© 2001 American Heart Association, Inc.
Cellular Biology |
Heterogeneity of Kv2.1 mRNA Expression and Delayed Rectifier Current in Single Isolated Myocytes From Rat Left Ventricle
From the Institut für Physiologie, Universität Hamburg, and Institut für Physiologie und Pathophysiologie, Ruprecht-Karls-Universität, Heidelberg, Germany.
Correspondence to Prof Dr Heimo Ehmke, Institut für Physiologie, Universität Hamburg, Martinistrasse 52, 20246 Hamburg, Germany. E-mail ehmke{at}uke.uni-hamburg.de
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Abstract |
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Abstract—Expression of the voltage-gated K+ channel Kv2.1, a possible molecular correlate for the cardiac delayed rectifier current (IK), has recently been shown to vary between individual ventricular myocytes. The functional consequences of this cell-to-cell heterogeneity in Kv2.1 expression are not known. Using multiplex single-cell reverse transcriptase–polymerase chain reaction (RT-PCR), we detected Kv2.1 mRNA in 47% of isolated midmyocardial myocytes from the rat left ventricular free wall that were positive for
-myosin
heavy chain mRNA (n=74). Whole-cell patch-clamp recordings demonstrated
marked differences in the magnitude of
IK (200
to 1450 pA at
VPip=40
mV) between individual myocytes of the same origin. Furthermore, the
tetraethylammonium (TEA)–sensitive outward current
(ITEA),
known to be partly encoded by Kv2.1 in mice, revealed a wide range of
current magnitudes between single cells (150 to 1130 pA at
VPip=40
mV). Combined patch-clamp recordings and multiplex single-cell RT-PCR
analysis of the same myocytes, however, showed no differences in
IK or
ITEA
magnitude or inactivation kinetics between myocytes expressing Kv2.1
mRNA and those that did not express Kv2.1 mRNA. In contrast, in all
midmyocardial myocytes expressing the transient outward potassium
current
(Ito1),
Kv4 mRNA, which has been shown to underlie
Ito1,
was detected (n=10). These results indicate that
IK
heterogeneity among individual left ventricular myocytes cannot be
explained by the distribution pattern of Kv2.1 mRNA. Other mechanisms
besides Kv2.1 mRNA expression appear to determine magnitude and
kinetics of
IK in
rat ventricular
myocytes.
Key Words: voltage-gated K+ channels • single-cell reverse transcriptase–polymerase chain reaction • Kv2.1 mRNA expression • K+ currents • delayed rectifier
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Depolarization-activated Ca2+-independent outward K+ currents recorded from rat ventricular myocytes have been separated into 2 components, as follows1 : first, a transient outward current (Ito1) that activates and inactivates rapidly and is inhibited by millimolar concentrations of 4-aminopyridine (4-AP), and second, a delayed rectifier current (IK) that also activates rapidly but inactivates slowly and is blocked by millimolar concentrations of tetraethylammonium (TEA). Recent work has suggested that IK does not represent a uniform entity but is composed of at least 3 pharmacologically and kinetically different components.2 Similarly, the molecular correlate of IK has not been clearly determined. Possible candidates for IK are members of the Kv1, Kv2, and Kv3 gene families (Kv1.2, Kv1.5, Kv2.1, Kv2.2, Kv3.1, and Kv3.2). These
subunits have all been detected at
significant levels in rat left
ventricle3 and share
functional characteristics with the native
IK when
expressed in heterologous
systems.4 5 6 7
Recent investigations of transgenic mice expressing a dominant negative
Kv2.1 subunit showed a reduction of the inactivating component
(IKslow)
of IK in
left ventricular myocytes.8
These observations strongly suggest that Kv2.1 contributes to
IK. K+ channels are not uniformly distributed in the heart (for review, see Nerbonne9 ). Extensive diversity in K+ channel gene expression as well as in K+ currents has been identified between atria and ventricles, but also in circumscribed regions such as the left ventricular free wall, where Ito1 and its underlying genes are differentially expressed between endo- and epicardial regions.3 10 11 12 Furthermore, K+ channel gene expression may even vary from cell to cell. Using in situ hybridization on single isolated myocytes of the ferret left ventricle, Brahmajothi et al13 detected Kv2.1 mRNA in only 65% and Kv2.2 mRNA in only 15% of individual myocytes. The present study was undertaken to further characterize possible cell-to-cell differences in Kv2 channel gene expression and its functional consequences by combining patch-clamp experiments with single-cell reverse transcriptase–polymerase chain reaction (RT-PCR) analysis in isolated rat left ventricular myocytes.
