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(Circulation Research. 1997;80:261-268.)
© 1997 American Heart Association, Inc.

Articles

Tissue and Species Distribution of mRNA for the I_Kr-like K⁺ Channel, erg

Randy S. Wymore, Gary A. Gintant, Rigel T. Wymore, Jane E. Dixon, David McKinnon, Ira S. Cohen

From the Department of Physiology and Biophysics (R.S.W., R.T.W., I.S.C.) and the Department of Neurobiology and Behavior (J.E.D., D.M.), State University of New York at Stony Brook, and the Cardiology Division (Department of Internal Medicine) and the Department of Pharmacology (G.A.G.), Wayne State University School of Medicine, Detroit, Mich.

Correspondence to Randy S. Wymore, Department of Physiology and Biophysics, Health Science Center, State University of New York at Stony Brook, Stony Brook, NY 11794. E-mail rwymore{at}brain.bio.sunysb.edu

	Abstract

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Abstract The human K⁺ channel gene, HERG, has been linked to the type 2 form of the autosomal dominant long-QT syndrome and has been suggested to encode the fast component of the delayed rectifier K⁺ current (I_Kr) found in heart. To date, the published electrophysiological and pharmacological data on the Xenopus-expressed HERG are very similar but are not identical to those of the endogenous I_Kr. In an effort to provide a different type of correlative data on the relationship between erg and I_Kr, cDNA fragments of erg homologues from guinea pig, rabbit, human, dog, and rat were cloned and used to test for the presence of erg mRNA in cardiac tissue. RNase protection assays reveal that erg message is found in the hearts of all five species and that it is expressed uniformly throughout the heart. The erg transcript is expressed at relatively high levels, being 50% more abundant than the most prevalent Kv-class K⁺ channel transcript in canine ventricle (Kv4.3). erg transcripts were found to have a wide tissue distribution in rat and are abundant in the brain, retina, thymus, and adrenal gland and are also found in skeletal muscle, lung, and cornea. Since there were no published reports of an I_Kr-like current in the rat heart, electrophysiological studies were performed to test whether the significant level of erg message in rat heart was correlated with the presence of an I_Kr-like current in rat. In isolated rat ventricular myocytes, an E-4031–sensitive current was observed, which is consistent with the presence of I_Kr. These results strengthen the link between erg and the native I_Kr in heart and suggest that erg may play an important role in other noncardiac tissues.

Key Words: K⁺ channel • delayed rectifier K⁺ current • cardiac muscle • mRNA expression

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The cardiac action potential is generated by a shifting balance of inward and outward currents. Whereas depolarization is caused by an influx of either Na⁺ or Ca²⁺ ions, depending on the tissue type, repolarization ensues when an activating efflux of K⁺ ions exceeds the inactivating inward currents. In most atrial muscle, a significant portion of this repolarization is produced by a rapidly activating and inactivating K⁺ current known as I_to.^{1 2} In the ventricular cardiac tissue types (endocardium, epicardium, and Purkinje) of some species, however, only phase 1 repolarization is due to a similar I_to, whereas final repolarization is dependent on the magnitude and kinetics of the delayed rectifier, IK.^{3 4} This delayed rectifier has been separated, on the basis of biophysical and pharmacological experiments, into both rapidly and slowly activating components, known as I_Kr and I_Ks, respectively.^{5 6 7}

Until recently, no candidate gene existed for the rapidly activating component of the delayed rectifier. Two laboratories have now heterologously expressed HERG, the human homologue of the K⁺ channel gene erg,⁸ in Xenopus oocytes, and have shown that it has properties similar to those of I_Kr, suggesting that the erg gene encodes I_Kr.^{9 10} That this gene plays a role in cardiac repolarization seems probable, as defects in HERG have been previously linked to electrophysiological abnormalities associated with one form of the long-QT syndrome.¹¹

In an attempt to further test the hypothesis that erg encodes I_Kr, we identified erg message in cardiac tissue from different species (guinea pig, rabbit, canine, human, and rat) as well as different cardiac tissue types in the same species. If erg does encode I_Kr, then erg mRNA should be prevalent in all tissues that express significant I_Kr. Conversely, it would be expected that there should be minimal (if any) erg mRNA in species, or tissues, that have no detectable rectifier.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning of erg Probes from Rabbit, Guinea Pig, Dog, Human, and Rat
It is possible to use highly conserved probes in Northern blot analyses across various species; this is not, however, the case with probes for RNase protection assays. Because of the intrinsic specificity of this assay system, it is necessary to obtain probes for each species to be examined. To obtain erg homologues from each of the five species in question, PCR amplification was used. Degenerate oligonucleotide primers were designed to correspond with amino acid sequences that are conserved across the entire known eag/erg superfamily.⁸ It was necessary to use regions conserved throughout the whole eag family, as there was only one published erg sequence.

