Barbara A. Wible

Antisense Oligodeoxynucleotides Directed Against Kv1.5 mRNA Specifically Inhibit Ultrarapid Delayed Rectifier K+ Current in Cultured Adult Human Atrial Myocytes

Several cloned K+ channel subunits are candidates to underlie macroscopic currents in the human heart, but direct evidence bearing on their role is lacking. The Kv1.5 K+ channel subunit has been suggested to play a potential role in human cardiac ultrarapid delayed rectifier (IKur) and transient outward (Ito) currents. To evaluate the role of proteins encoded by the Kv1.5 gene, we incubated cultured human atrial myocytes for 48 hours in medium containing antisense phosphorothioate oligodeoxynucleotides directed against octodecameric segments of the Kv1.5 mRNA coding sequence, the same concentration of homologous oligodeoxynucleotides with four mismatch mutations, or vehicle (control group). Cells exposed to antisense showed a highly significant (approximately 50%) reduction in IKur whether measured by step current at the end of a 400-millisecond depolarizing pulse, tail current at -20 mV, or current sensitive to a concentration of 4-aminopyridine (50 mumol/L) that is highly selective for IKur compared with control cells or cells exposed to mismatch oligodeoxynucleotides. In contrast, Ito was not different among the three experimental groups. When cultured human ventricular myocytes were exposed to Kv1.5 antisense oligodeoxynucleotides with the same controls, no changes occurred in either Ito or the sustained current at the end of a depolarizing pulse. We conclude that Kv1.5 channel subunits are essential to the expression of IKur and do not play a role in Ito in cultured human atrial myocytes. These studies provide the first direct evidence with an antisense approach for the equivalence between a macroscopic cardiac K+ current and a cloned K+ channel subunit and offer insights into the molecular electrophysiology of the human heart.

Molecular Physiology and Pharmacology of HERG Single-Channel Currents and Block by Dofetilide

BACKGROUND The human ether-a-go-go-related gene (HERG) is one locus for the hereditary long-QT syndrome. A hypothesis is that HERG produces the repolarizing cardiac potassium current IKr with the consequence that mutations in HERG prolong the QT interval by reducing IKr. The elementary properties of HERG are unknown, and as a test of the hypothesis that HERG produces IKr, we compared their elementary properties. METHODS AND RESULTS We injected HERG cRNA into Xenopus oocytes and measured currents from single channels or current variance from the noise produced by ensembles of channels recorded from macro patches. Single-channel conductance was dependent on the extracellular potassium concentration ([K]o). At physiological [K]o, it was 2 picosiemens (pS), and at 100 mmol/L [K]o, it was 10 pS. Openings occurred in bursts with a mean duration of 26 ms at -100 mV. Mean open time was 3.2 ms and closed times were 1.0 and 26 ms. In excised macro patches, HERG currents were blocked by the class III antiarrhythmic drug dofetilide, with an IC50 of 35 nmol/L. Dofetilide block was slow and greatly attenuated at positive potentials at which HERG rectifies. CONCLUSIONS The microscopic physiology of HERG and IKr is similar, consistent with HERG being an important component of IKr. The pharmacology is also similar; dofetilide appears to primarily block activated channels and has a much lower affinity for closed and inactivated channels.

Transmural heterogeneity of calcium handling in canine.

Abstract— Spatial heterogeneity of the action potential and its influence on arrhythmia vulnerability is known. However, heterogeneity of intracellular calcium handling and, in particular, its effect on the electrophysiological substrate is less clear. Using optical mapping techniques, calcium transients and action potentials were recorded simultaneously from ventricular sites across the transmural wall of the arterially perfused canine left ventricular wedge preparation during steady-state baseline pacing and rapid pacing. During baseline pacing, the decay of intracellular calcium to diastolic levels and calcium transient duration were slower (70%, P <0.005) and longer (20%, P <0.005), respectively, closer to the endocardial surface compared with the epicardial surface. Tissue samples isolated from the left ventricular wall demonstrate that sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) expression was significantly less in the subendocardial and midmyocardial layers compared with the subepicardial layer. In contrast, no significant difference in the transmural expression of Na+-Ca2+ exchanger was observed. During rapid pacing, calcium transient alternans and increased levels of diastolic intracellular calcium were significantly greater (P <0.01) closer to the endocardium (101%±62% and 41%±15%, respectively) compared with the epicardium (12%±7% and 12%±14%, respectively). In conclusion, cells closer to the endocardium exhibit a slower decay of intracellular calcium compared with cells near the epicardium, which may be due in part to reduced expression of SERCA2a. As a possible consequence, calcium transient alternans and increased diastolic levels of intracellular calcium may occur preferentially closer to the endocardial surface.

