold, D. B. (2003). An evolutionarily conserved di-leucine motif in Shal K+ channels mediates dendritic targeting. Nat. Neurosci. 6,243 -250.[CrossRef][Medline]
Roberds, S. L. and Tamkun, M. M. (1991).
Cloning and tissue-specific expression of five voltage-gated potassium channel
cDNAs expressed in rat heart. Proc. Natl. Acad. Sci.
USA 88,1798
-1802.
Roden, D. M., Balser, J. R., George, A. L., Jr and Anderson, M. E. (2002). Cardiac ion channels. Annu. Rev. Physiol. 64,431 -475.[CrossRef][Medline]
Romer, A. S. and Parsons, T. S. (1986). The Vertebrate Body, 6th edn. Philadelphia: Saunders.
Rosati, B., Pan, Z., Lypen, S., Wang, H. S., Cohen, I., Dixon,
J. E. and McKinnon, D. (2001). Regulation of
KChIP2 potassium channel ß subunit gene expression underlies the
gradient of transient outward current in canine and human ventricle.
J. Physiol. 533,119
-125.
Scannevin, R. H., Wang, K., Jow, F., Megules, J., Kopsco, D. C.,
Edris, W., Carroll, K. C., Lü, Q., Xu, W., Xu, Z. et al.
(2003). A fundamental role for KChIPs in determining the
molecular properties and trafficking of Kv4.2 potassium channels.
J. Biol. Chem. 278,36445
-36454.
Serôdio, P., Kentros, C. and Rudy, B.
(1994). Identification of molecular components of A-type channels
activating at subthreshold potentials. J.
Neurophysiol. 72,1516
-1529.
Serôdio, P., Vega-Saenz de Miera, E. and Rudy, B.
(1996). Cloning of a novel component of A-type K+
channels operating at subthreshold potentials with unique expression in heart
and brain. J. Neurophysiol.
75,2174
-2179.
Shibata, R., Misonou, H., Campomanes, C. R., Anderson, A. E.,
Schrader, L. A., Doliveira, L. C., Carroll, K. I., Sweatt, J. D.,
Rhodes, K. J. and Trimmer, J. S. (2003). A fundamental role
for KChIPs in determining the molecular properties and trafficking of Kv4.2
potassium channels. J. Biol. Chem.
278,36445
-36454.
Sidow, A. (1996). Gen(om)e duplications in the evolution of early vertebrates. Curr. Opin. Genet. Dev. 6,715 -722.[CrossRef][Medline]
Spreafico, F., Barski, J. J., Farina, C. and Meyer, M. (2001). Mouse DREAM / Calsenilin / KChIP3: Gene structure, coding potential, and expression. Mol. Cell. Neurosci. 17, 1-16.[CrossRef][Medline]
Spring, J. (1997). Vertebrate evolution by interspecific hybridization-are we polyploid? FEBS Lett. 400,2 -8.[CrossRef][Medline]
Van Hoorick, D., Raes, A., Keysers, W., Mayeur, E. and Snyders, D. J. (2003). Differential modulation of Kv4 kinetics by KCHIP1 splice variants. Mol. Cell. Neurosci. 24,357 -366.[CrossRef][Medline]
Wang, S., Patel, S. P., Qu, Y., Hua, P., Strauss, H. C. and Morales, M. J. (2002). Kinetic properties of Kv4.3 and their modulation by KChIP2b. Biochem. Biophys. Res. Comm. 295,223 -229.[CrossRef][Medline]
Zhang, Y., MacLean, J. N., An, W. F., Lanning, C. C. and
Harris- Warrick, R. M. (2003). KChIP1 and frequenin
modify shal-evoked potassium currents in pyloric neurons in the
lobster stomatogastric ganglion. J. Neurophysiol.
89,1902
-1909.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in JEB:
This article has been cited by other articles:
|
|
 |
|
  TUNICATES SET TREND FOR POTASSIUM CHANNEL J. Exp. Biol., February 15, 2006; 209(4): ii - ii. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