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Materials and Methods |
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Isolation of Myocytes
Female Wistar rats weighing 180 to 240 g were anesthetized by IP injection of Trapanal (thiopental-sodium, Byk Gulden), 100 mg/kg body mass. The heart was quickly excised and midmyocardial myocytes were isolated as described previously.14 Cells were stored at 22°C to 26°C in modified Tyrode’s solution containing 100 µmol/L Ca2+. Only single rod-shaped cells with clear cross striations and no spontaneous contractions were used for experiments.
Patch-Clamp Technique
The ruptured-patch whole-cell configuration was used
as described
previously.12 14 15
Myocytes were transferred to an elongated chamber (2.5x20 mm) mounted
on the stage of an inverted microscope (Axiovert 25, Zeiss). Patch
pipettes were pulled from borosilicate glass (GC150-15, Clark
Electromedical Instruments) using a P-87 Puller (Sutter Instruments).
Currents were recorded using an EPC-9 amplifier (HEKA Elektronik)
controlled by a Power-Macintosh computer (Apple Computers) and the
Pulse Software (HEKA Elektronik). Membrane capacitance
(Cm) and
series resistance
(Rs)
were calculated using the automated capacitance compensation procedure
of the EPC-9 amplifier. When filled with potassium glutamate, pipette
resistance averaged 3.9±0.1 M
(n=129).
Rs
averaged 6.2±0.2 M (n=130) and was compensated by 85% leading to
an average effective
Rs of
0.9 M.
Cm
averaged 140±3 pF (n=129). Pipette potential
(VPip)
and membrane potential
(Vm)
were corrected for a liquid junction potential (13.3 mV for pipette
solution versus control solution). Whole-cell data were analyzed using
the PulseFit software (HEKA Elektronik) and IGOR (WaveMetrics). Data
are given as mean±SEM unless stated otherwise. Statistical
significance was calculated using the appropriate version of the
Student t test using the
software PRISM (Graph-Pad Inc). Differences with
P<0.05 were considered
statistically significant. All experiments were performed at room
temperature (22°C to 26°C).
Solutions and Chemicals
Seals (G) were obtained in modified Tyrode’s
solution (control solution), containing (in mmol/L) NaCl 138, KCl 4,
MgCl2 1,
NaH2PO4 0.33,
CaCl2 2, glucose 10, and HEPES 10, titrated to
pH 7.30 with NaOH. To inhibit Ca2+ currents,
CdCl2 0.3 mmol/L was added.
Cd2+ slightly increased (
10%, n=8) the
slowly inactivating component of
IK but
had no effect on its TEA-sensitive component (n=23, data not shown).
TEA (10 mmol/L) was added to control solution to estimate the
TEA-sensitive current. The pipette solution contained (in mmol/L)
glutamic acid 120, KCl 10, MgCl2 2, EGTA 10,
Na2-ATP 2, and HEPES 10, titrated to pH 7.20
with KOH.
