|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online June 13, 2008
Journal of Experimental Biology 211, 2162-2171 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.016121
A novel inwardly rectifying K+ channel, Kir2.5, is upregulated under chronic cold stress in fish cardiac myocytes
Faculty of Biosciences, University of Joensuu, PO Box 111, 80101 Joensuu, Finland
* Author for correspondence (e-mail: matti.vornanen{at}joensuu.fi)
Accepted 10 April 2008
 |
Summary |
|---|
 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Key words: temperature acclimation, fish heart, repolarisation
|
INTRODUCTION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
; Vandenberg,
1987; Ficker et al.,
1994; Lopatin et al.,
1994; Fakler et al.,
1995; Kurata et al.,
2006). The small outward current is, however, physiologically
important in stabilising resting membrane potential close to the K+
equilibrium potential and in regulating the duration of the cardiac action
potential by phase-3 repolarisation
(Shimoni et al., 1992;
Lopatin and Nichols,
2001).
The Kir2 subfamily has four known members (Kir2.1–2.4), three of
which (Kir2.1–2.3) are expressed in the heart. Kir2.1 is the dominating
subunit in the mammalian heart, although Kir2.2 and Kir2.3 channels are
variably expressed depending on the animal species and cardiac chamber
(Liu et al., 2001;
Preisig-Müller et al.,
2002; Zobel et al.,
2003; Dhamoon et al.,
2004; Hume and Uehara,
1985; Wang et al.,
1998; Melnyk et al.,
2002). Each homotetrameric Kir2 channel has distinct kinetics,
conductance and sensitivity to polyamines, enabling the regulation of
IK1 density and inward rectification by variable
expression and coassembly of the Kir2 subunits
(Périer et al., 1994;
Takahashi et al., 1994;
Ishihara and Yan, 2007). Human
diseases due to Kir2 mutations and knockout animal models of the Kir2 channels
have demonstrated a vital role of IK1 in normal cardiac
function (Plaster et al.,
2001; Zaritsky et al.,
2001). Furthermore, expression and function of the cardiac
IK1 is altered in hypoxia, ischaemia and disease states
(Piao et al., 2007;
Liu et al., 2007;
Ten Eick et al., 1992)
indicating that Kir2 channels are plastic entities and are probably involved
in cardiac remodelling in pathophysiological conditions.
Unlike mammalian hearts, the hearts of ectothermic vertebrates are
naturally exposed to large temperature changes, which impose special
requirements for cardiac ion channel function to maintain proper excitability
and to prevent cardiac arrhythmias. For example, crucian carp (Carassius
carassius L.) can tolerate temperatures between 0 and 38°C
(Horoszewicz, 1973) and in
their natural environment face seasonal temperature changes of over 20°C
(Vornanen and Paajanen, 2004).
Owing to its excellent thermal tolerance, crucian carp heart is an interesting
subject for testing the plasticity of vertebrate cardiac phenotype and its
molecular basis, in particular the responses of cardiac ion channels to
temperature change. The objective of this study was to examine the
contribution of different Kir2 subunits to putative temperature-induced
changes in the cardiac IK1. Three Kir2 channel genes were
found in crucian carp heart, two of them being homologues of the mammalian
Kir2.1 and Kir2.2 channel genes. The third one was a new, previously unknown
member of the Kir2 subfamily and was designated as ccKir2.5
(cc for Carassius carassius). The expression of ccKir2.5 was
strongly increased in the cold-acclimated fish (4°C), suggesting that the
novel ccKir2.5 is intimately involved in cardiac adjustment to low temperature
conditions by increasing the density of IK1.
|
MATERIALS AND METHODS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Molecular methods
Extraction of RNA and DNA
Atrium, ventricle and brains and a piece of gill, muscle, liver and kidney
were homogenised under liquid nitrogen and RNA was extracted with TRIzol
reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's
instructions. DNA was extracted from liver by the method of Sambrook et al.
(Sambrook et al., 1989). The
quality and quantity of RNA and DNA were monitored by agarose gel
electrophoresis and UV spectrophotometry, respectively.
Molecular cloning of cardiac Kir genes
The open reading frames (ORFs) for crucian carp Kir2.1, Kir2.2 and
Kir2.5 and a 437 bp fragment for crucian carp DnaJA2 gene
were cloned by reverse transcriptase-PCR (RT-PCR). First strand cDNA synthesis
was carried with RNAseH+ (Finnzymes, Espoo, Finland) using random
hexamers or oligo(dT) primers. Degenerative primers designed to the conserved
regions of mammalian Kir2.1, Kir2.2 and Kir2.3 genes were
used to get partial cDNA clones of crucian carp Kir2 genes
(Table 1). In spite of several
trials, no Kir2.3 products were found in the crucian carp heart. The
first fragment of an unidentified Kir gene was obtained with
degenerative primers to mammalian Kir2.1. New primers for further
cloning were designed for each clone on the basis of sequences obtained. PCR
was performed using a PTC-200 DNA Engine Cycler (MJ Research, Waltham, MA,
USA) and conditions described previously
(Hassinen et al., 2007).
