Role of the Kv4.3 K+ Channel in Ventricular Muscle
A Molecular Correlate for the Transient Outward Current
The expression of 15 different K+ channels in canine heart was examined, and a new K+ channel gene (Kv4.3), which encodes a rapidly inactivating K+ current, is described. The Kv4.3 channel was found to have biophysical and pharmacological properties similar to the native canine transient outward current (Ito). The Kv4.3 gene is also expressed in human and rat heart. It is concluded that the Kv4.3 channel underlies the bulk of the Ito in canine ventricular myocytes, and probably in human myocytes. Both the Kv4.3 and Kv4.2 channels are likely to contribute to the Ito in rat heart, and differential expression of these two channels can account for observed differences in the kinetic properties of the Ito in different regions of rat ventricle. There are significant differences in the pattern of K+ channel expression in canine heart, compared with rat heart, and these differences may be an adaptation to the different requirements for cardiac function in mammals of markedly different sizes. It is possible that the much longer ventricular action potential duration observed in canine heart compared with rat heart is due, in part, to the lower levels of Kv1.2, Kv2.1, and Kv4.2 gene expression in canine heart.
We have previously suggested that the Kv4.2 gene encodes one of the main outward currents in rat myocytes, Ito.1 Surprisingly, we have found that neither the Kv4.2 nor the Kv4.1 gene is expressed at significant levels in canine or human ventricular myocytes, even though these cells express an Ito that has, in general, biophysical and pharmacological properties that are very similar to the rat Ito.2 3 4 5 Using degenerate oligonucleotides and PCR, we have identified a new K+ channel gene, Kv4.3, which is expressed at relatively high levels in the hearts of the three different species that we have tested: rat, canine, and human. The Kv4.3 channel, when expressed in Xenopus oocytes, encodes a channel that is rapidly inactivating and has biophysical and pharmacological properties that are similar to the native Ito. The results suggest that the Kv4.3 channel underlies a significant fraction of the Ito found in the hearts of several species, including rat, canine, and human.
The action potential waveforms of ventricular myocytes can be markedly different in different species.6 This difference is functionally significant and probably reflects the different requirements for cardiac function in species with different body sizes and widely varying resting and maximal heart rates. It is likely that at least part of the difference in ventricular action potential shape in different species is caused by the expression of different K+ channels or by the expression of different amounts of the same K+ channels.7 8 Rat myocytes have a relatively brief action potential with a “triangular” shape.9 This abbreviated waveform is necessary because the resting and maximal heart rates of rats are very high and could not be maintained with a more prolonged action potential. We wished to compare K+ channel expression in the rat with a species that has a traditional “spike-and-dome” cardiac action potential and a slower resting heart rate. For this purpose, we chose canine heart, which is a widely used model of cardiac function and is functionally quite similar to human heart. The average action potential duration in canine (≈200 ms)3 and human (≈350 ms)10 ventricular myocytes is significantly longer than in rat myocytes (≈40 ms).9 Similarly, the resting heart rate of dogs (≈110 bpm)11 and humans (≈70 bpm)12 is low compared with that of rats (≈450 bpm).13 Cellular physiological studies suggest that there is less rapidly activating sustained outward current in canine and human ventricular myocytes compared with rat myocytes,7 14 and our results suggest a molecular basis for these species differences.
Materials and Methods
Preparation of DNA Templates
Because of the inherent specificity of the RNase protection assay, which generally prevents using cross-species cDNA templates for the production of RNA probes, it was necessary to clone canine and human equivalents for all the cDNA templates used in the present study. In most cases, the canine or human cDNA had not been cloned previously, and for this reason, it was necessary to use degenerate oligonucleotides for PCR amplification (the exceptions were canine Kv1.215 and Kv1.516 ). The required cDNA fragments (180 to 390 bp) were isolated by reverse transcription and PCR amplification from total cellular RNA isolated from canine or human heart or brain. DNA templates were prepared by subcloning small cDNA fragments into pBluescript II SK (Stratagene) and then linearizing with the appropriate restriction enzyme. All constructs were confirmed by sequencing two to four independent isolates.
The standard nomenclature for K+ channel genes17 is used throughout. For each template, the PCR primers that were used for amplification and the deduced amino acid sequence of the region encompassed by the probe are given.