C-TERMINUS DETERMINANTS FOR MG2+ AND POLYAMINE BLOCK OF THE INWARD RECTIFIER K+ CHANNEL IRK1

Critical loci for ion conduction in inward rectifier K+ channels are only now being discovered. The C‐terminal region of IRK1 plays a crucial role in Mg2+i blockade and single‐channel K+ conductance. A negatively charged aspartate in the putative second transmembrane domain (position 172) is essential for time‐dependent block by the cytoplasmic polyamines spermine and spermidine. We have now localized the C‐terminus effect in IRK1 to a single, negatively charged residue (E224). Mutation of E224 to G, Q and S drastically reduced rectification. Furthermore, the IRK1 E224G mutation decreased block by Mg2+i and spermidine and, like the E224Q mutation, caused a dramatic reduction in the apparent single‐channel K+ conductance. The double mutation IRK1 D172N+ E224G was markedly insensitive to spermidine block, displaying an affinity similar to ROMK1. The results are compatible with a model in which the negatively charged residue at position 224, E224, is a major determinant of pore properties in IRK1. By means of a specific interaction with the negatively charged residue at position 172, D172, E224 contributes to the formation of the binding pocket for Mg2+ and polyamines, a characteristic of strong inward rectifiers.

Potassium channels Kv1.1, Kv1.2 and Kv1.6 influence excitability of rat visceral sensory neurons.

Voltage‐gated potassium channels, Kv1.1, Kv1.2 and Kv1.6, were identified as PCR products from mRNA prepared from nodose ganglia. Immunocytochemical studies demonstrated expression of the proteins in all neurons from ganglia of neonatal animals (postnatal days 0‐3) and in 85‐90 % of the neurons from older animals (postnatal days 21‐60). In voltage clamp studies, α‐dendrotoxin (α‐DTX), a toxin with high specificity for these members of the Kv1 family, was used to examine their contribution to K+ currents of the sensory neurons. α‐DTX blocked current in both A‐ and C‐type neurons. The current had characteristics of a delayed rectifier with activation positive to −50 mV and little inactivation during 250 ms pulses. In current‐clamp experiments α‐DTX, used to eliminate the current, had no effect on resting membrane potential and only small effects on the amplitude and duration of the action potential of A‐ and C‐type neurons. However, there were prominent effects on excitability. α‐DTX lowered the threshold for initiation of discharge in response to depolarizing current steps, reduced spike after‐hyperpolarization and increased the frequency/pattern of discharge of A‐ and C‐type neurons at membrane potentials above threshold. Model simulations were consistent with these experimental results and demonstrated how the other major K+ currents function in response to the loss of the α‐DTX‐sensitive current to effect these changes in action potential wave shape and discharge.

Cloning and Functional Expression of an Inwardly Rectifying K+ Channel From Human Atrium

The cardiac inward rectifier current (IK1) contributes to the shape and duration of the cardiac action potential and helps to set the resting membrane potential. Although several inwardly rectifying K+ channels (IRKs) from different tissues have been cloned recently, the nature and number of K+ channels contributing to the cardiac IK1 are presently unknown. To address this issue in human heart, we have used the reverse-transcriptase-polymerase chain reaction (PCR) technique with human atrial total RNA as a template to identify two sequences expressed in heart that are homologous to previously cloned IRKs. One of the PCR products we obtained was virtually identical to IRK1 (cloned from a mouse macrophage cell line); the other, which we named hIRK, exhibited < 70% identity to IRK1. A full-length clone encoding hIRK was isolated from a human atrial cDNA library and functionally expressed in Xenopus oocytes. This channel, like IRK1, exhibited strong inward rectification and was blocked by divalent cations. However, hIRK differed from IRK1 at the single-channel level: hIRK had a single-channel conductance of 36 pS compared with 21 pS for IRK1. We have identified single channels of 41, 35, 21, and 9 pS in recordings from dispersed human atrial myocytes. However, none of these atrial inward rectifiers exhibited single-channel properties exactly like those of cloned hIRK expressed in oocytes. Our findings suggest that the cardiac IK1 in human atrial myocytes is composed of multiple inwardly rectifying channels distinguishable on the basis of single-channel conductance, each of which may be the product of a different gene.

Cardiac Glycosides as Novel Inhibitors of Human Ether-a-go-go-Related Gene Channel Trafficking

Direct block of the cardiac potassium channel human ether-a-go-go-related gene (hERG) by a large, structurally diverse group of therapeutic compounds causes drug-induced QT prolongation and torsades de pointes arrhythmias. In addition, several therapeutic compounds have been identified more recently that prolong the QT interval by inhibition of hERG trafficking to the cell surface. We used a surface expression assay to identify novel compounds that interfere with hERG trafficking and found that cardiac glycosides are potent inhibitors of hERG expression at the cell surface. Further investigation of digitoxin, ouabain, and digoxin revealed that all three cardiac glycosides reduced expression of the fully glycosylated cell surface form of hERG on Western blots, indicating that channel exit from the endoplasmic reticulum is blocked. Likewise, hERG currents were reduced with nanomolar affinity on long-term exposure. hERG trafficking inhibition was initiated by cardiac glycosides through direct block of Na+/K+ pumps and not via off-target interactions with hERG or another closely associated protein in its processing or export pathway. In isolated guinea pig myocytes, long-term exposure to 30 nM of the clinically used drugs digoxin or digitoxin reduced hERG/rapidly activating delayed rectifier K+ current (IKr) currents by approximately 50%, whereas three other cardiac membrane currents—inward rectifier current, slowly activating delayed rectifier K+ current, and calcium current—were not affected. Importantly, 100 nM digitoxin prolonged action potential duration on long-term exposure consistent with a reduction in hERG/IKr channel number. Thus, cardiac glycosides are able to delay cardiac repolarization at nanomolar concentrations via hERG trafficking inhibition, and this may contribute to the complex electrocardiographic changes seen with compounds such as digitoxin.

