Jane E. Dixon

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 (I(to)). 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 I(to) in canine ventricular myocytes, and probably in human myocytes. Both the Kv4.3 and Kv4.2 channels are likely to contribute to the I(to) in rat heart, and differential expression of these two channels can account for observed differences in the kinetic properties of the I(to) 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.

Molecular Basis of Transient Outward Potassium Current Downregulation in Human Heart Failure A Decrease in Kv4.3 mRNA Correlates With a Reduction in Current Density

BACKGROUND Despite advances in medical therapy, congestive heart failure remains a major cause of death in the developed world. A disproportionate number of the deaths of patients with heart failure are sudden and presumed to be arrhythmic. Heart failure in humans and in animal models is associated with prolongation of the action potential duration (APD), the result of downregulation of K+ currents-prominently, the Ca2+-independent transient outward current (Ito). The mechanism for the reduction of Ito in heart failure is unknown. The K+ channel alpha-subunit Kv4.3, a homolog of the Drosophila Shal family, is most likely to encode all or part of the native cardiac Ito in humans. METHODS AND RESULTS We used ribonuclease protection assays and whole-cell electrophysiological recording to study changes in the level of Kv4.3 mRNA and Ito in human tissues and isolated ventricular myocytes, respectively. We found that the level of Kv4.3 mRNA decreased by 30% in failing hearts compared with nonfailing controls. Furthermore, this reduction correlated with the reduction in peak Ito density measured in ventricular myocytes isolated from adjacent regions of the heart. There was no significant change in the steady-state level of any other mRNA studied (HERG, Kv1.4, Kir2.1, Kvss1.3, and the alpha1C subunit of the Ca2+ channel). mRNAs encoding Kv1.2, Kv1.5, and Kv2.1 were found in low abundance in human ventricle. CONCLUSIONS These data provide further support for the hypothesis that Kv4.3 encodes all or part of the native cardiac Ito in humans and that part of the downregulation of this current in heart failure may be transcriptionally regulated.

Tissue and species distribution of mRNA for the IKr-like K+ channel, erg.

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 (IKr) 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 IKr. In an effort to provide a different type of correlative data on the relationship between erg and IKr. 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 approximately 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 IKr-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 IKr-like current in rat. In isolated rat ventricular myocytes, an E-4031-sensitive current was observed, which is consistent with the presence of IKr. These results strengthen the link between erg and the native IKr in heart and suggest that erg may play an important role in other noncardiac tissues.

Transient Outward Current, Ito1, Is Altered in Cardiac Memory

BACKGROUND Cardiac memory refers to an altered T-wave morphology induced by ventricular pacing or arrhythmias that persist for variable intervals after resumption of sinus rhythm. METHODS AND RESULTS We induced long-term cardiac memory (LTM) in conscious dogs by pacing the ventricles at 120 bpm for 3 weeks. ECGs were recorded daily for 1 hour, during which time pacing was discontinued. At terminal study, the heart was removed and the electrophysiology of left ventricular epicardial myocytes was investigated. Control (C) and LTM ECG did not differ, except for T-wave amplitude, which decreased from 0.12+/-0.18 to -0.34+/-0.21 mV (+/-SEM, P<0.05), and T-wave vector, which shifted from -37+/-12 degrees to -143+/-4 degrees (P<0.05). Epicardial action potentials revealed loss of the notch and lengthening of duration at 20 days (both P<0.05). Calcium-insensitive transient outward current (Ito) was investigated by whole-cell patch clamp. No difference in capacitance was seen in C and LTM myocytes. Ito activated on membrane depolarization to -25+/-1 mV in C and -7+/-1 mV (P<0.05) in LTM myocytes, indicating a positive voltage shift of activation. Ito density was reduced in LTM myocytes, and a decreased mRNA level for Kv4.3 was observed. Recovery of Ito from inactivation was significantly prolonged: it was 531+/-80 ms (n=10) in LTM and 27+/-6 ms (n=9) in C (P<0.05) at -65 mV. CONCLUSIONS Ito changes are associated with and can provide at least a partial explanation for action-potential and T-wave changes occurring with LTM.

Expression of the trk gene family of neurotrophin receptors in prevertebral sympathetic ganglia.