For cDNA synthesis the following reagents and solutions were used: 200 U/µL reverse transcriptase Moloney murine leukemia virus (GIBCO), 40 U/µL ribonuclease inhibitor (Promega), 100 mmol/L DTT, and 5x buffer-primer-dNTP mix (25 µmol/L hexamer random primers [Boehringer] and 2.5 mmol/L of each deoxyribonucleotide [Pharmacia] in 10 mmol/L Tris-HCL [pH 8.3], 1.5 mmol/L MgCl2, and 0.1 mol/L diethylpyrocarbonate [DEPC]). For PCR amplification the following solutions were used (in mmol/L): 10x buffer (Tris-HCl 200 ([pH 8.3] and KCl 500; GIBCO), MgCl2 50, and dNTP mix 10, and 5 U/µL Taq DNA polymerase (GIBCO).
Single-Cell RT-PCR
Under visual control (using 100x magnification), a
single myocyte was sucked into a micropipette (tip opening 30 µm)
filled with 6 µL of 0.1 mol/L DEPC. The pipette content was ejected
into a reaction cup (200-µL cups) and after short centrifugation the
following (in µL) were added, up to a total volume of 10 µL: RNase
inhibitor 0.5, buffer/primer/dNTP mix 2, DTT 1, and reverse
transcriptase 0.5 (for stock solution concentrations see Solutions and
Chemicals, above). After reverse transcription at 37°C for 60 minutes
in a water bath, the cup was stored at -20°C until PCR
amplification.
Single-cell RT-PCR (PE-2400, Applied Biosystems) was performed as described previously.16 17 The cDNA reaction (10 µL) was used directly for PCR amplification. For each PCR reaction, the following (in µL) were mixed and filled up to a reaction volume of 100 µL: 10x buffer 10, MgCl2 solution 3, dNTP mix 3, each amplification primer (10 µmol/L solution) 6, and Taq DNA polymerase 0.5 (see Solutions and Chemicals). The cycle conditions were as follows: 94°C for 5 minutes, hot start, 35 step cycles (94°C for 30 seconds, 57°C for 30 seconds, and 68°C for 40 seconds), and 68°C for 7 minutes. Subsequently, 2 consecutive rounds of PCR with heminested primer pairs were performed. Degenerate primer pairs were used to amplify both members of the Kv2 family in the same reaction. The template for the second PCR was 1 µL derived from the first PCR. For each round of PCR, amplification controls for possible contamination were performed. If controls were positive (<10% of all amplifications), the complete RT-PCR analysis was discarded. To verify specificity of amplification, PCR products were probed by sequencing after subcloning or Southern blotting with radiolabeled oligonucleotides. Primer pairs were intron-overspanning to prevent amplification of genomic DNA. Positive controls for primer efficiency were run using plasmids at several dilutions (down to 0.1 fg plasmid DNA). After 2 rounds of PCR (35 cycles each), Kv2.1 and Kv2.2 mRNAs were amplified at similar efficiency. Sequences and locations of the following primers for Kv2.1 (X16476) and Kv2.2 (M77482) refer to sequences and locations published in GenBank (National Center for Biotechnology Information, available at http://www.ncbi.nlm.nih.gov): upper primer, 5'-TACTGGGGCATCGATGAGA-3' (372–394 and 701–723); lower primer, 5'-CC(GT)(GA)AGGATGCGCATGAT-3' (104–1126 and 1443–1465); and lower nested primer, 5'-CGAAAGATCTGGACCACGCG-3' (889–908 and 1218–1237). Oligonucleotides for Southern blotting were the following: Kv2.1, 5'-CTCTGGCCGAACTCGTCTAGGCTC-3' (654–677), and Kv2.2, 5'-GGTTGCCCAAATTCATCGTTTTCT-3' (983–1006). The washing conditions were 0.5x SSC at 55°C. To determine whether the fraction of Kv2 mRNA–positive cells declines as a function of time after cell isolation, single-cell RT-PCR was performed immediately (<1 hour) and 8 to 10 hours after cell isolation. Kv2 mRNA expression rates were similar in both groups (0 to 1 hour, 40% [n=30]; 8 to 10 hours, 40% [n=40]).