Canine cDNA Clones
Canine cDNA clones are as follows:
Oligos: Same as for Kv3.1
Oligos: Same as for Kv3.1
Kv4.2 (second probe)
Oligos: Same as for Kv4.2
Human cDNA Clones
Human cDNA clones are as follows:
Oligos: Same as for Kv4.1
Oligos: Same as for Kv4.1
Preparation of RNA
Tissue samples were quick-frozen in liquid N2 and then homogenized in guanidinium thiocyanate. Total RNA was prepared by pelleting the homogenate over a CsCl step gradient. All RNA samples were carefully quantified by spectrophotometric analysis. Canine ventricle samples were dissected as a section of tissue across the width of the left ventricular free wall. Human ventricular RNA was prepared from small samples of frozen human ventricular tissue from normal heart (provided by Gordon Tomaselli, Johns Hopkins University School of Medicine). Similar results were obtained with human heart mRNA obtained from a commercial supplier (Clontech). Rat RNA was prepared as described previously.1
RNase Protection Assay
RNA probes were prepared as described previously.1 In all cases, a significant amount of nonhybridizing sequence (≈50 bp) was included in the probe to allow easy distinction between the probe and the specific protected band. The specificity of the assay was such that there was no evidence for unwanted cross-reaction between any probe and another nonspecific K+ channel transcript. Most of the templates for the RNA probes were prepared using degenerate oligonucleotides, and there was often some nibbling of the ends of the probe-target mRNA hybridization complex, resulting in more than one protected band. These double bands typically differed in size by ≈5 to 20 nucleotides and did not normally affect the interpretation of the results.
RNase protection assays were performed as described previously.1 For each sample point, 5 or 10 μg of total RNA was used in the assay. A species-specific cyclophilin probe was included in the hybridization as an internal control to confirm that the sample was not lost or degraded during the assay. Yeast tRNA (5 μg) was used as a negative control to test for the presence of probe self-protection bands. RNA expression was quantified directly from dried RNase protection gels using a PhosphorImager (Molecular Dynamics).
Isolation of Full-Length Kv4.3 cDNA
A partial cDNA for the Kv4.3 gene was initially obtained from heart mRNA using standard RT-PCR procedures with oligonucleotides that were specific for members of the Kv4 gene family (see above). A full-length rat Kv4.3 cDNA clone was obtained by first performing a modified 5′ and 3′ RACE protocol (essentially as described by Frohman18 ) using anchor oligonucleotides complementary to the partial Kv4.3 clone. Once cDNAs were obtained that extended beyond both the 3′ and 5′ ends of the open reading frame, oligonucleotides complementary to noncoding regions just outside either end of the coding sequence were designed, and a full-length clone was obtained by PCR using the proof-reading enzyme Vent (NEB) for amplification. The following oligonucleotides were used for amplification: forward, GCC CAA AAG CTG GAG TCA C; reverse, CAC CCA CCA ACA TGC CAG. DNA sequencing was performed on plasmid DNA using Sequenase (USB), and sequence alignment was performed using the Clustal W program.19
Expression in Xenopus Oocytes
Oocytes were prepared from mature female Xenopus laevis using established procedures.20 Oocytes were injected with 50 nL of Kv4.3 mRNA (≈0.3 ng/nL) using a microdispenser (Drummond) and a micropipette with a tip diameter of 10 to 20 μm. Injected oocytes were incubated at 18°C for 24 to 48 hours before analysis.
Oocytes were voltage clamped using a two-microelectrode voltage clamp (either an Axoclamp 2A [Axon Instruments] or a TEV-200 [Dagan]). Intracellular electrodes filled with 3 mol/L KCl with resistances of 0.5 to 3 MΩ were used. Data collection and analysis were performed using Axoclamp software (Axon Instruments). Drugs were obtained from Sigma Chemical Co.