Oligo(dT) primers and a 3'-RACE kit (Invitrogen) were used to clone the
3' end of the ccKir2.2 and ccKir2.5 gene, respectively
(Table 1). Genome Walker kit
(Clontech, Palo Alto, CA, USA) was used for cloning the 5' ends. Four
genomic libraries from crucian carp DNA were constructed and used as templates
in PCR as previously described (Hassinen et
al., 2007). Finally, the sequences were confirmed by cloning the
whole coding region of the ccKir2.1, ccKir2.2 and ccKir2.5
genes.
|
All PCR products were analysed by agarose gel electrophoresis, extracted from the gel by Qiaex II gel extraction kit (Qiagen, Valencia, CA, USA) and cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA). DNA sequencing was conducted using the ABI PRISM BigDye Terminator cycle sequencing kit v2.0 (Applied Biosystems, Foster City, CA, USA) and the reactions were analysed using ABI PRISM 310 Genetic Analyzer (Applied Biosystems).
Phylogenetic analysis
The whole coding sequences of ccKir2 proteins, zebrafish Kir proteins and
all known mouse Kir proteins were aligned by ClustalW and a phylogenetic tree
was constructed in ClustalX
(http://www.clustal.org/download/current/)
using the neighbour-joining method. Sequence positions containing gaps in any
of the Kir genes were ignored from the analysis. KirBac3.1 was used
as an outgroup member and the analysis was performed with 1000 bootstrap
replicates. Graphical presentation of the tree was produced in Treeview
(http://taxonomy.zoology.gla.ac.uk/rod/treeview.html/).
Quantitative PCR
Atrium, ventricle and brains and a piece of gill, muscle, liver and kidney
were pooled from several fish for RNA sample preparation (N=3 for
both acclimation groups). First strand cDNA synthesis was performed from
DNAse-treated RNA (Hassinen et al.,
2007) and DNA contamination was tested by a control cDNA synthesis
containing all other reaction components except the RT enzyme. Quantitative
RT-PCR was performed using Chromo4 Continuous Fluorescence Detector (MJ
Research) under previously described conditions
(Hassinen et al., 2007).
Primers were designed to the non-conserved N- or C-termini of the
ccKir2.1, ccKir2.2 and ccKir2.5 and to the cloned region
(nucleotides 121–557; GenBank accession number EU191947) of the crucian
carp DnaJA2 (Table 2).
DnaJA2 was used as a reference gene because it is more stable in
thermal acclimation than the conventional reference genes
(Vornanen et al., 2005).
Transcript abundance of the ccKir2 genes was normalised to the
DnaJA2 expression level.
|
Electrophysiological methods
Heterologous expression of the ccKir2 proteins
ORF sequences for the putative ion channel-forming genes ccKir2.1,
ccKir2.2 and ccKir2.5 were subcloned into the pcDNA3.1/Zeo (+)
vector (Invitrogen) for expression in a COS-1 cell line
(Hassinen et al., 2007).
Electrophysiological experiments were made 48–72 h after
transfection.
Whole-cell patch clamp
Ventricular myocytes were enzymatically isolated
(Vornanen, 1997) and used
within 8 h of isolation. Whole-cell voltage clamp experiments were done using
an EPC9 patch clamp amplifier (HEKA Instruments Inc., Lambrecht/Pfalz,
Germany), a PC-16 solution exchanger (Bioscience Tools, San Diego, CA, USA)
and Pulse acquisition software (HEKA Instruments). External saline solution
contained (mmol l–1): 150 NaCl, 5.4 KCl, 1.8
CaCl2, 1.2 MgCl2, 10 glucose and 10 Hepes (pH 7.6).
Patch pipettes were pulled from borosilicate glass (Garner, Claremont, CA,
USA) and filled with K+-based electrode solution (mmol
l–1: 140 KCl, 1 MgCl2, 5 EGTA, 4 MgATP and 10
Hepes at pH 7.2). For experiments with ventricular myocytes, tetrodotoxin (0.5
µmol l–1, Tocris Cookson, Bristol, UK)
(Haverinen et al., 2007),
nifedipine (10 µmol l–1, Sigma, Helsinki, Finland),
glibenclamide (10 µmol l–1, Sigma) and E-4031 (1 µmol
l–1, Alomone labs, Jerusalem, Israel) were added to the
extracellular solution to block Na+, Ca2+, ATP-sensitive
K+ and delayed-rectifier K+ (IKr)
current, respectively. The mean resistance of the pipettes was
2.52±0.07 M
.
IK1 was elicited every 10 s by repolarising ramps or
square-wave pulses from the holding potential of –80 mV. Barium
inhibition of IK1 was determined in the presence of cumulatively added
concentrations of BaCl2 (10–9 to
3x10–4 mol l–1).