Multiplex Single-Cell RT-PCR
For multiplex single-cell RT-PCR, an
intron-overspanning primer pair specific for -myosin heavy chain
(-MHC) was added to the reaction cup in addition to the primers for
the Kv2 subfamily (1 µL of each primer out of 10 µm/L solution).
Conditions for the first round of PCR were identical to those described
above for single-cell RT-PCR. The second round of PCR amplification was
performed separately for Kv2 and -MHC with cycling conditions as
described above. Primers for -MHC (KO1464) were the following: upper
primer, 5'-AGCCCCATACCTCCGCAAGT-3' (1624–1644); lower primer,
5'-TTGACGGTGACACAGAAGAGGC-3' (3269–3277); and upper nested primer,
5'-CCTTTGACATCCGCACAGAGT-3' (1678–1698).
To estimate the detection threshold of the multiplex
single-cell RT-PCR, we performed control reactions using cloned Kv2.1,
Kv2.2, and -MHC plasmids. After mixing in one reaction cup, all
genes were consistently amplified from an amount of 0.1 fg, which
corresponds to <10 copies of each transcript. A similar sensitivity of
single-cell RT-PCR has been reported
previously.16 17
To exclude a possible amplification of mRNA fragments from the
surrounding bath solution, tissue components of myocytes were analyzed
by single-cell RT-PCR under identical conditions. All of these controls
gave negative results (n=8, data not shown).
Multiplex single-cell RT-PCR for Kv4 and -MHC was
performed under the same cycle conditions as described above.
Degenerate primer pairs were used to amplify all members of the Kv4
family (Kv4.1, Kv4.2, and Kv4.3) in the same reaction as described
previously.17 Primers for
Kv4.1 (M64226), Kv4.2 (MS64320), and Kv4.3 (U75448) were the following:
upper primer, 5'-T(CT)ATCGA(TC)GTG-GTGGCCATC-3' (797–816,
1342–1361, and 802–821); upper nested primer,
5'-TACAC(AC)CT(CG)AAGAGCTGTGC-3' (943–962, 1513–1532, and 954–973);
and lower primer,
5'-TGGTAGAT(CG)-C(GT)(AG)CT(AG)AAGTT-3'
(1228–1247, 1773–1792, and 1223–1242).
Combined Patch-Clamp Recordings and Single-Cell
RT-PCR
Using the whole-cell patch-clamp configuration,
depolarization-activated outward currents were recorded in the absence
and in the presence of TEA, which required 5 minutes. Subsequently,
a second pipette filled with DEPC-water (0.1 mol/L) was forwarded to
the myocyte. After flushing the cell and its surroundings with DEPC,
the myocyte was lifted from the bottom of the chamber by elevating the
patch pipette and then sucked into the second pipette. The amount of
the remaining DEPC was 6 µL. The content of the second pipette was
expelled into a reaction cup and transferred to multiplex single-cell
RT-PCR analysis.
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Results |
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Detection of Kv2 mRNA in Individual Myocytes
Figure 1A
shows the results of a single-cell RT-PCR
analysis performed on 6 individual myocytes. In 2 myocytes (2 and 3), a
PCR product with the expected length of 540 bp was amplified. In the
remaining myocytes as well as in the 2 controls (C1 and C2),
amplification yielded no detectable products. Overall, a PCR product
was amplified in 6 out of a total of 18 myocytes (33%) investigated.
Figures 1B and 1C display Southern blots of the single-cell
RT-PCR products shown in
Figure 1A using specific probes for Kv2.1
(Figure 1B) and Kv2.2
(Figure 1C). In both myocytes, only Kv2.1 mRNA was
identified. The same result was obtained in all other myocytes with a
positive PCR amplification for Kv2 mRNA.
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Multiplex Single-Cell RT-PCR
In 12 of 18 myocytes (67%), no Kv2-related mRNA could
be detected. A myocyte that yielded no amplification product by
single-cell RT-PCR could actually not express Kv2 channel genes;
alternatively, the amplification process might not have been
successful. We therefore decided to coamplify -MHC mRNA, which is
constitutively expressed in cardiac myocytes, as an internal control
for the process of cell transfer and amplification.