The upstream primer was directed toward part of the S1 transmembrane segment encompassing the amino acid sequence FKAVWDW; the oligonucleotide sequence was 5'-TT(T/C) AA(A/G) (A/G)C(A/C/G/T) (A/G)(T/C)(A/C/G/T) TGG GA(T/C) TGG-3'. The downstream primer was directed to the S5 transmembrane region encompassing the amino acid sequence WKACIWY; the oligonucleotide sequence was 5'-(A/G)TA CCA (A/G/T)AT (A/G)CA (A/C/G/T)AG CCA (A/G)TG-3'.

Heart RNA was reverse-transcribed, and the subsequent cDNA was used as a template for the PCR reactions. The above two primers were used successfully for PCR amplification of erg homologues from each of the five species, using either Taq polymerase in dimethyl sulfoxide containing buffer (guinea pig, dog, rabbit, and human, Perkin-Elmer Corp) or the Expand system (rat, Boehringer-Mannheim). The 480-bp PCR products were isolated by agarose get electrophoresis, purified using the Glass-Max system (GIBCO BRL), then cloned into pBluescript (Stratagene), and transformed into X1-1 blue Escherichia coli (Stratagene). Insert-bearing individual clones were identified, and the plasmid DNA was purified using the Wizard mini-prep system (Promega) and then sequenced with Sequenase (USB).

Preparation of RNA
Total RNA was prepared by quick freezing of the tissues isolated from adult animals in liquid N₂, followed by homogenization in guanidinium thiocyanate. RNA was prepared essentially as described by Chirgwin et al¹² and included a final step of pelleting the homogenate over a CsCl step gradient. Canine ventricular samples were dissected as a section of tissue across the width of the left ventricular free wall. Canine right atria were dissected as a section of tissue through the atrial wall. Free-running canine Purkinje fibers were dissected from both ventricles. Rabbit ventricle was dissected as a section of tissue through the left ventricle. Rabbit atrium was dissected as a section of tissue through the right atrium. Rabbit SA nodes were isolated from 10 rabbits. The rat and guinea pig tissues were homogenized after dissection and were not frozen. Rat and guinea pig ventricles were taken as left ventricular sections through the tissue and near the apex of the heart. Rat tissues (kidney, adrenal gland, thymus, cornea, lens, and retina) were isolated as whole organs and homogenized. Approximately one half of a gram of tissue was isolated from lung, liver, and small intestine for the homogenization. Both rat and guinea pig brains were whole hemispheric dissections. Human heart and brain RNA were mRNA preparations obtained commercially from Clontech. Poly-A⁺ RNA was isolated from adult animals using paramagnetic poly-dT beads (Dynal Inc); after isolation, canine Purkinje fibers were homogenized fresh in reagents supplied with the Dynal kit. Rabbit SA node tissue was homogenized frozen, and the poly-A⁺ RNA was extracted as described using the Dynal mRNA extraction kit.

RNase Protection Assays
RNA probes were prepared as described previously.¹³ In all experiments, a significant stretch of nonhybridizing sequence of 50 bp was included with the probe to allow easy distinction between the probe and the specific protected band. The specificity of the assay is such that there was no evidence for cross-reactivity between the probe and any other transcripts, with the possible exception of the guinea pig heart experiments. The guinea pig erg probe protects a predominant band in both brain and ventricle of the size expected; however, there are numerous smaller protected bands that appear primarily in the heart sample. This was observed with two different probes in two different RNA preparations (one total RNA and one poly-A⁺); it is unclear whether the probe is cross-reacting with other closely related genes or with alternative splices of the guinea pig erg gene.

RNase protection assays were performed essentially as described previously.¹³ For each experiment, 5 µg of total or 1 µg of poly-A⁺ RNA was used. Cyclophilin probes were included in the hybridization reaction to confirm that the sample was not lost during the course of the experiment. Rat and canine cyclophilin probes were PCR-cloned, using available sequence data to design primers for the PCR. The human cyclophilin probe was purchased from Ambion. In the guinea pig and rabbit experiments, the rat cyclophilin probe was used. The probe protected fragments smaller than the full-length band found in rat RNA. The same protected fragments appeared replicably, regardless of the tissue used. Five micrograms of yeast tRNA was used as a negative control for probe self-protection bands. The rat RNase protection assay figures are 2-day exposures; all of the other figures were generated from 1-day exposures.