Concentration–response curves were fitted with a Hill equation:
 |
The voltage dependence of inward rectification was measured separately
using a single high dose of Ba2+ (0.1–0.3 mmol
l–1) for a complete and reversible block of the current
within 45 s of the onset of the whole-cell configuration.
Ba2+-sensitive current was compared with the unblocked
(non-rectifying) current, which was obtained from the current–voltage
relationship between –120 mV and the reversal potential
(Vrev) of IK1 and extrapolated to the
voltage area of inward rectification. Scattering data points around
Vrev (±1.5 mV) were omitted and the current was
fitted with a Boltzmann function:
 |
Single-channel patch clamp
Single-channel properties of the cloned ccKir2 channels were recorded at
room temperature (21±1°C) in inside-out configuration with an EPC-9
amplifier and Pulse software and analysed with TAC, TACFit (Bruxton
Corporation, Seattle, WA, USA) and SigmaPlot 6.0 (SPSS, Chicago, IL, WA)
software (Paajanen and Vornanen,
2004). The pipette solution was composed of (mmol
l–1): 134 KCl, 1.8 CaCl2, 2 MgCl2, 10
glucose and 10 Hepes adjusted to pH 7.6 with KOH ([K+]=141 mmol
l–1). The composition of the bath solution in the inside-out
experiments was (mmol l–1): 140 KCl, 2 EGTA, 1 EDTA and 5
Hepes adjusted to pH 7.6 with KOH. EGTA and EDTA were included to prevent
endogenous Ca2+-dependent currents of the COS-1 cells. Inside-out
patches with any outward current were excluded from further analyses.
Native Kir2 channels of fish ventricular myocytes were measured in
inside-out and cell-attached configurations. Inside-out experiments were made
under the same experimental conditions as the experiments with the cloned
channels. Cell-attached recordings were conducted at 11°C (temperature in
the middle of the acclimation temperatures of 4 and 18°C) by using the
same external saline in the bath as in the whole-cell experiments. Pipettes
were pulled (PP-83 puller, Narishige, Tokyo, Japan) from thick-walled
borosilicate glass (Garner), coated with Sylgard (WPI, Stevenage, UK), fire
polished on a microforge (MF-83, Narishige) and filled with the same high
K+ solution that was used for the inside-out patches. The mean
resistance of the pipettes was 25.1±1.8 M.Because low external
K+ causes a voltage offset, the Nernst potential of K+
ions (–80 mV) was added to the membrane voltage.
All single-channel recordings were sampled at 4 kHz and low-pass filtered at 2 kHz. Single-channel conductance was determined by applying 5 s square pulses from –120 to +80 or –200 to –20 mV in 20 mV increments every 10 s for inside-out and cell-attached patches, respectively. Distributions of open and closed times were obtained from 20 to 120 s recordings at –100 mV. Open and closed time analyses were performed on patches that had only a single open current level. Open and closed times were detected with time-course fitting, and probability density functions (pdf) were analysed (TACFit) from idealised data with the log-likelihood method on log(event times). Histograms of single-channel conductance were constructed from cell-attached and inside-out recordings of the endogenous inward-rectifier channels from cold- and warm-acclimated fish to see the natural variation of conductance levels.
Statistics
Differences between mean current values from warm- and cold-acclimated carp
ventricular IK1 were assessed by Student's
t-test, whereas mean current values of the channels encoded by the
cloned ccKir2.1, ccKir2.2 and ccKir2.5 genes were evaluated
by using one-way analysis of variance. Differences in Kir2 transcript
abundances were tested using Student's t-test. A P value of
0.05 was regarded as the limit of statistical significance.
|
|
RESULTS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
ORFs for ccKir2.1, ccKir2.2 and ccKir2.5 consisted of 1284, 1314 and 1269 bp, coding for 427, 437 and 422 amino acids, respectively (supplementary material Fig. S1). To establish evolutionary relationships between the ccKir2 channels and the known vertebrate Kir channels, we constructed a phylogenetic tree with members from all seven Kir subfamilies. Phylogenetic analysis showed that ccKir2.1 and ccKir2.2 are, indeed, homologues of Kir2.1 and Kir2.2, respectively (Fig. 2). From the known Kir family members, the closest relative of ccKir2.5 was Kir2.2 with an 81% bootstrap value. Importantly, ccKir2.5 shares a common ancestor with other Kir2 genes, providing strong evidence that ccKir2.5 is a new member of the Kir2 subfamily. Taken together, crucian carp heart expresses ccKir2.1, ccKir2.2 and ccKir.2.5, but not a homologue of vertebrate Kir2.3.
|
Sequence structure of the ccKir2 genes
All vertebrate Kir proteins consist of a conserved pore-forming region (P)
flanked by two transmembrane alpha helices (M1 and M2). These functional
domains were identified from all cloned ccKir2 proteins and were more
conserved than the cytoplasmic N- and C-termini
(Fig. 1A, supplementary
material Fig. S2). In mammalian Kir2.1, Mg2+ binds to serine S165
(numbering according to mouse Kir2.1)
(Fujiwara and Kubo, 2002),
whereas glutamates E224 and E299 function as an intermediate binding site for
polyamines before entering to the pore-blocking site D172
(Xie et al., 2003). All these
residues also exist in ccKir2.5, ccKir2.2 and ccKir2.1, suggesting a similar
inward-rectification mechanism for mammalian and fish Kir2 channels. In
contrast, some variability appeared in amino acids important for
Ba2+ binding. In mammalian Kir2.1, glutamate E125 between M1 and P
binds Ba2+ and facilitates its movement to the plugging site
threonine T141 of the narrow pore region
(Alagem et al., 2001).