Figure 2 shows the results of a multiplex single-cell RT-PCR
analysis performed on 7 myocytes (lanes 1 through 7). Six of these
myocytes (lanes 1 through 4, 6, and 7) were positive for -MHC
(300 bp,
Figure 2A), indicating that the process of cell transfer and
amplification did work. In 3 of the myocytes positive for -MHC
(lanes 2, 3, and 6), a PCR product of the expected size for Kv2 cDNA
was detected and later identified as Kv2.1 by sequencing. In total, 74
of the 77 myocytes (96%) investigated were positive for -MHC. Kv2.1
mRNA was identified in 35 of the 74 myocytes positive for -MHC
(47%). Kv2.1 mRNA was not detected in myocytes negative for -MHC.
These data indicate that Kv2.1 mRNA is expressed in only 40% to 50%
of midmyocardial myocytes of the rat left ventricular free
wall.
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Pattern of Delayed Rectifier Current
To investigate whether the difference in Kv2.1 mRNA
expression between individual myocytes is associated with differences
in functional properties, we analyzed depolarization-activated outward
currents recorded from myocytes isolated from the same region as above.
Figure 3A shows current traces recorded by depolarizing
voltage steps from a holding potential of
VPip=-90
mV to potentials ranging from
VPip=+60
to -40 mV in steps of -20 mV. The initial phase of the current is
dominated by
Ito1.
After the inactivation of
Ito1,
ie, after 200 ms,
IK,
which inactivates much more slowly, becomes visible. The rat
IK has
recently been further divided into 3 components, as follows: a rapidly
inactivating component (
400 ms), a more slowly inactivating
component ( in the range of seconds), and a steady-state
component.2 In agreement with
that study, 3 inactivating components of the outward current could be
identified in most myocytes by fitting the current decay with the sum
of 3 exponential functions: a short time constant
(1) in the range of 40 ms, an intermediate
time constant (2) in the range of 300 ms, and
a long time constant (3) in the range of 2500
ms. 1 describes the decay of
Ito1,
2 may represent the rapidly inactivating
IK
component described by Himmel et
al,2 whereas
3 describes the more slowly inactivating
component of
IK. In
28% of the myocytes, only the current components inactivating with
1 and 3 could be
detected. Similar results have been obtained in ventricular myocytes of
mice, in which the depolarization-activated outward current was divided
into
Itof
(70 ms),
Itos
(200 ms, not present in all myocytes), and
IKslow
(1200 ms).18 It is
therefore likely that the current component inactivating with
3 corresponds to
IKslow
observed in mice. This current has been shown to be partially encoded
by Kv2.1.8 We therefore
further analyzed this component.
Figure 3B shows an average current-voltage relation of
recordings similar to those shown in
Figure 3A. Activation of the current started at -20 mV,
and the current magnitude increased linearly with depolarization.
Similar properties have been described for recombinant Kv2.1
heterologously expressed in
Xenopus
oocytes19 or in mammalian
cells.4
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If Kv2.1 contributed to
IK and
was present in only about half of the myocytes; as suggested by the
results above, one should expect a broad distribution of
IK
magnitudes. The average current-voltage relation shown in
Figure 3B
indeed reveals a large variation of
IK
magnitude among individual myocytes.
Figure 3C depicts a histogram that plots the number of
occurrences versus the magnitude of
IK at
VPip=40
mV. The histogram shows a broad distribution of
IK
magnitudes with no clear indication for separate populations of
myocytes. It is likely, however, that >1 current component is coding
for
IKslow,8
and it might well be that, in myocytes in which Kv2.1 mRNA is absent,
an increase of the remaining component(s) may compensate for at least
part of the current component normally encoded by Kv2.1. We therefore
determined the TEA-sensitive current component
(ITEA)
to further isolate Kv2 currents.