For the comparison of erg and Kv4.3 mRNA levels in canine ventricle, specific signals were quantified directly from dried RNase protections gels using a PhosphorImager (Molecular Dynamics). In this experiment, three independent samples of RNA were used, and the erg and Kv4.3 samples were assayed separately so that no erg signal would contaminate the Kv4.3 protected band. Cloning and preparation of the Kv4.3 probe have been described previously.¹⁴

Electrophysiology
Myocyte Isolation Procedures
Adult rat ventricular myocytes were isolated according to techniques reported previously.¹⁵ Briefly, hearts were cannulated under sterile conditions and superfused with a Ca²⁺-free solution containing collagenase (CLS 2, Worthington) and hyaluronidase (type II, Sigma Chemical Co). Subsequently, the hearts were minced and digested with trypsin IX (Sigma) and deoxyribonuclease II (Worthington). Myocytes were filtered through nylon mesh, sedimented through Ca²⁺-containing Krebs-Henseleit bicarbonate buffer solution supplemented with albumin (bovine fraction V, Sigma), and stored in culture medium (medium 199) at room temperature. Myocytes were examined within 12 hours of harvest.

Electrophysiological Recordings
Myocytes were allowed to settle on the bottom of a Peltier-based temperature-controlled perfusion bath mounted on an inverted microscope and studied as described previously.¹⁶ Cells were superfused (0.8 mL/min) with a HEPES-buffered Tyrode's solution containing (mmol/L) NaCl 132, MgSO₄ 1.2, HEPES 20, glucose 11.1, KCl 4, and CaCl₂ 2 (pH 7.4 with HCl; bath temperature 37°C).

Myocytes were accessed using whole-cell patch-clamp techniques and an Axopatch 200A amplifier (Axon Instruments). Patch pipettes were filled with a high-[K⁺] intracellular solution containing (mmol/L) potassium aspartate 125, KCl 20, EGTA 10, ATP (magnesium salt) 5, MgCl₂ 1, and HEPES-free acid 5, adjusted to 7.3 with 5N KOH. Junction potentials were compensated by offset adjustment of -10 mV immediately after cell break-in. Series resistance compensation was typically adjusted to values between 60% and 70%. Capacitance values of myocytes were determined from settings on the patch-clamp amplifier obtained after clamp tune-up at a holding potential of -40 mV.

Extracellular solution contained nimodipine (4 to 5 µmol/L) to block L-type Ca²⁺ current; Na⁺ current was partially inactivated by holding potential (-75 mV) and further reduced by the use of slowly depolarizing ramp pulses to facilitate accommodation. E-4031 was added from a 5 mmol/L aqueous stock solution, for a final concentration of 5 µmol/L; this concentration has been shown to block nearly all of I_Kr in isolated ventricular myocytes from guinea pig (IC₅₀, 0.4 µmol/L),⁶ dog,³ and neonatal mouse.¹⁷ Dofetilide was prepared from a 5 mmol/L stock solution dissolved in ethyl alcohol and used at a final concentration of 1 µmol/L. Dofetilide has been shown to selectively block I_Kr in rabbit myocytes (K_d, 3.9x10^-9 mol/L).¹⁸

Preliminary experiments demonstrated that it was difficult to measure I_Kr in adult rat ventricular myocytes during depolarizing square test pulses because of the coincident activation of large rapidly activating transient and sustained outward currents and the lack of an agent to block these currents without blocking I_K. Furthermore, the small amplitude of I_Kr tail currents upon repolarization to -40 mV suggested that I_Kr would be exceedingly small during strongly depolarizing test pulses, given the inwardly rectifying characteristics of this current in other species. Consequently, we used a three-phase clamp protocol to assess I_Kr consisting of (1) a slowly depolarizing ramp from a holding potential of -75 mV to +25 mV (duration of either 1 second or 600 milliseconds), followed by (2) a 200-millisecond "pedestal" step to 0 mV, followed by (3) a test pulse to -40 mV to elicit tail currents. The I_Kr was identified as a time-dependent drug-sensitive tail current recorded upon repolarization to -40 mV. The amplitude of I_Kr was assessed by measuring the amplitude of E-4031–sensitive or dofetilide-sensitive time-dependent current obtained by digital subtraction (ie, control minus drug). Recordings within a brief 5- to 6-minute period were compared to minimize possible nonspecific changes in membrane currents. Because of the small amplitude of the tail currents, averages from a minimum of three recordings in the absence and presence of drug were used for digital subtractions. The clamp protocols were repeated once every 10 seconds.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning of RNase Protection Assay Probes
The RNase protection assay provides a means for determining the presence of mRNA transcripts that is both more specific and more sensitive than Northern blot analysis. To perform such experiments requires the use of a probe that is specific not only for the gene of interest but also for the species in question. It was necessary, therefore, to generate fragments of erg DNA from each of the five species examined in the present study. Degenerate oligonucleotide primers were designed that were conserved across the entire eag/erg superfamily and were used in the PCR, along with cDNA made from ventricular RNA, to generate 480-bp erg clones. Each of these clones was sequenced and compared with the published human sequence.⁸

An alignment of the sequences is shown in Fig 1. These probes encompass most of the hydrophobic core of the channel from the beginning of the first transmembrane segment (S1) through the end of the fifth transmembrane region (S5). The canine and rabbit clones are 99% identical to the human sequence at the amino acid level; the rat and guinea pig clones are 96% identical to the human sequence. The main area of variability is in the S1-S2 extracellular loop; this same lack of conservation is found across the entire eag/erg superfamily at this position. The S4 voltage-sensing domain contains absolute identity across the various species.