Threonine T141 is conserved in all mammalian and fish cardiac Kir2 channels.
In contrast, glutamate E125 exists in mammalian Kir2.1, but not in mammalian
Kir2.2 and Kir2.3 channels. In crucian carp, the glutamate E125 is replaced by
other amino acids, not only in ccKir2.1, but also in ccKir2.5 and ccKir2.2.
Yet, the cloned ccKir2.5 and ccKir2.2 channels were about 10 times more
sensitive to Ba2+ than ccKir2.1. While this agrees with the finding
that mammalian Kir2.2 is 5–10 times more sensitive to Ba2+
than Kir2.1 (Liu et al., 2001;
Preisig-Müller et al.,
2002), it strongly suggests that other residues in addition to
E125 are important for Ba2+ binding.
Tissue distribution of ccKir2.5 mRNA
Transcript abundance of ccKir2 subunits was determined by quantitative PCR
in seven tissues of the cold-acclimated crucian carp. All three
ccKir2 genes were expressed to some extent in heart, brain, gill,
kidney, liver and skeletal muscle (Fig.
3). ccKir2.5 was a major ccKir2 channel component in atrium
(59.1±2.1%) and ventricle (65.6±3.2%) of the heart and in the
skeletal muscle (88.6±3.4%), while in other tissues it was weakly
expressed (<25%), suggesting that it is a muscle-specific isoform.
|
90%) of COS-1 cells transfected with ccKir2
genes and a separate eGFP vector had a large inward-rectifying current with a
slope conductance of 100±15 nS, which reversed direction near the
equilibrium potential of K+ ions (–73.3±1.0 mV). The
current was reversibly blocked by 0.1–0.3mmoll–1
[Ba2+]o (Fig.
4A), indicating that the cloned genes encoded functional
inward-rectifying K+ channels of the Kir2 family.
|
When expressed in COS-1 cells, ccKir2.5 was almost 10-times more sensitive to [Ba2+]o (Kd, 2.43±0.37 µmol l–1) than the ccKir2.1 channel (22.25±5.37 µmol l–1; P<0.05), and it also rectified much more strongly (z=2.40±0.08, V1/2=–85.62±3.50 mV) than ccKir2.1 (z=1.73±0.04, V1/2=–54.20±0.57 mV; P<0.05; Fig. 4B–D). With regard to Ba2+ sensitivity, ccKir2.5 current was similar to the current generated by ccKir2.2 (3.48±0.90 µmol l–1; P>0.05), but more strongly rectifying than ccKir2.1 (V1/2=–76.29±2.21 mV; P<0.05). Accordingly, at the whole-cell level ccKir2.5 differs from the other ccKir2 channels with regard to either Ba2+ sensitivity or inward rectification, or both.
Even at the single-channel level, ccKir2.5 channels were functionally closer to ccKir2.2 than to ccKir2.1 channels (Fig. 5). Single-channel conductance of ccKir2.5 (44.19±1.89pS) was 3 times as large as that of ccKir2.1 (14.62±1.95 pS; P<0.05), but only 1.6 times as large as the conductance of the ccKir2.2 (27.54±5.3 pS; Fig. 5B). The three channels also had variable single-channel kinetics. The mean open time at –100 mV was 45.73±10.89, 22.93±9.11 and 9.21±1.63 ms for ccKir2.5, ccKir2.2 and Kir2.1, respectively. Furthermore, ccKir2.5 had 4.17 times as high an open probability as ccKir2.2 (0.401±0.056 vs 0.096±0.005; P<0.05; Fig. 5E), and is therefore likely to contribute more to the whole-cell IK1 than ccKir2.2. Taken together, the functional properties of ccKir2.5 are strikingly different from those of ccKir2.1 and closer to, although still distinct from, those of the ccKir2.2.
|
|
ccKir2.5 and ccKir2.2 were clearly the main ccKir2 subunits in the crucian carp heart, forming together over 99% of all ccKir2 transcripts. ccKir2.1 accounted for less than 0.8% of the transcripts, which was considered to be physiologically insignificant. Thermal acclimation for 4 weeks had a striking impact on ccKir2 mRNA expression. In cold-acclimated fish, ccKir2.5 was the dominating isoform representing 59.1±2.1% and 65.6±3.2% of the total ccKir2 transcripts in atrium and ventricle, respectively. In contrast, in warm-acclimated fish hearts ccKir2.2 was the main component, accounting for 83.6±1.6% of atrial and 77.7±1.7% of ventricular Kir2 mRNA, respectively (Fig. 6). These findings indicate that in cold-acclimated carp the major part of the ccKir2.2 transcripts is replaced by ccKir2.5, suggesting that ccKir2.5 is the cold-adapted isoform and is probably important for acclimation of the heart to low temperatures. Interestingly, the total amount of ccKir2 transcripts was slightly higher in warm- than cold-acclimated crucian carp heart, both in atrium and ventricle (P<0.05).