Recording From Canine Ventricular Myocytes
Single ventricular myocytes were isolated from canine ventricle using a trituration method described previously.21 The disaggregated cells were kept in KB medium at room temperature for at least 1 hour before experiments. Recordings of Ito were made using the whole-cell patch-clamp technique in modified Tyrode's solution containing Ca2+ channel blockers (mmol/L: NaCl 137.7, NaOH 2.3, MgCl2 1, glucose 10, HEPES 5, KCl 5.4, CaCl2 1.8, MnCl2 2, and CdCl2 0.2, pH 7.4). The pipette resistance was 2 to 4 MΩ, and the internal solution contained (mmol/L) NaCl 6, potassium aspartate 130, MgCl2 2, CaCl2 5, EGTA 11, HEPES 10, Na2-ATP 2, and Na-GTP 0.1, pH 7.2. Recording bath temperature was maintained at 30°C to 32°C.
Cloning of the Kv4.3 K+ Channel
In preliminary experiments, we found that there was little or no Kv4.2 or Kv4.1 mRNA expressed in canine ventricle muscle. This was a surprising observation because the Kv4.2 gene appears to encode a significant fraction of the Ito in rat heart,1 and this current is quite similar in its biophysical and pharmacological properties to the Ito in canine heart. To determine whether another member of the Kv4 family was expressed in canine heart, degenerate oligonucleotides, which were specific to regions conserved in the Kv4.1 and Kv4.2 channels, were used to amplify cDNAs from canine heart cDNA using PCR. Sequencing of individual cDNA clones indicated that some isolates encoded a new K+ channel that we have called Kv4.3. A cDNA encompassing the entire coding region of the Kv4.3 gene was subsequently obtained from rat, and this cDNA was used for sequence comparisons and expression.
Fig 1⇓ shows an alignment of the deduced amino acid sequences of the three Kv4 family genes. There is strong similarity between all three sequences. There is 75% identity between Kv4.3 and Kv4.2, 65% identity between Kv4.3 and Kv4.1, and 65% identity between Kv4.2 and Kv4.1. The Kv4.3 and Kv4.2 sequences are more similar to each other than either is to the Kv4.1 sequence. Typical for this class of proteins, the central core of the Kv4 channels is very highly conserved, and the amino- and carboxy-terminal ends are less well conserved. The leucine zipper motif between residues 307 and 328 is absolutely conserved in all three channels, and the pore region (residues 354 to 370) has only one conservative substitution (A356S) in Kv4.3. Of the 12 putative protein kinase C phosphorylation sites in Kv4.3, five are conserved in all three channels. One of these is the site between S4 and S5 that is conserved in all rapidly activating voltage-gated channels.17
Expression of the Kv4.3 K+ Channel
Injection of Kv4.3 mRNA into Xenopus oocytes results in the expression of a rapidly activating and inactivating K+ current (Fig 2A⇓), with properties generally similar to those of the Kv4.1 and Kv4.2 channels when expressed in oocytes.22 23 The threshold for activation of the current is between −50 and −40 mV (Fig 2B⇓). The steady state inactivation curve (Fig 2C⇓) has a midpoint and slope of −59±0.6 mV and 4.4±0.06 mV, respectively (n=13). Recovery from inactivation of the Kv4.3 channel is relatively rapid (Fig 2D⇓), with a time course that is well fitted with a single exponential (Fig 2E⇓). The time constant of recovery is voltage dependent (Fig 2F⇓), with values ranging from 58 to 202 ms for membrane potentials ranging from −120 to −90 mV.
The Kv4.3 current is unaffected by 5 mmol/L TEA (Fig 3A⇓). The inactivation of the Kv4.3 channel is only slightly slowed by 1 mmol/L H2O2 (Fig 3B⇓), in marked contrast to the effect of this reagent on the inactivation properties of the Kv1.4 and Kv3.4 channels.24 25 The Kv4.3 channel is quite sensitive to blockade by the antiarrhythmic drug flecainide (Fig 3C⇓), with a Kd of 26±2 μmol/L (n=4).
K+ Channel mRNA Expression in Canine Left Ventricle
Initial experiments suggested that there were large differences in K+ channel gene expression between canine and rat heart. To determine the extent of these differences, we used RNase protection analysis to examine the abundance of transcripts encoding 15 different voltage-activated K+ channels in canine ventricle.