View larger version (20K): [in this window] [in a new window]   Figure 1. Amino acid alignment of the erg fragments used as RNase protection assay probes. The published human sequence is compared with PCR-generated fragments from guinea pig (g. pig), dog, rabbit, and rat. These fragments are highly conserved in all regions except for the putative S1-S2 extracellular loop; they are 96% to 99% identical at the amino acid level and 89% to 91% identical at the nucleotide level.

There was no evidence for alternative splicing within the region encompassed by these clones. Amino- or carboxy-terminal splicing variants would not have been detected using these probes. No other eag-related genes were detected by PCR in the hearts of the species examined, even though these primers were able to amplify related cDNAs in other tissues.

Species Distribution of erg mRNA in Heart
There have been published reports of I_Kr in the hearts of several species. Guinea pig, rabbit, dog, and human hearts have been shown to have significant E-4031–sensitive I_K.^{3 7 19 20} To our knowledge, there is no previous report of this current in the rat. Since it has been proposed that the erg K⁺ channel gene encodes a subunit of the I_Kr channel, we examined the amount of erg mRNA in cardiac tissues from each of these species to determine whether message levels paralleled the presence of I_Kr.

Fig 2 shows the results of RNase protection assays performed on RNA from the hearts of guinea pig, rabbit, dog, human, and rat, using the erg probes described above. The RNA used in all sample lanes is from the left ventricle, except for human RNA, which was whole-heart RNA (Clontech). A strong erg-protected signal is observed in the heart RNA from all of the species tested, including rat ventricle.

View larger version (50K): [in this window] [in a new window]   Figure 2. Species distribution of erg in the heart, as determined by RNase protection assays. Total left ventricular RNA from guinea pig, rabbit, dog, and rat heart, as well as poly-A+ RNA from human heart, were probed with the erg fragments and cyclophilin (cyc) from the corresponding species. A strong erg-protected signal is observed in all of the heart lanes.

Distribution of erg mRNA in Rabbit and Dog Hearts
I_Kr has been well characterized in rabbit SA node, atrium, and ventricle.^{19 21} Fig 3A shows that erg mRNA is approximately equally abundant in left ventricle, left atrium, and SA node of the rabbit heart.

View larger version (42K): [in this window] [in a new window]   Figure 3. Distribution of erg in rabbit and canine heart, as determined by RNase protection assays. A, Poly-A+ RNA from left ventricle (l vent), left atrium (l atrium), and SA node was probed with the rabbit (rb) erg probe. A uniformly strong erg-protected band is visible in all three sample lanes. B, Poly-A+ RNA from l vent, Purkinje fibers (Purk), and right ventricle (r vent) was probed with the canine (c) erg probe. Comparably strong erg-protected bands are visible in all three sample lanes. cyc indicates cyclophilin.

I_Kr has also been well characterized in canine ventricle.^{3 4} RNase protection experiments were performed to determine whether erg mRNA was present in other regions of canine heart. Fig 3B reveals that there are similar levels of erg transcripts in the left ventricle, Purkinje fibers, and right ventricle. These results show that there is relatively uniform erg mRNA expression within the various specialized tissues of rabbit and dog hearts.

Tissue Distribution of erg mRNA
In contrast to the characterizations of I_Kr in the hearts of several species, no reports could be found suggesting that an E-4031–sensitive I_K exists in noncardiac tissues. A variety of tissues from rats was examined for the presence of erg mRNA. Fig 4 indicates that the strongest signal (per microgram of RNA) is found in whole brain, but strong signals are also observed in RNA from left ventricle, adrenal gland, thymus, and retina; weaker erg-protected bands are seen in soleus, tibialis, lung, cornea, and lens. Although not visible in the autoradiographs shown, after a week-long exposure, a very faint erg-protected band is visible in kidney, liver, and small intestine.

View larger version (37K): [in this window] [in a new window]   Figure 4. Tissue distribution of erg in adult rat, as determined by RNase protection assays. Poly-A+ RNA from various tissues was probed with the rat (r) erg probe. A, The erg-protected band is strongest in brain, with a strong band in heart and less intense bands in soleus and tibialis. No erg-protected band is visible in kidney, although exposure for 1 week reveals a very faint band. cyc indicates cyclophilin; l vent, left ventricle. B, Relatively strong erg-protected bands are seen in adrenal gland, thymus, and retina, with faint but detectable bands in lung, cornea, and lens. No protected bands are visible in liver or small intestine (sm int), although a faint signal appears after a 1-week exposure.

Since rat brain had an abundance of erg mRNA, we examined two other species to determine the generality of this observation. erg mRNA was abundantly expressed in human brain, similar to what was found in rat brain (Fig 5B). Surprisingly, however, erg mRNA was significantly less abundant in guinea pig brain, suggesting marked species differences (Fig 5A).