Ba2+ sensitivity of IK1 was similar in cold- and warm-acclimated fish, which is not unexpected considering the similar Ba2+ sensitivities of ccKir2.5 and ccKir2.2 (Fig. 4B). The voltage dependence of inward rectification of IK1 was also similar in cold- and warm-acclimated carp ventricular myocytes (–79.60±0.85 vs –81.74±1.72), but IK1 had a slightly shallower slope of rectification (z=2.5±0.1 vs 3.0±0.1; P<0.05) in cold- than warm-acclimated fish. The rectification properties of IK1 from cold- and warm-acclimated fish do not conform well to the rectification characteristics of the cloned ccKir2 channels and the transcript levels of ccKir2.5 and ccKir2.2 in ventricular myocytes, suggesting that temperature acclimation must also modify the regulation of Kir2 channel function.
Single-channel currents with similar characteristics to the currents of
cloned ccKir2 channels were observed in crucian carp ventricular myocytes
(Fig. 5)
(Paajanen and Vornanen, 2003).
Channels with ccKir2.5 characteristics (the slow IK1) were
found in 8 out of 19 and 1 out of 16 cell-attached patches from cold- and
warm-acclimated fish, respectively, whereas ccKir2.2-type channels (the fast
IK1) were found in 9 out of 19 and 15 out of 16
cell-attached patches from cold- and warm-acclimated fish, respectively. These
frequencies are in line with mRNA expression levels of ccKir2.5 and ccKir2.2
in warm- and cold-acclimated fish. Single-channel amplitudes and mean open
times of slow IK1 and fast IK1
channels of ventricular myocytes correspond well with the values of cloned
ccKir2.5 and ccKir2.2 channels (Fig.
5D,E), suggesting that they are composed of ccKir2.5 and ccKir2.2
subunits, respectively. Although Kir2.5 channels were more frequent in cold-
than warm-acclimated fish hearts, the mean conductance of the endogenous Kir2
channels did not differ between acclimation groups (P>0.05;
Fig. 5B). A small amplitude
(14 pS) current (small IK1) was found only in a few
cell-attached patches (Fig.
5C). Similarly, the probability of finding a small amplitude
channel was low in inside-out patches (in 6 out of 100 and 5 out of 52 patches
from cold- and warm-acclimated fish, respectively). These findings suggest
that the small ccKir2.1-like channels have low expression levels in
ventricular myocytes of cold- and warm-acclimated crucian carp.
|
DISCUSSION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
;
Lopatin and Nichols, 2001). In
mammalian hearts, this task is accomplished by Kir2.1, Kir2.2 and Kir2.3
channels, Kir2.1 being the dominant isoform
(Lopatin and Nichols, 2001;
Liu et al., 2001;
Wang et al., 1998). We have
shown here that in crucian carp ventricular myocytes too, three
inward-rectifier channels are involved in the formation of
IK1, two of them being homologues of the known mammalian
cardiac Kir2.1 and Kir2.2 channels. The third gene of the crucian carp cardiac
Kir assembly seems to belong to the Kir2 subfamily, even though it is
not clearly homologous to any of the known Kir2 genes, and
accordingly is proposed to be a new, fifth member for the vertebrate
Kir2 subfamily, ccKir2.5. Indeed, the phylogenetic analysis
showed that ccKir2.5 has a common ancestor with all four known
Kir2 genes (Kir2.1, Kir2.2, Kir2.3, Kir2.4), thus providing
firm evidence that it belongs to the Kir2 subfamily. Interestingly, a
predicted protein (XM_001335914) sharing 98.3% similarity with ccKir2.5 was
found from the zebrafish sequence database, suggesting the Kir2.5 channels
might be more generally expressed in different fish species. In contrast, no
homologues of ccKir2.5 were found in mammalian genomes. Thus, it is
likely that the novel ccKir2.5 is unique to ectothermic
vertebrates.
All vertebrate Kir2 channels rectify strongly due to a high-affinity
voltage-dependent block by free polyamines and Mg2+, and thus in a
given cell type the magnitude of the physiologically important outward current
is primarily determined by the sensitivity of Kir2 channels to polyamines
(Dhamoon et al., 2004;
Panama and Lopatin, 2006).