Separable Kvβ Subunit Domains Alter Expression and Gating of Potassium Channels

Kvβ subunits have been shown to affect kinetic properties of voltage-gated K+ channel Kv1α subunits and increase the number of cell surface dendrotoxin-binding sites when coexpressed with Kv1.2. Here, we show that Kvβ1.2 alters both current expression and gating of Kvα1 channels and that each effect is mediated by a distinct Kvβ1.2 domain. The Kvβ1.2 N terminus or Kvα1-blocking domain introduced steady state current block, an apparent negative shift in steady state activation, and a slowing of deactivation along with a dramatic reduction in single channel open probability. N-terminal deletions of Kvβ1.2 no longer altered channel kinetics but promoted dramatic increases in Kv1.2 current. The conserved Kvβ1 C terminus or Kvα1 expression domain alone was sufficient to increase the number of functional channels. The same effect was observed with the normally noninactivating subunit, Kvβ2. By contrast, Kv1.5 currents were reduced when coexpressed with either the Kvβ1 C terminus or Kvβ2, indicating that the Kvα1 expression domain has Kvα1 isoform-specific effects. Our results demonstrate that Kvβ subunits consist of two domains that are separable on the basis of both primary structure and functional modulation of voltage-gated K+ channels.

Comparison of Binding and Block Produced by Alternatively Spliced Kvβ1 Subunits

Voltage-gated K+ (Kv) channels consist of α subunits complexed with cytoplasmic Kvβ subunits. Kvβ1 subunits enhance the inactivation of currents expressed by the Kv1 α subunit subfamily. Binding has been demonstrated between the C terminus of Kvβ1.1 and a conserved segment of the N terminus of Kv1.4, Kv1.5, and Shaker α subunits. Here we have examined the interaction and functional properties of two alternatively spliced human Kvβ subunits, 1.2 and 1.3, with Kvα subunits 1.1, 1.2, 1.4, and 1.5. In the yeast two-hybrid assay, we found that both Kvβ subunits interact specifically through their conserved C-terminal domains with the N termini of each Kvα subunit. In functional experiments, we found differences in modulation of Kv1α subunit currents that we attribute to the unique N-terminal domains of the two Kvβ subunits. Both Kvβ subunits act as open channel blockers at physiological membrane potentials, but hKvβ1.2 is a more potent blocker than hKvβ1.3 of Kv1.1, Kv1.2, Kv1.4, and Kv1.5. Moreover, hKvβ1.2 is sensitive to redox conditions, whereas hKvβ1.3 is not. We suggest that different Kvβ subunits extend the range over which distinct Kv1α subunits are modulated and may provide a variable mechanism for adjusting K+ currents in response to alterations in cellular conditions.

Mutations in the Kvβ2 Binding Site for NADPH and Their Effects on Kv1.4

Kvβ2 enhances the rate of inactivation and level of expression of Kv1.4 currents. The crystal structure of Kvβ2 binds NADP+, and it has been suggested that Kvβ2 is an oxidoreductase enzyme (1). To investigate how this function might relate to channel modulation, we made point mutations in Kvβ2 in either the NADPH docking or putative catalytic sites. Using the yeast two-hybrid system, we found that these mutations did not disrupt the interaction of Kvβ2 with Kvα1 channels. To characterize the Kvβ2 mutants functionally, we coinjected wild-type or mutant Kvβ2 cRNAs and Kv1.4 cRNA in Xenopus laevis oocytes. Kvβ2 increased both the amplitude and rate of inactivation of Kv1.4 currents. The cellular content of Kv1.4 protein was unchanged on Western blot, but the amount in the plasmalemma was increased. Mutations in either the orientation or putative catalytic sites for NADPH abolished the expression-enhancing effect on Kv1.4 current. Western blots showed that both types of mutation reduced Kv1.4 protein. Like the wild-type Kvβ2, both types of mutation increased the rate of inactivation of Kv1.4, confirming the physical association of mutant Kvβ2 subunits with Kv1.4. Thus, mutations that should interfere with NADPH function uncouple the expression-enhancing effect of Kvβ2 on Kv1.4 currents from its effect on the rate of inactivation. These results suggest that the binding of NADPH and the putative oxidoreductase activity of Kvβ2 may play a role in the processing of Kv1.4.