View larger version (42K): [in this window] [in a new window]   Figure 5. Expression of erg mRNA in guinea pig and human heart and brain. Guinea pig total RNA and human poly-A+ RNA were probed for the presence of erg mRNA using the guinea pig (gp) probe or the human (h) probe. A, erg-protected bands reveal that erg is found in guinea pig brain, but at low levels compared with left ventricle (l vent). B, The erg-protected band in human brain mRNA is as intense as the protected band in heart. cyc indicates cyclophilin.

Comparison of erg and Kv4.3 mRNA in Canine Heart
Kv4.3, which encodes the primary subunit of I_to in canine and human heart, has previously been shown to be the most abundant Kv-class K⁺ channel transcript expressed in canine heart.¹⁴ In preliminary experiments, we observed that erg transcript levels in canine heart appeared to be quite high relative to Kv4.3. Using quantitative RNase protection experiments, we directly compared the levels of erg mRNA with Kv4.3 and found that erg mRNA levels are 1.5-fold more abundant than those of Kv4.3 in canine right ventricle (Fig 6). The erg transcript appears to be the most prevalent K⁺ channel mRNA expressed in heart.

View larger version (25K): [in this window] [in a new window]   Figure 6. Comparison of Kv4.3 and erg mRNA levels in canine right ventricular (r vent) muscle. A, RNase protection assay of canine r vent muscle, combining Kv4.3, erg, and cyclophilin (cyc) probes. B, Histogram showing that canine erg mRNA is 46±1% more abundant than Kv4.3 transcripts. Data are normalized relative to the abundance of Kv4.3 and are corrected to reflect the different specific activities of the two probes. A.U. indicates arbitrary units. Values are the means of three independent mRNA samples, and error bars are SEM.

Delayed Rectifier in Rat Ventricle
The high level of erg message in rat heart was unexpected because I_Kr had not been previously reported in this species, although inferences have been made on the basis of the effect of class III antiarrhythmics on fetal rats.^{22 23} Biophysical and pharmacological experiments were carried out to determine whether a corresponding I_Kr-like current is present in rat cardiac myocytes.

Fig 7 displays superimposed averaged current traces obtained from a typical adult rat ventricular myocyte in the absence (open circles) and presence (closed circles) of 5 µmol/L E-4031. E-4031 rapidly and consistently blocked the slowly decaying outward current elicited upon repolarization to -40 mV. Using this clamp paradigm, the average peak current density was 0.19±0.04 pA/pF (mean±SEM, n=9 myocytes, obtained from four hearts). The same protocol applied to two canine ventricular myocytes also revealed an E-4031–sensitive decaying tail current at -40 mV (authors' unpublished data, 1996), consistent with the previous demonstration of I_Kr in canine heart.^{3 4} E-4031 had no consistent effect on membrane currents during the depolarizing ramps (assessed at -20, 0, and +20 mV). Although averaged data showed a trend toward less outward current at the more positive ramp potentials, this did not achieve statistical significance (authors' unpublished data, 1996). Such an effect is consistent with the inwardly rectifying properties of I_Kr, which likely results from differences in the voltage- and time-dependent kinetics of activation versus fast inactivation.²⁴

View larger version (16K): [in this window] [in a new window]   Figure 7. Demonstration of IKr in a rat ventricular myocyte. A, The top trace of the panel shows the clamp protocol, consisting of a depolarizing ramp and "pedestal" to 0 mV, followed by repolarization to -40 mV to elicit the tail current. E-4031 rapidly and consistently blocked the slowly decaying tail current measured at -40 mV. In contrast, E-4031 had no consistent effect on membrane current during the conditioning depolarizing ramp. Each trace represents the average of four recordings. Holding potential was -75 mV; the bottom of the calibration represents +80 pA. E-4031 concentration was 5 µmol/L. B, Illustration (on an expanded time scale) of an E-4031–sensitive outward current, obtained by digital subtraction of traces in the upper panel (ie, control minus E-4031), is shown. A time-dependent, slowly decaying, E-4031–sensitive outward current was routinely observed. The horizontal calibration is positioned at 0 pA.

Dofetilide is another pharmacological agent that preferentially blocks I_Kr.¹⁸ To confirm the presence of I_Kr in rat myocytes, the effect of dofetilide was examined using the same protocol as in Fig 7. The slow tail current component was abolished by 1 µmol/L dofetilide, consistent with the presence of I_Kr in rat myocytes; the amplitude of the dofetilide-sensitive tail current was 0.31±0.22 pA/pF (mean±SEM, n=6, obtained from two hearts).