Although all three cardiac ccKir2 channels of the crucian carp heart have a
distinct negative slope conductance and they completely rectify at 0 mV,
notable differences exist between the channel isoforms in terms of inward
rectification. ccKir2.5 is clearly the strongest inward rectifier followed by
ccKir2.2 and ccKir2.1. Among the mammalian cardiac Kir2 channels, Kir2.1 is
the weakest and Kir2.2 the strongest inward rectifier
(Dhamoon et al., 2004;
Panama and Lopatin, 2006).
Kir2.3 is intermediate between Kir2.1 and Kir2.2, but it rectifies
incompletely. Evidently, ccKir2.5 is the strongest inward-rectifier
K+ channel of the vertebrate heart and passes little outward
current. Consequently, its membrane potential stabilising and repolarising
effects are relatively weak.
The present findings indicate that under chronic thermal stress the
phenotype of crucian carp cardiac IK1 is changed by a
compensatory increase in the density of IK1 in the cold.
In the chronic cold, ccKir2.5 transcripts were strongly upregulated,
suggesting that the ccKir2.5 isoform might be important in producing the
cold-acclimated phenotype of the cardiac IK1.
Concomitantly with the increased ccKir2.5 expression, transcripts of ccKir2.2
were strongly suppressed, suggesting the possibility that the cold-induced
increase in the cardiac IK1 was obtained by an isoform
shift from ccKir2.2 towards ccKir2.5. But how is the cold-induced increase in
IK1 achieved by the strongly rectifying ccKir2.5 channels,
especially when the total amount of ccKir2 transcripts is simultaneously
reduced? Two single-channel properties of ccKir2.5 could be contributing.
First, open probability and mean open time of ccKir2.5 are 4.17 and 4.96
times, respectively, as large as those of ccKir2.2. Second, ccKir2.5 has a
larger single-channel conductance than ccKir2.2. Thus, in spite of the strong
voltage-dependent block by polyamines, ccKir2.5 channels might allow more
outward current than ccKir2.2 channels, mainly because they stay longer in the
open state and because they have a larger conductance than ccKir2.2 channels.
While the larger slope conductance of IK1 in
cold-acclimated crucian carp is consistent with this, the rectification of the
endogenous IK1 does not match the rectification properties
of the cloned ccKir2.2 and ccKir2.5 channels and the expression levels of
these channels in cold- and warm-acclimated fish. Rectification of
IK1 in cold-acclimated fish is weaker than would be
assumed on the basis of the high ccKir2.5 expression level. Therefore, other
mechanisms in addition to ccKir2 isoform change must be involved in
temperature-dependent regulation of the crucian carp cardiac
IK1. The present results do not provide an explanation for
the divergence in rectification properties between cloned and endogenous
currents, but it can be speculated that in cardiac myocytes ccKir2.2 and
ccKir2.5 channels are located in different membrane compartments and therefore
might face different free polyamine concentrations and/or compositions. A
different distribution of Kir2.1 and Kir2.3 channels between cholesterol-rich
and cholesterol-poor membrane domains has recently been suggested
(Tikku et al., 2007), raising
the possibly that regulation of Kir2 isoforms may differ depending on their
subcellular location.
Although isoform change of ccKir2 channels does not alone explain
temperature-induced changes in the IK1 of the crucian carp
heart, it probably has a central role in thermal modification of the IK1.
Recently, we showed that Kir2.2 is upregulated by warm acclimation in another
fish species, the rainbow trout (Hassinen
et al., 2007). Together these studies suggest that
temperature-induced changes in Kir2 isoforms may be a common way for
temperature acclimation of the cardiac IK1 in fish.
Evidently, ccKir2.2 is a warm-adapted and ccKir2.5 a cold-adapted isoform of
the cardiac inward-rectifying K+ channel and the cardiac phenotype
of IK1 is determined by their relative abundance.
It is interesting that temperature compensation of IK1
density was produced by an isoform shift and not by parallel upregulation of
all three Kir2 channel isoforms in the cold. The potential benefit of isoform
switching in comparison to a simple increase in the number of the ccKir2
population remains unexplained, especially since the electrophysiological
properties of ccKir2.2 and ccKir2.5 do not radically differ (with the
exception of mean open time and open probability). It can be speculated that
there might be some constraints on genome function that would prevent
upregulation of ccKir2.2 and ccKir2.1 isoforms at low temperatures. In this
regard it is interesting that rainbow trout heart, which responds to constant
cold by a decrease in the density of the IK1, does not
express Kir2.5 (Vornanen et al.,
2002; Hassinen at al.,
2007). Thus, it is possible that cold-induced compensation of
cardiac IK1 is dependent on the expression of the Kir2.5
isoform, and species that lack Kir2.5 or cannot express it in the heart are
unable to upregulate IK1 in the cold. Future research
should find out how widely the novel Kir2.5 isoform is distributed among fish
species and other ectotherms, and to clarify its importance in thermal
adaptation of heart and muscle tissues.