Kv1 Gene Family
The Kv1.2 gene was not expressed at all in canine ventricle (Fig 4⇓). All five of the other Kv1 genes were expressed at varying levels, with Kv1.5 being the most abundant. This pattern of expression is significantly different from what has been found previously in rat heart (Table⇓). The most striking difference was the absence of Kv1.2 expression, because both the Kv1.2 mRNA and protein26 27 are quite abundant in rat ventricle. The relatively high level of expression of Kv1.5 mRNA is similar to that in rat heart1 and also human heart.28
Kv2 Gene Family
The Kv2.1 gene was expressed at very low levels, and the Kv2.2 gene was not expressed at all in canine ventricle (Fig 5⇓). This pattern of expression was also strikingly different from that in rat heart, where both Kv2.1 mRNA and protein are abundant, and there is some evidence suggesting that this channel underlies the ultrarapid delayed rectifier current expressed in these cells.27
Kv3 Gene Family
Two members of this family, Kv3.1 and Kv3.2, were not expressed at detectable levels in canine left ventricle (Fig 6⇓). Transcripts from the Kv3.3 gene were very rare and could be detected only after long exposures to x-ray film (7 days). Kv3.4 mRNA was expressed at low levels in canine heart and could be detected weakly after a 1- to 2-day exposure. The abundance of this transcript in ventricle muscle was quite low relative to skeletal muscle (Fig 6⇓).
The pattern of Kv3 gene expression in canine heart was generally similar to that seen in rat heart. Transcripts from the Kv3.1, Kv3.2, and Kv3.3 genes were all expressed at negligibly low levels in the hearts of both species. The one difference was the Kv3.4 gene, which was expressed at higher levels in canine ventricle than in rat ventricle.
Kv4 Gene Family
Neither Kv4.1 nor Kv4.2 mRNA is expressed at detectable levels in canine ventricular muscle. In marked contrast, Kv4.3 is quite abundant (Fig 7⇓). The most striking contrast with rat heart is the absence of Kv4.2 expression in canine heart (Table⇑). This was particularly surprising, since there is evidence that the Kv4.2 channel underlies a significant fraction of the Ito in rat heart.1 To confirm this observation, a second Kv4.2 template was made to a different region of the gene (see “Materials and Methods”). When this second probe was used in RNase protection assays, a similar result was obtained: high expression in brain and none in heart (data not shown).
Comparison of Kv1.4, Kv3.4, and Kv4.3 mRNA Abundance
The relative abundance of the Kv1.4, Kv3.4, and Kv4.3 transcripts was compared, because all three of these mRNAs encode channels that are rapidly inactivating and could potentially encode the Ito. The Kv4.3 mRNA was considerably more abundant than the other two transcripts, with the Kv1.4 transcript being 16±1% and the Kv3.4 transcript being 8±3% as abundant as the Kv4.3 transcript (Fig 8⇓).
Properties of Ito in Canine Left Ventricular Myocytes
Three of the K+ channel genes expressed in canine heart are known to encode rapidly inactivating channels: Kv1.4, Kv3.4, and Kv4.3. We examined the biophysical and pharmacological properties of Ito in canine heart to determine if we could eliminate some of these channels as candidates for the native current.
It has been shown that the inactivation rate of both the Kv1.4 and Kv3.4 channels is very sensitive to the oxidation state of cysteine residues in the amino-terminal inactivation domain of both channels.24 29 Treatment of the channels with an oxidizing agent such as H2O2 results in a large decrease in the inactivation rate of the Kv1.4 and Kv3.4 channels but leaves the Kv4 family channels unaffected.24 25 If the channels underlying the native Ito are encoded by either the Kv1.4 or Kv3.4 genes, it would be expected that the inactivation rate of the current would be significantly reduced by treatment with H2O2. In marked contrast to this prediction, the inactivation rate of the native Ito was only slightly changed by treatment with 1 mmol/L H2O2 for 5 minutes (Fig 9A⇓). Higher concentrations of H2O2 (up to 10 mmol/L) and longer incubations (10 minutes) did not produce any further effect. Similar results were observed in four epicardial ventricular myocytes. This result strongly suggests that the native channel is not encoded by members of either the Kv1 or Kv3 family of channels.
The sensitivity of all members of the Kv3 family of channels to blockade by TEA is very high, with Kds in the range of 0.1 to 0.3 mmol/L.17 This is in marked contrast to the native Ito, which was unaffected by 5 mmol/L TEA (Fig 9B⇑), suggesting that the Kv3.4 channel is unlikely to encode the native current.