I_Kr is also paradoxically reduced by lowering [K⁺]_o.^{3 25} To further characterize the E-4031–sensitive current in rat myocytes as I_Kr, the effects of lowered [K⁺]_o on the E-4031–sensitive current were examined. After initial recording in 4 mmol/L [K⁺]_o, the solution was switched to one containing nominally 0 mmol/L [K⁺]_o. This produced a reduction in the amplitude of the time-dependent tail current upon repolarization to -40 mV. The effects of E-4031 were then evaluated in the presence of 0 mmol/L [K⁺]_o. In each of four myocytes tested, a time-dependent E-4031–sensitive current was negligible (0.026±0.005 pA/pF, mean±SEM), consistent with the near absence of I_Kr, under these recording conditions.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
I_Kr has several unique properties, which include a sensitivity to E-4031, dependence on external K⁺, and inward rectification.⁷ None of the previously cloned voltage-gated (Kv), or inward rectifier (K_ir) K⁺ channels strongly resembles I_Kr. When HERG was heterologously expressed in oocytes, it was clear that this channel was a candidate for I_Kr; it activated with increasing depolarization, showed marked voltage-dependent inward rectification, and had strong dependence on [K⁺]_o. However, the activation kinetics of the expressed current were slower than the native I_Kr, and the sensitivity and characteristics of block by E-4031 differed as well from the cardiac myocyte I_Kr, causing some uncertainty. ^{9 10} More recently, with modification of the protocols used on native myocytes, HERG expressed in oocytes has been shown to be blocked by the methanesulfonanilides initially used to characterize I_Kr, further supporting the hypothesis that I_Kr is encoded by erg.²⁶ One purpose of the experiments described in the present study was to provide an alternative test of the hypothesis that erg encodes the native I_Kr in various cardiac tissues.

The abundant expression of erg message in the hearts of guinea pig, rabbit, dog, and human corresponds well with existing characterizations of the delayed rectifier.^{3 4 7 16 19 20 21} The presence of erg in all regions of the hearts examined is also consistent with reports of I_Kr in ventricle, atrium, and SA node in rabbit heart¹⁹ as well as ventricle and atrium in dog heart.^{3 4} Our results are also consistent with the finding that erg mRNA was detected in dispersed ventricle, SA node, and atrial myocytes isolated from ferret.²⁷ The finding that erg mRNA is nearly as abundant in rat heart as in the other species examined was surprising in light of the fact that literature searches could identify no previous reports describing I_K in rat heart, although there have been reports of class III antiarrhythmic effects in fetal rats.^{22 23} With the data from these RNase protection experiments in mind, we examined rat ventricular myocytes for the presence of delayed rectifier and found that the rat does indeed possess an I_Kr-like current, with properties similar to those described in other species.

Since I_Kr has been exclusively characterized as a cardiac channel, the original design of these experiments was to examine the erg mRNA content from noncardiac tissues in the expectation that there would be little expression outside the heart. Instead of limited expression, however, we found that erg mRNA is widely expressed in rat, and although it is not possible to directly compare RNA levels with functional currents, it would not be surprising to detect currents corresponding to erg mRNA in brain, adrenal glands, thymus, retina, and possibly skeletal muscle. The widespread distribution of erg gene expression suggests that pharmacological agents that suppress I_Kr in heart may produce unwanted effects in numerous other tissues and that careful consideration of these effects may be warranted when evaluating the pharmacological effects of these agents.

Northern blot analysis has shown that erg is expressed at low levels in the human brain.¹¹ In contrast to this previous report, we have reproducibly detected an abundant erg message in the human brain, which is approximately equal in strength to that found in human heart. In every species we examined, erg is a ubiquitous heart channel; the presence of erg mRNA in the brain, however, was species variable. The guinea pig has considerably more erg mRNA in the ventricle than in the brain, in contrast to the rat, which, like the human, has high levels of erg mRNA levels in the brain.

Prior studies with ventricular myocytes have identified and characterized I_Kr as an inwardly rectifying K⁺ current blocked by the methanesulfonanilides E-4031 or dofetilide in ventricular cells from numerous species including dogs,^{3 16} guinea pigs,⁶ neonatal mice,^{17 28} and humans.²⁰ The present study concludes that I_Kr is present in rat ventricular myocytes on the basis of (1) the presence of a slowly decaying outward tail current upon repolarization to minus40 mV, (2) block of the tail current by E-4031 and dofetilide, (3) the reduction of tail current by superfusion with 0 mmol/L [K⁺]_o, and (4) the lack of effect by E-4031 on tail currents in the presence of 0 mmol/L [K⁺]_o. Together, these observations are consistent with the presence of a small I_Kr in adult rat ventricular myocytes sensitive to methanesulfonanilides. The small amplitude of I_Kr precluded more detailed studies of the characteristics of I_Kr in this species. Earlier reports of the absence of I_Kr in adult rat myocytes likely resulted from the small amplitude of this current relative to larger time-dependent currents. The small amplitude of I_Kr is also consistent with the lack of effect of dofetilide on the action potentials of rat papillary muscles.²⁹