Figure 4A shows current traces recorded using the same
voltage-pulse protocol as applied in
Figure 3A. The recordings shown in
Figure 4B were obtained in the presence of 10 mmol/L TEA in
the bath solution.
IK was
markedly reduced, whereas
Ito1
remained unaffected by TEA.
Figure 4C depicts
ITEA,
calculated by subtracting the current traces shown in
Figure 4B from those shown in
Figure 4A.
Ito1 is
eliminated completely as a result of its insensitivity to TEA.
ITEA
activates rapidly and inactivates slowly within seconds, until a steady
state is reached. Inactivation of
ITEA
followed a monoexponential time course with an average inactivation
time constant of TEA=2157±86 ms
(VPip=40
mV; n=63). In some recordings, in which a part of
Ito1 had
escaped subtraction (probably because of an incompletely compensated
RS), the
current decay was fitted with a double-exponential function to exclude
a contribution of
Ito1.
The magnitude of
ITEA was
estimated as the current component ascribed to
TEA and averaged 540±27 pA
(VPip=40
mV, n=63). Because TEA was similar to
3 and
ITEA
reached 80% of the current that inactivated with
3, it is likely that
ITEA
largely underlies the slowly inactivating component of
IK.
Currents were intentionally not corrected for cell capacitance to allow
for a direct correlation with the presence of Kv2.1 mRNA expression in
individual myocytes, which cannot be corrected for cell size (see next
section).
Figure 4D shows the average current-voltage relation of
ITEA,
calculated from recordings similar to those shown in
Figure 4C. Similarly to
Figure 3B, the current activated at
VPip=-20
mV and increased rather linearly with depolarization.
Figure 4E depicts a histogram of the magnitudes of
ITEA.
ITEA was
distributed over a wide range of magnitudes from 150 to 1130 pA
(VPip=40
mV). Xu et al8 reported that
the inactivating component of
ITEA was
largely reduced in left ventricular myocytes isolated from mice
expressing a dominant negative mutant of Kv2.1. Accordingly, an
explanation for the wide range of
ITEA may
be that myocytes that express Kv2.1 mRNA display a large
ITEA,
whereas myocytes that do not express Kv2.1 mRNA have a small
ITEA.
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Mice expressing a dominant negative Kv2.1 subunit displayed
an accelerated inactivation of
IKslow.8
We therefore correlated the inactivation time constant of
ITEA
with its magnitude. This relation is depicted in
Figure 4F for a total of 63 myocytes. Linear regression
analysis revealed a weak but significant positive correlation
(P=0.045,
r2=0.064)
between
ITEA and
TEA. Thus, in some myocytes a faster
inactivation of
ITEA may
be explained by a lack of Kv2.1 expression.
Collectively, these data are consistent with the concept that expression of Kv2.1 mRNA in myocytes may cause a larger magnitude and a slower inactivation of IK.
Combined Patch-Clamp Recordings and Multiplex
Single-Cell RT-PCR
To test this hypothesis directly, we combined analysis
of current properties and Kv2 mRNA expression in the same myocytes.
ITEA was
estimated in the whole-cell configuration; thereafter, the myocyte was
sucked into a second pipette and transferred to multiplex single-cell
RT-PCR analysis for Kv2 mRNA (see Materials and Methods).
Figure 5 shows current recordings and gene expression in 2
representative ventricular myocytes.
ITEA was
similar and -MHC mRNA expression was detected in both myocytes, but
only the myocyte shown on the left expressed Kv2.1 mRNA. This combined
analysis was repeated in a total of 27 myocytes. -MHC mRNA
expression was detected in all myocytes investigated, whereas Kv2 mRNA
was found in 9 cells (33%).