In conclusion, a new member for the Kir2 subfamily of the inward-rectifying K+ channels has been isolated and characterised from the heart of crucian carp. Increased expression of the novel ccKir2.5 channel, at the expense of the ccKir2.2 isoform, is assumed to contribute to the cold-induced increase of atrial and ventricular IK1, which partly compensates for the depressive effects of low temperature on the current that maintains negative resting membrane potential and accelerates the phase-3 repolarisation of the cardiac action potential. These electrophysiological effects may be necessary to limit action potential prolongation and stabilise membrane potential to prevent cardiac arrhythmias in the cold winter waters.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Alagem, N., Dvir, M. and Reuveny, E. (2001).
Mechanism of Ba2+ block of a mouse inwardly rectifying
K+ channel: differential contribution by two discrete residues.
J. Physiol. 534,381
-393.
Dhamoon, A. S., Pandit, S. V., Sarmast, F., Parisian, K. R.,
Guha, P., Li, Y., Bagwe, S., Taffet, S. M. and Anumonwo, J. M. B.
(2004). Unique Kir2.x properties determine regional and species
differences in the cardiac inward rectifier K+ current.
Circ. Res. 94,1332
-1339.
Fakler, B., Brändle, U., Glowatzki, E., Weidemann, S., Zenner, H.-P. and Ruppersberg, J. P. (1995). Strong voltage-dependent inward rectification of inward rectifier K+ channels caused by intracellular spermine. Cell 80,149 -154.[CrossRef][Medline]
Ficker, E., Taglialatela, M., Wible, B. A., Henley, C. M. and
Brown, A. M. (1994). Spermine and sermidine as gating
molecules for inward rectifier K+ channels.
Science 266,1068
-1072.
Fujiwara, Y. and Kubo, Y. (2002). Ser165 in the
second transmembrane region of the Kir2.1 channel determines its
susceptibility to blockade by intracellular Mg2+. J.
Gen. Physiol. 120,677
-692.
Hassinen, M., Paajanen, V., Haverinen, J., Eronen, H. and Vornanen, M. (2007). Cloning and expression of cardiac Kir2.1 and Kir2.2 channels in thermally acclimated rainbow trout. Am. J. Physiol. 292,R2328 -R2339.
Haverinen, J., Hassinen, M. and Vornanen, M. (2007). Fish cardiac sodium channels are tetrodotoxin sensitive. Acta Physiol. 191,197 -204.[CrossRef]
Horoszewicz, L. (1973). Lethal and `disturbing' temperatures in some fish species from lakes with normal and artificially elevated temperature. J. Fish Biol. 5, 165-181.[CrossRef]
Hume, J. R. and Uehara, A. (1985). Ionic basis
of the different action potential configurations of single-guinea-pig atrial
and ventricular myocytes. J. Physiol.
368,525
-544.
Ishihara, K. and Yan, D. H. (2007).
Low-affinity spermine block mediating outward currents through Kir2.1 and
Kir2.2 inward rectifier potassium channels. J.
Physiol. 583,891
-908.
Kurata, H. T., Marton, L. J. and Nichols, C. G.
(2006). The polyamine binding site in inward rectifier
K+ channels. J. Gen. Physiol.
127,467
-480.
Liu, G. X., Derst, C., Schlichthörl, G., Heinen, S.,
Seebohm, G., Brüggemann, A., Kummer, W., Veh, R. W., Daut, J. and
Preisig-Müller, R. (2001). Comparison of cloned Kir2
channels with native inward rectifier K+ channels from guinea-pig
cardiomyocytes. J. Physiol.
532,115
-126.
Liu, X.-S., Jiang, M., Zhang, M., Tang, D., Clemo, H. F., Higgins, R. S. D. and Tseng, G.-N. (2007). Electrical remodeling in a canine model of ischemic cardiomyopathy. Am. J. Physiol. 292,H560 -H571.
Lopatin, A. N. and Nichols, C. G. (2001). Inward rectifiers in the heart: an update on IK1. J. Mol. Cell. Cardiol. 33,625 -638.[CrossRef][Medline]
Lopatin, A. N., Makhina, E. N. and Nichols, C. G. (1994). Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectifcation. Nature 372,366 -369.[CrossRef][Medline]
Matsuda, H., Saigusa, A. and Irisawa, H. (1987). Ohmic conductance through the inwardly rectifying K channel and blocking by internal Mg2+. Nature 325,156 -159.[CrossRef][Medline]
Melnyk, P., Zhang, L., Shrier, A. and Nattel, S. (2002). Differential distribution of Kir2.1 and Kir2.3 subunits in canine atrium and ventricle. Am. J. Physiol. 283,H1123 -H1133.