It has been shown that the Kv4.3 gene encodes the 4-aminopyridine–sensitive component of I_to in canine ventricle¹⁴ and that Kv4.3 transcripts are the most abundant Kv-class K⁺ transcripts expressed in heart. We have shown in the present study that the mRNA level for erg in canine ventricular myocardium actually exceeds that for Kv4.3 by 50%. It is difficult to meaningfully compare densities of different currents unless similar clamp protocols and experimental conditions are used. However, when identical disaggregation procedures and comparable internal and external solutions are used, it is evident that the density of I_to is much greater than that of I_Kr in canine ventricle. Specifically, the reported current densities for I_to in canine epicardium and midmyocardium are 30 pA/pF, declining to 5.6 pA/pF in canine endocardium.³⁰ In contrast, the density of I_Kr in canine myocytes is only 0.22 pA/pF.³ Thus, despite the prevalence of erg mRNA over Kv4.3 mRNA, I_Kr current density is 25- to 136-fold smaller than that of I_to. This suggests that posttranscriptional events can contribute to determining the final amplitude of the erg currents in heart and points to limitations in comparing message levels with membrane currents in the absence of measurements of membrane protein levels. Although unlikely, it is possible that differences in the single-channel conductances of the I_to and I_Kr channels could account for some of the difference in current densities

In conclusion, our experiments demonstrate that although erg is unlikely to encode a cardiac-specific channel, it is prevalent in all cardiac regions of all the species tested. The mechanism of posttranscriptional regulation of erg channel expression remains a fruitful area for further investigation, as does the identification of the role of the erg channel in the noncardiac tissues (brain, thymus, adrenal glands, and skeletal muscle) in which the erg message is expressed at significant levels.

	Selected Abbreviations and Acronyms

 

IK = delayed rectifier K+ current IKr = rapidly activating component of IK IKs = slowly activating component of IK Ito = transient outward current PCR = polymerase chain reaction SA = sinoatrial

	Acknowledgments

 
This study was supported in part by National Institutes of Health grants HL-20558 and HL-28958 (Dr Cohen), NS-29755 (Dr McKinnon), and HL-49918 (Dr Gintant); by an American Heart Association Grant-in-Aid (Dr McKinnon); and by a SUNY, Stony Brook, pharmacology postdoctoral fellowship (T32-DK07521, Dr Wymore). Dofetilide was supplied courtesy of Pfizer Central Research. Sequences of the erg clones have been submitted to Genbank under the following accession numbers: U75210 (rat erg), U75211 (guinea pig erg), U75212 (rabbit erg), and U75213 (canine erg).

Received September 16, 1996; accepted November 27, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Giles WR, Imaizumi Y. Comparison of potassium currents in rabbit atrial and ventricular cells. J Physiol (Lond). 1988;405:123-145.[Abstract/Free Full Text]

2. Gintant GA, Cohen IS, Datyner NB, Kline RP. Time dependent outward currents in the heart. In: Fozzard HA, Haber E, Katz AM, Morgan HE, eds. The Heart and Cardiovascular System. New York, NY: Raven Press Publishers; 1992.

3. Gintant GA. Regional differences in I_k density in canine left ventricle: role of I_k,s in electrical heterogeneity. Am J Physiol. 1995;268:H604-H613.[Abstract/Free Full Text]

4. Liu D-W, Antzelevitch C. Characteristics of the delayed rectifier current (I_Kr and I_Ks) in canine ventricular epicardial, midmyocardial, and endocardial myocytes: a weaker I_Ks contributes to the longer action potential of the M cell. Circ Res. 1995;76:351-365.[Abstract/Free Full Text]

5. Noble D, Tsien RW. Outward membrane currents activated in the plateau range of potential in cardiac Purkinje fibers. J Physiol (Lond). 1969;200:205-231.[Abstract/Free Full Text]

6. Sanguinetti MC, Jurkiewicz NH. Two components of cardiac delayed rectifier K⁺ current: differential sensitivity to block by class III antiarrhythmic agents. J Gen Physiol. 1990;96:195-215.[Abstract/Free Full Text]

7. Sanguinetti MC, Jurkiewicz NH. Delayed rectifier outward K⁺ current: differential sensitivity to block by class III antiarrhythmic agents. Am J Physiol. 1991;260:H393-H399.[Abstract/Free Full Text]

8. Warmke JW, Ganetzky B. A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci U S A. 1994;91:3428-3442.[Abstract/Free Full Text]

9. Sanguinetti MC, Jiang C, Curran ME, Keating MT. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the I_kr potassium channel. Cell. 1995;81:299-307.[Medline] [Order article via Infotrieve]

10. Trudeau MC, Warmke JW, Ganetzky B, Robertson GA. HERG, a human inward rectifier in the voltage-gated potassium channel family. Science. 1995;269:92-95.[Abstract/Free Full Text]

11. Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, Keating MT. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell. 1995;80:795-603.[Medline] [Order article via Infotrieve]

12. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979;24:5294-5298.