Figure 6 compares the distribution of the magnitude and
inactivation of
ITEA in
both groups. Both variables were equally distributed in Kv2.1
mRNA–positive and Kv2.1 mRNA–negative myocytes
(ITEA,
576±62 versus 570±52 pA; TEA, 2494±203
versus 2439±171 ms). Note that
Cm
(Figure 6C) and the current ascribed to
3 of the total outward current (data not
shown) were also equally distributed in both groups. These data
indicate that the expression pattern of Kv2.1 mRNA among individual
myocytes does not account for the differences in
ITEA or
its inactivation time constant.
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) expressing Kv2.1 mRNA. B, Distribution of
To confirm that a direct correlation of ionic currents with
the expression of specific mRNAs in individual cardiac myocytes is
possible, we analyzed
Ito1 and
Kv4 mRNA expression, which has been shown to encode for
Ito1
(for review, see Reference 99 ). Multiplex single-cell RT-PCR amplifying
both Kv4 mRNA and -MHC mRNA revealed that 87% of myocytes positive
for -MHC also expressed Kv4. mRNA
Figure 7 displays the results of combined patch-clamp
recordings and multiplex single-cell RT-PCR analysis.
Ito1 was
present in all myocytes with a magnitude of 1140 to 5880 pA (estimated
as peak current-current after 600 ms at
VPip=40
mV), which is in good agreement with results we have previously
obtained in midmyocardial
myocytes.14 In all of the
myocytes investigated, PCR fragments of the expected size for Kv4 and
-MHC were amplified.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Methodological Limitations
In a substantial fraction of myocytes (
59%),
single-cell RT-PCR failed to detect Kv2 mRNA transcripts. The
successful amplification of -MHC in 96% of all myocytes revealed
that the process of cell transfer, reverse transcription, and PCR
amplification was successful. A difficulty of single-cell RT-PCR is
mRNA degradation by
RNases.20 Because the total
amount of -MHC mRNA is probably larger than that of Kv2 mRNA, the
latter may become degraded to an extent that does not allow its
detection by single-cell RT-PCR, whereas -MHC mRNA is still
detectable. This might lead to an underestimation of Kv2 mRNA–positive
myocytes. If this were the case, however, the number of Kv2
mRNA–positive myocytes should be reduced considerably further in
experiments in which single-cell RT-PCR was preceded by
electrophysiological measurements, because in these experiments the
procedure of cell harvesting was substantially prolonged. The finding
of similar fractions of Kv2 mRNA–positive myocytes in all experimental
series suggests that mRNA degradation does not account for the
heterogeneity of Kv2 mRNA expression among left ventricular myocytes.
Furthermore, our data are in agreement with previous results obtained
from ferret left ventricular myocytes using in situ
hybridization.13 Thus, Kv2.1
mRNA appears to be present in only 40% to 50% of midmyocardial
myocytes from the rat left ventricular free wall.
Identification of Kv2.1 Currents
When expressed in heterologous systems, Kv2.1 currents
are activated by depolarizing voltage steps exceeding
Vm=-20
mV; display a linear current-voltage
relationship4 19 ;
and are sensitive to several substances such as
TEA,21
4-AP,4
hanatoxins,22 and
propafenone.23
Unfortunately, Kv2.1 shares these properties with several other Kv
channels expressed in the mammalian heart. Further candidates possibly
underlying
IK, such
as Kv1.5, Kv3.1, and Kv3.2 are also inhibited by
TEA.24 25 Members
of the Kv4 family, which probably underlie
Ito1,
are also inhibited by 4-AP and propafenone at similar concentrations as
Kv2.1,23 and hanatoxins are
known to block
Kv4.2.22
In rat left ventricular myocytes, we have found
ITEA to
share many properties with heterologously expressed Kv2.1 currents; the
current-voltage relation and inactivation time constants were similar,
and 75% of the current component inactivating with the slow time
constant was inhibited by 10 mmol/L TEA, which would also be expected
for Kv2.1 currents
(Kd4
mmol/L).21 26 In
an independent series of experiments, we have found steady-state
inactivation and recovery from inactivation of
ITEA to
be in the range observed for Kv2 currents (data not shown). Most
importantly, the inactivating component of
ITEA has
been reported to be absent in mice expressing a dominant negative Kv2.1
subunit.8 However, because
TEA also inhibits other ionic currents present in cardiac myocytes,
which share kinetic properties with Kv2, we cannot exclude that
ITEA may
also contain current components other than Kv2.