Paajanen, V. and Vornanen, M. (2003). Effects of chronic hypoxia on inward rectifier K+ current (IK1) in ventricular myocytes of crucian carp (Carassius carassius) heart. J. Membr. Biol. 194,119 -127.[CrossRef][Medline]
Paajanen, V. and Vornanen, M. (2004). Regulation of action potential duration under acute heat stress by IK,ATP and IK1 in fish cardiac myocytes. Am. J. Physiol. 286,R405 -R415.
Panama, B. K. and Lopatin, A. N. (2006).
Differential polyamine sensitivity in inwardly rectifying Kir2 potassium
channels. J. Physiol.
571,287
-302.
Périer, F., Radeke, C. M. and Vandenberg, C. A.
(1994). Primary structure and characterization of a
small-conductance inwardly rectifying potassium channel from human
hippocampus. Proc. Natl. Acad. Sci. USA
91,6240
-6244.
Piao, L., Li, J., McLerie, M. and Lopatin, A. N. (2007). Cardiac IK1 underlies early action potential shortening during hypoxia in the mouse heart. J. Mol. Cell. Cardiol. 43,27 -38.[CrossRef][Medline]
Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canún, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R. et al. (2001). Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome. Cell 105,511 -519.[CrossRef][Medline]
Preisig-Müller, R., Schlichthörl, G., Goerge, T.,
Heinen, S., Brüggemann, A., Rajan, S., Derst, C., Veh, R. W. and Daut,
J. (2002). Heteromerization of Kir2.x potassium channels
contributes to the phenotype of Andersen's syndrome. Proc. Natl.
Acad. Sci. USA 99,7774
-7779.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Analysis and cloning of eukaryotic genomic DNA. InMolecular cloning: A Laboratory Manual. 2nd edn , pp.9.14 -9.19. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Shimoni, Y., Clark, R. B. and Giles, W. R.
(1992). Role of an inwardly rectifying potassium current in
rabbit ventricular action potential. J. Physiol.
448,709
-727.
Takahashi, N., Morishige, K.-I., Jahangir, A., Yamada, M.,
Findlay, I., Koyama, H. and Kurachi, Y. (1994). Molecular
cloning and functional expression of cDNA encoding a second class of inward
rectifier potassium channels in the mouse brain. J. Biol.
Chem. 269,23274
-23279.
Ten Eick, R. E., Whalley, D. W. and Rasmussen, H. H. (1992). Connections: heart disease, cellular electrophysiology, and ion channels. FASEB J. 6,2568 -2580.[Abstract]
Tikku, S., Epshtein, Y., Collins, H., Travis, A. J., Rothblat, G. H. and Levitan, I. (2007). Relationship between Kir2.1/Kir2.3 activity and their distributions between cholesterol-rich and cholesterol-poor membrane domains. Am. J. Physiol. 293,C440 -C450.[CrossRef]
Vandenberg, C. A. (1987). Inward rectification
of a potassium channel in cardiac ventricular cells depends on internal
magnesium ions. Proc. Natl. Acad. Sci. USA
84,2560
-2564.
Vornanen, M. (1997). Sarcolemmal Ca influx through L-type Ca channels in ventricular myocytes of a teleost fish. Am. J. Physiol. 272,R1432 -R1440.[Medline]
Vornanen, M. and Paajanen, V. (2004). Seasonality of dihydropyridine receptor binding in the heart of an anoxia-tolerant vertebrate, the crucian carp (Carassius carassius L.). Am. J. Physiol. 287,R1263 -R1269.
Vornanen, M., Ryökkynen, A. and Nurmi, A. (2002). Temperature-dependent expression of sarcolemmal K+ currents in rainbow trout atrial and ventricular myocytes. Am. J. Physiol. 282,R1191 -R1199.
Vornanen, M., Hassinen, M., Koskinen, H. and Krasnov, A. (2005). Steady-state effects of temperature acclimation on the transcriptome of the rainbow trout heart. Am. J. Physiol. 289,R1177 -R1184.
Wang, Z., Yue, L., White, M., Pelletier, G. and Nattel, S.
(1998). Differential distribution of inward rectifier potassium
channel transcripts in human atrium versus ventricle.
Circulation 98,2422
-2428.
Xie, L.-H., John, S. A. and Weiss, J. N.
(2003). Inward rectification by polyamines in mouse Kir2.1
channels: synergy between blocking components. J.
Physiol. 550,67
-82.
Zaritsky, J. J., Redell, J. B., Tempel, B. L. and Schwarz, T.
L. (2001). The consequences of disrupting cardiac inwardly
rectifying K+ current (IK1) as revealed by the targeted
deletion of the murine Kir2.1 and Kir2.2 genes. J.
Physiol. 533,697
-710.
Zobel, C., Cho, H. C., Nguyen, T.-T., Pekhletski, R., Diaz, R.
J., Wilson, G. J. and Backx, P. H. (2003). Molecular
dissection of the inward rectifier potassium current (IK1) in
rabbit cardiomyocytes: evidence for heteromeric co-assembly of Kir2.
1 and Kir2. 2. J. Physiol.
550,365
-372.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
o) and
open probability (Po).