Kv4 K+ Channel mRNA Expression in Human Left Ventricle
To determine whether the results that we obtained in canine heart were more generally true for large mammals, we examined the expression of Kv4 channel genes in human heart. The pattern of expression of Kv4 genes in human ventricle was generally similar to that in canine ventricle (Fig 10⇓). The Kv4.2 gene was not expressed, and the Kv4.3 mRNA was relatively abundant. The only difference in canine ventricle was that there was a small amount of Kv4.1 transcript in human ventricle, whereas this transcript was undetectable in canine heart. The Kv4.1 mRNA constituted <5% of the total Kv4 message, which is similar to what is found in rat heart.1
Kv4 K+ Channel mRNA Expression in Rat Left Ventricle
We have previously observed that there is a gradient of expression of Kv4.2 mRNA across the left ventricle wall of rat heart1 that is similar to the gradient of Ito expression.30 Both Kv4.2 mRNA and the Ito are more abundant in epicardial muscle than in endocardial cells, suggesting that the Kv4.2 gene encodes a significant fraction of the Ito in rat heart. One problem with this hypothesis is that it cannot account for the different kinetics of Ito in myocytes obtained from the epicardial and endocardial surfaces.31 The Ito in endocardial myocytes recovers from inactivation at a significantly slower rate than does the epicardial current, although the other properties of the current are relatively similar.
We examined Kv4.2 and Kv4.3 mRNA expression in the left ventricle wall of rat heart and found that they have distinct distribution patterns (Fig 11⇓). The Kv4.2 mRNA is expressed at significantly lower levels in endocardial than epicardial muscle, as described previously.1 In marked contrast, the Kv4.3 mRNA is found in essentially equal abundance throughout the ventricle wall.
The data presented in the present study support two main conclusions. First, it is likely that the Kv4.3 channel underlies a significant fraction of the Ito in canine ventricular myocytes and human myocytes and also contributes to the Ito in rat ventricle. Second, there are significant differences in the pattern of K+ channel expression in the hearts of larger mammals compared with rat heart, and these differences may be an adaptation to the different requirements for cardiac function in mammals of markedly different sizes.
Of the K+ channel genes expressed in canine heart, three (Kv1.4, Kv3.4, and Kv4.3) are known to encode rapidly inactivating channels and could theoretically encode the native Ito. Messenger RNA encoding the Kv4.3 channel is significantly more abundant than either the Kv1.4 or Kv3.4 transcripts (Fig 8⇑). The Kv4.3 mRNA is, in fact, the most abundant K+ channel transcript expressed in canine heart. This result suggests that the Kv4.3 channel may underlie the bulk of the Ito found in canine heart.
There are a number of other observations that support this conclusion. In particular, the functional properties of the native Ito are inconsistent with the possibility that either the Kv1.4 or Kv3.4 genes encode a channel contributing a significant fraction of the Ito in ventricular myocytes. The inactivation properties of both the Kv1.4 and Kv3.4 channels have previously been shown to be modified by changes in the oxidization state of cysteines in their amino-terminal inactivation domains, whereas the Kv4 family channels, which do not have the equivalent cysteine residues, are essentially unaffected.24 25 29 H2O2, which can rapidly cross membranes and oxidize cysteine residues, did not alter the kinetic properties of the native Ito, in direct contrast to the results expected if the Kv1.4 or Kv3.4 channels contribute significantly to this current.24 25
The native Ito is blocked by flecainide at concentrations32 33 very similar to those required to block the Kv4.3 channel, whereas the Kv1.4 channel is not blocked by flecainide in this concentration range.34 Members of the Kv3 family, including the Kv3.4 channel, have a very high sensitivity to the channel blocker TEA.35 36 This is markedly different from the native Ito, which is virtually insensitive to TEA (Fig 9⇑). It seems relatively unlikely that an ancillary subunit could modify the properties of the pore of the Kv1.4 or Kv3.4 channels sufficiently to account for the markedly different effects of these compounds on the native channel.