When the phenotype of neurons in pre- and paravertebral sympathetic ganglia are compared, there are marked differences in NGF dependence, neuropeptide content, connectivity and electrophysiological properties. The trophic interactions that induce these differences are currently poorly understood. One explanation is that prevertebral neurons receive a second neurotrophic signal, other than NGF, from their target of innervation. If this is the case, neurons in the prevertebral ganglia should express another neurotrophin receptor, in addition to the NGF receptor (trkA). To test this prediction, the level of expression of three neurotrophin receptors, trkA, trkB and trkC, were examined in one paravertebral sympathetic ganglia, the SCG, and two prevertebral ganglia, the celiac and superior mesenteric ganglia. It was found that mRNA encoding the full-length form of the trkB receptor was barely expressed in the SCG. Significantly higher levels of full-length trkB mRNA expression were found in the prevertebral ganglia. Ligands of the trkB receptor may, therefore, contribute to the differentiation and/or survival of some prevertebral sympathetic neurons.

Unexpected and Differential Effects of Cl− Channel Blockers on the Kv4.3 and Kv4.2 K+ Channels

The Kv4.3 K+ channel is thought to underlie the Ca(2+)-insensitive transient outward current (I(to1)) in ventricular myocytes of canine and human heart and to contribute to the I(to1) in rat myocytes. It has been suggested that there is a second component of the transient outward current in some species that is contributed by a Ca(2+)-activated Cl- current (known as I(to2)). The evidence for the existence of the I(to2) current is based, in part, on the pharmacological effects of various Cl- channel blockers. To test for possible interactions between these compounds and I(to1), the effect of several different Cl- channel blockers on the Kv4.3 channel was examined. The fenamates (niflumic and flufenamic acid) were found to have large effects on the position of the steady state inactivation curve of the Kv4.3 channel. The disulfonic stilbenes (DIDS and SITS) had markedly different effects and were found to greatly reduce the rate of recovery from inactivation of the Kv4.3 channel without large changes in the position of the activation and steady state inactivation curves. Both classes of drugs produced an apparent blockade of the Kv4.3 channel under some recording conditions. Surprisingly, the closely related Kv4.2 channel was found to be markedly less sensitive to these drugs. Caffeine was found to block both the Kv4.3 and Kv4.2 channels to a similar extent. These nonspecific drug effects have implications for the study of the two components of the transient outward current and suggest that purely pharmacological criteria cannot be used to define the physiological role of I(to2).

Cloning of a mammalian elk potassium channel gene and EAG mRNA distribution in rat sympathetic ganglia

1 Three new members of the EAG potassium channel gene family were identified in rat and the complete coding sequence of one of these genes (elk1) was determined by cDNA cloning. 2 The elk1 gene, when expressed in Xenopus oocytes, encodes a slowly activating and slowly deactivating potassium channel. 3 The elk1 gene is expressed in sympathetic ganglia and is also expressed in sciatic nerve. 4 Six of the seven known EAG genes were found to be expressed in rat sympathetic ganglia, suggesting an important functional role for these channels in the sympathetic nervous system.

Potassium Channel mRNA Expression in Prevertebral and Paravertebral Sympathetic Neurons

The expression of eighteen different voltage‐activated potassium channel genes in rat sympathetic ganglia was quantitatively analysed using an RNase protection assay. Eleven α‐subunit genes and two β‐subunit genes were expressed in sympathetic ganglia. The relative level of potassium channel mRNA expression was compared between the superior cervical ganglion (SCG) and two prevertebral sympathetic ganglia, the coeliac ganglion (CG) and the superior mesenteric ganglion (SMG). Four mRNAs were differentially expressed: Kv1.2, Kv1.4, Kv2.2 and Kvβ1. Transcripts from all four genes were more abundant in the prevertebral ganglia. From comparisons with previous electrophysiological studies it was concluded that genes encoding the channels underlying the M‐current and D2‐current, which are both prominent in sympathetic neurons, have yet to be identified. It was also concluded that members of the Kv4 family are likely to underlie the low‐threshold A‐current in sympathetic neurons.

Alternative splicing of KCNQ2 potassium channel transcripts contributes to the functional diversity of M-currents

1 The region of alternative splicing in the KCNQ2 potassium channel gene was determined by RNase protection analysis of KCNQ2 mRNA transcripts. 2 Systematic analysis of KCNQ2 alternative splice variant expression in rat superior cervical ganglia revealed multiple variant isoforms. 3 One class of KCNQ2 splice variants, those that contained exon 15a, was found to have significantly different kinetics to those of the other isoforms. These transcripts encoded channel subunits that, when co‐expressed with the KCNQ3 subunit, activated and deactivated approximately 2.5 times more slowly than other isoforms. Deletion of exon 15a in these isoforms produced a reversion to the faster kinetics. 4 Comparison of the kinetic properties of the cloned channel splice variants with those of the native M‐current suggests that alternative splicing of the KCNQ2 gene may contribute to the variation in M‐current kinetics seen in vivo.