Discordance Between Kv2.1 mRNA Expression
Pattern and
ITEA
Magnitude
Although it has been shown in mice that
ITEA is
significantly altered in ventricular myocytes expressing a dominant
negative Kv2.1 subunit,8 we
could not detect a correlation between magnitude or kinetics of
ITEA and
the expression of Kv2.1 mRNA in the present study. Several factors
might account for this discrepancy. The presence of Kv2.1 mRNA might
not reflect the amount of the corresponding protein present in the cell
membrane. In neurons, however, the method of combined
electrophysiological characterization and single-cell RT-PCR has
repeatedly and successfully been used to ascribe specific electrical
behavior to the presence or absence of ion
channels.16 17
Furthermore, we could demonstrate the presence of both
Ito1 and
Kv4 mRNA in all 10 myocytes in which a combined analysis of
Ito1 and
Kv4 mRNA was achieved. This suggests that also in cardiac myocytes a
relation of channel mRNA and corresponding ionic current is likely. We
cannot exclude, however, that protein turnover of Kv2.1 in cardiac
myocytes is very slow and thus might lead to an mRNA level too low to
be detected by single-cell RT-PCR even in myocytes expressing a large
IK.
Another possibility is that in rat left ventricular myocytes Kv2.1 mRNA
is expressed at levels too low to significantly contribute to
IK. In
view of the demonstration of significant Kv2 expression levels in the
rat left ventricle,3 however,
this seems to be unlikely. Thus, the large difference in
ITEA
between individual rat midmyocardial myocytes may rather be ascribed to
factors other than Kv2.1 mRNA expression. Another delayed rectifier
component encoded by Kv1.5, Kv3.1, or Kv3.2, or an as-yet-unidentified
subunit, could vary in magnitude or could account for a larger
fraction of total
IK, thus
making it impossible to identify the contribution of Kv2.1 mRNA to
IK
magnitude. Alternatively, intracellular signaling mechanisms as well as
expression of additional subunits may primarily determine magnitude and
kinetics of Kv2.1-generated currents. Kv2.1 polypeptides are
extensively serine phosphorylated at their C-terminal
domain,27 and it has been
demonstrated that Kv2.1 channels can coassemble with other Kv channel
subunits such as
Kv5.1,28
Kv6.1,26
Kv6.2,29 or Kv9.1 to
Kv9.3.28 30
Heteromultimerization with these subunits could significantly modulate
the electrical and pharmacological properties of Kv2.1
currents.
Conclusions
By using single-cell RT-PCR, which is the current most
sensitive technique to identify mRNA in single cells, the present study
demonstrates that Kv2.1 mRNA can be detected in 41% (78/189) of
individual midmyocardial myocytes isolated from the rat left
ventricular free wall. Although the TEA-sensitive component of
IK,
which is known to be at least partly encoded for by Kv2.1 in mice,
showed considerable differences among individual myocytes, a combined
molecular and electrophysiological approach failed to detect a
correlation of
ITEA
magnitude or inactivation kinetics with the expression of Kv2.1 mRNA.
In contrast, Kv4 mRNA was detected in all myocytes in which
Ito1 was
identified. These results indicate that additional mechanisms besides
expression of Kv2.1 mRNA shape
IK in
the rat left
ventricle.
|
Acknowledgments |
|---|
This work was supported by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg "Experimentelle Nieren- und Kreislaufforschung"). We thank Rudolf Dussel and Anita Kühner for expert technical assistance.
|
Footnotes |
|---|
Original received September 6, 2000; revision received January 17, 2001; accepted January 17, 2001.
1 Both authors contributed equally to this study. 
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References |
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