The native canine Ito recovers from inactivation relatively quickly (≈50 to 100 ms).2 In marked contrast, the half-time for recovery from inactivation of the Kv1.4 channel is more than an order of magnitude slower (3 to 5 s).37 38 Coexpression of the Kv1.4 channel with a β subunit only serves to further decrease the rate of recovery.39 In contrast, the Kv4.3 channel recovers from inactivation relatively rapidly (Fig 2⇑), with a strong dependence of the time constant for recovery on membrane potential, which is similar to the native Ito.2
Taken together, these data strongly suggest that the native Ito is not encoded by members of either the Kv1 or Kv3 family of channels. One other result that supports this conclusion is the observation that the Kv1.4 protein is expressed very inefficiently in rat cardiac muscle, even though the mRNA is relatively abundant.26 27 Inefficient expression of Kv1.4 protein may be a general property of cardiac myocytes. At present, however, the molecular basis for this result is poorly understood.
The pattern of expression of Kv4 genes in human heart is similar to that of canine heart, with the relatively minor difference that there is a detectable, but low, amount of Kv4.1 transcripts in human heart (<5% of the total Kv4 family transcripts). The Kv4.3 mRNA is expressed in human ventricle at high levels similar to those found in canine ventricle; therefore, it is likely that the Kv4.3 channels also underlie a significant fraction of the Ito found in human heart. There is evidence that some properties of the Ito are heterogeneous between different regions of human ventricle, although the extent of these differences has not been completely resolved (compare References 4 and 5). This result suggests either that two different channels contribute to the Ito in human heart or that there are region-specific modifications of the properties of the same channel. Presently, there are insufficient data to decide between these two possibilities.
The pattern of expression of the Kv4 genes in rat ventricle is complex. Both the Kv4.2 and Kv4.3 genes are expressed, but their distribution patterns are quite different. The Kv4.3 mRNA is expressed almost uniformly across the left ventricle wall, whereas the Kv4.2 mRNA is expressed in a marked gradient. It is likely that both transcripts encode components of the Ito in rat heart. It has been reported that Ito recovers from inactivation faster in epicardial myocytes than in endocardial myocytes.31 The differential expression of the Kv4.2 and Kv4.3 genes suggests a molecular basis for this observation, if it is assumed that the two channels have slightly different kinetic properties.
There are other significant differences in the pattern of K+ channel expression in rat and canine heart. Neither the Kv1.2 nor the Kv2.1 gene is expressed at significant levels in canine heart, whereas both the mRNA and protein products of these genes are quite abundant in rat heart.26 27 It is likely that one or both of these channels contribute to the noninactivating component of the rapidly activating K+ channels found in rat heart.26 27 The absence of these channels in canine heart makes functional sense, because the canine ventricular action potential has a long plateau phase, during which very little outward current flows. The much shorter action potential in rats is probably produced, in part, by the higher level of Kv1.2 and Kv2.1 channel expression, as well as by the relatively high level of Kv4.2 channel expression.
One limitation of the present study is that we have not directly identified the cell types that express the transcripts detected in the mRNA prepared from bulk ventricular muscle. Further studies, using histochemical techniques,26 40 are required to determine whether any of these channels are expressed in cell types other than ventricular myocytes.
In conclusion, we have presented evidence that suggests that the Kv4.3 gene encodes a channel that underlies a significant fraction, if not all, of the Ito in canine and human heart. Both the Kv4.3 and Kv4.2 channels are likely to contribute to the Ito in rat heart, and differential expression of these two channels may account for observed differences in the kinetic properties of the Ito in different regions of rat ventricle. There are significant differences in the pattern of K+ channel expression in canine heart, compared with rat heart, and these differences may be an adaptation to the different requirements for cardiac function in mammals of markedly different sizes. It is possible that the much longer ventricular action potential duration observed in canine heart compared with rat heart is due, in part, to the lower levels of Kv1.2, Kv2.1, and Kv4.2 gene expression in canine heart.
Selected Abbreviations and Acronyms
|Ito||=||transient outward current|
|PCR||=||polymerase chain reaction|
This study was supported by grants NS-29755 and HL-20558 from the National Institutes of Health and from the American Heart Association, New York State Affiliate, Inc. We would like to thank Gordon Tomaselli for the gift of human heart tissue and Mike Frohman for reagents and advice on using the RACE procedure.
Winner of the American Heart Association's 1996 Council on Circulation Boots Cardiovascular Research Prize.
- Received February 26, 1996.
- Accepted June 4, 1996.
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