13. Dixon JE, McKinnon D. Quantitative analysis of potassium channel mRNA expression in atrial and ventricular muscle of rats. Circ Res. 1994;75:252-260.[Abstract/Free Full Text]

14. Dixon JE, Shi W, Wang H-S, McDonald C, Yu H, Wymore RS, Cohen IS, McKinnon D. Role of the Kv4.3 K⁺ channel in ventricular muscle: a molecular correlate for the transient outward current. Circ Res. 1996;79:659-668.[Abstract/Free Full Text]

15. Ellingsen O, Davidoff AJ, Prasad SK, Berger HJ, Springhorn JP, Marsh JD, Kelly RA, Smith TW. Adult rat ventricular myocytes cultured in defined medium: phenotype and electromechanical function. Am J Physiol. 1993;265:H747-H754.[Abstract/Free Full Text]

16. Gintant GA. Two components of delayed rectifier current in canine atrium and ventricle: does I_Ks play a role in the reverse rate dependence of class III agents? Circ Res. 1996;78:26-37.[Abstract/Free Full Text]

17. Nuss HB, Marban E. Electrophysiologic properties of neonatal mouse cardiac myocytes in primary culture. J Physiol (Lond). 1994;479:265-279.[Abstract/Free Full Text]

18. Carmeliet EE. Voltage- and time-dependent block of the delayed K⁺ current in cardiac myocytes by dofetilide. J Pharmacol Exp Ther. 1992;262:809-817.[Abstract/Free Full Text]

19. Ito H, Ono K. A rapidly activating delayed rectifier K⁺ channel in rabbit sinoatrial node cells. Am J Physiol. 1995;269:H443-H452.[Abstract/Free Full Text]

20. Li G-R, Feng J, Yue L, Carrier M, Nattel S. Evidence for two components of delayed rectifier K⁺ current in human ventricular myocytes. Circ Res. 1996;78:689-696.[Abstract/Free Full Text]

21. Shibasaki T. Conductance and kinetics of delayed rectifier potassium channels in nodal cells of the rabbit heart. J Physiol (Lond). 1987;387:227-250.[Abstract/Free Full Text]

22. Spence SG, Vetter C, Hoe CM. Effects of the class III antiarrhythmic, dofetilide (UK-68,798), on the heart rate of midgestation rat embryos, in vitro. Teratology. 1994;49:282-292.[Medline] [Order article via Infotrieve]

23. Abrahamsson C, Palmer M, Ljung B, Duker G, Baarnhielm C, Carlsson L, Danielsson B. Induction of rhythm abnormalities in the fetal rat heart: a tentative mechanism for the embryotoxic effect of the class III antiarrhythmic agent almokalant. Cardiovasc Res. 1994;28:337-344.[Abstract/Free Full Text]

24. Smith PL, Baukeowitz T, Yellen G. The inward rectification mechanism of the HERG cardiac potassium channel. Nature. 1996;379:833-836.[Medline] [Order article via Infotrieve]

25. Sanguinetti MC, Jurkiewicz NH. Role of external Ca²⁺ and K⁺ in gating of the cardiac delayed rectifier K⁺ currents. Pflugers Arch. 1992;420:180-186.[Medline] [Order article via Infotrieve]

26. Spector PS, Curran ME, Keating MT, Sanguinetti MC. Class III antiarrhythmic drugs block HERG, a human cardiac delayed rectifier K⁺ channel: open-channel block by methanesulfonanilides. Circ Res. 1996;78:499-503.[Abstract/Free Full Text]

27. Brahmajothi MV, Morales MJ, Liu S, Rasmusson RL, Campbell DL, Strauss HC. In situ hybridization reveals extensive diversity of K⁺ channel mRNA in isolated ferret cardiac myocytes. Circ Res. 1996;78:1083-1089.[Abstract/Free Full Text]

28. Wang L, Feng Z-P, Konodo CS, Sheldon RS, Duff HJ. Developmental changes in the delayed rectifier K⁺ channels in mouse heart. Circ Res. 1996;79:79-85.[Abstract/Free Full Text]

29. Tande PM, Bjornstad H, Yant G, Refsum H. Rate-dependent class III antiarrhythmic action, negative chronotropy, and positive inotropy of a novel I_K blocking drug UK-68-798: potent in guinea-pig but no effect in rat myocardium. J Cardiovasc Pharmacol. 1990;16:401-410.[Medline] [Order article via Infotrieve]

30. Liu D-W, Gintant GA, Antzelevitch CA. Ionic bases for electrophysiological distinctions among epicardial, midmyocardial, and endocardial myocytes from the free wall of the canine left ventricle. Circ Res. 1993;72:671-687.[Abstract/Free Full Text]

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