Deletion of the Kv2.1 delayed rectifier potassium channel leads to neuronal and behavioral hyperexcitability.

The Kv2.1 delayed rectifier potassium channel exhibits high-level expression in both principal and inhibitory neurons throughout the central nervous system, including prominent expression in hippocampal neurons. Studies of in vitro preparations suggest that Kv2.1 is a key yet conditional regulator of intrinsic neuronal excitability, mediated by changes in Kv2.1 expression, localization and function via activity-dependent regulation of Kv2.1 phosphorylation. Here we identify neurological and behavioral deficits in mutant (Kv2.1(-/-) ) mice lacking this channel. Kv2.1(-/-) mice have grossly normal characteristics. No impairment in vision or motor coordination was apparent, although Kv2.1(-/-) mice exhibit reduced body weight. The anatomic structure and expression of related Kv channels in the brains of Kv2.1(-/-) mice appear unchanged. Delayed rectifier potassium current is diminished in hippocampal neurons cultured from Kv2.1(-/-) animals. Field recordings from hippocampal slices of Kv2.1(-/-) mice reveal hyperexcitability in response to the convulsant bicuculline, and epileptiform activity in response to stimulation. In Kv2.1(-/-) mice, long-term potentiation at the Schaffer collateral - CA1 synapse is decreased. Kv2.1(-/-) mice are strikingly hyperactive, and exhibit defects in spatial learning, failing to improve performance in a Morris Water Maze task. Kv2.1(-/-) mice are hypersensitive to the effects of the convulsants flurothyl and pilocarpine, consistent with a role for Kv2.1 as a conditional suppressor of neuronal activity. Although not prone to spontaneous seizures, Kv2.1(-/-) mice exhibit accelerated seizure progression. Together, these findings suggest homeostatic suppression of elevated neuronal activity by Kv2.1 plays a central role in regulating neuronal network function.

ERG proteins and functional cardiac I(Kr) channels in rat, mouse, and human heart.

The voltage-gated K(+) channel (Kv) pore forming alpha subunit, ERG1 (KCNH2), has been identified as the locus of mutations in one type of inherited long QT syndrome, LQT2. Heterologous expression of ERG1 reveals rapidly activating and inactivating K(+) currents, characterized by marked inward rectification at potentials positive to 0 mV, which are similar to the rapid component of cardiac delayed rectification I(Kr). There are, however, marked differences in the properties of expressed ERG1 and endogenous cardiac I(Kr), suggesting that functional I(Kr) channels reflect the coassembly of full-length ERG1 with splice variants and /or accessory subunits. Consistent with these hypotheses, N- and C-terminal variants of ERG1 have been identified, and it has been demonstrated that heterologously expressed ERG1 and minK (or MiRP1) coimmunoprecipitate. Recent biochemical studies, however, suggest that only full-length ERG1 is expressed in adult mouse, rat, or human heart. Clearly, further studies, focused on identifying the subunits that coassemble with ERG1 in vivo, as well as on post-translational processing of the full-length ERG1 protein will be necessary to define the molecular composition of functional cardiac I(Kr) channels.

Genetic manipulation of cardiac K(+) channel function in mice: what have we learned, and where do we go from here?

In the mammalian myocardium, potassium (K(+)) channels control resting potentials, action potential waveforms, automaticity, and refractory periods and, in most cardiac cells, multiple types of K(+) channels that subserve these functions are expressed. Molecular cloning has revealed the presence of a large number of K(+) channel pore forming (alpha) and accessory (beta) subunits in the heart, and considerable progress has been made recently in defining the relationships between expressed K(+) channel subunits and functional cardiac K(+) channels. To date, more than 20 mouse models with altered K(+) channel expression/functioning have been generated using dominant-negative transgenic and targeted gene deletion approaches. In several instances, the genetic manipulation of K(+) channel subunit expression has revealed the role of specific K(+) channel subunit subfamilies or individual K(+) channel subunit genes in the generation of myocardial K(+) channels. In other cases, however, the phenotypic consequences have been unexpected. This review summarizes what has been learned from the in situ genetic manipulation of cardiac K(+) channel functioning in the mouse, discusses the limitations of the models developed to date, and explores the likely directions of future research.

Functional expression of a GFP-tagged Kv1.5 alpha-subunit in mouse ventricle.

The experiments here were undertaken to determine the feasibility of increasing the cell surface expression of voltage-gated ion channels in cardiac cells in vivo and to explore the functional consequences of ectopic channel expression. Transgenic mice expressing a green fluorescent protein (GFP)-tagged, voltage-gated K+ (Kv) channel alpha-subunit, Kv1.5-GFP, driven by the cardiac-specific alpha-MHC promoter, were generated. In recent studies, Kv1.5 has been shown to encode the micromolar 4-aminopyridine (4-AP)-sensitive delayed rectifier K+ current (I(K,slow)) in mouse myocardium. Unexpectedly, Kv1.5-GFP expression is heterogeneous in the ventricles of these animals. Although no electrocardiographic abnormalities were evident, expression of Kv1.5-GFP results in marked decreases in action potential durations in GFP-positive ventricular myocytes. In voltage-clamp recordings from GFP-positive ventricular myocytes, peak outward K+ currents are significantly higher, and their waveforms are distinct from those recorded from wild-type cells. Pharmacological experiments revealed a selective increase in a micromolar 4-AP-sensitive current, similar to the 4-AP-sensitive component of I(K,slow) in wild-type cells. The inactivation rate of the "overexpressed" current, however, is significantly slower than the Kv1.5-encoded component of I(K,slow) in wild-type cells, suggesting differences in association with accessory subunits and/or posttranslational processing.

Molecular heterogeneity of the voltage-gated fast transient outward K+ current, I(Af), in mammalian neurons.

Recently, we identified four kinetically distinct voltage-gated K(+) currents, I(Af), I(As), I(K), and I(SS), in rat superior cervical ganglion (SCG) neurons and demonstrated that I(Af) and I(As) are differentially expressed in type I (I(Af), I(K), I(SS)), type II (I(Af), I(As), I(K), I(SS)), and type III (I(K), I(SS)) SCG cells. In addition, we reported that I(Af) is eliminated in most ( approximately 70%) SCG cells expressing Kv4.2W362F, a Kv4 subfamily-specific dominant negative. The molecular correlate(s) of the residual I(Af), as well as that of I(As), I(K), and I(SS), however, are unknown. The experiments here were undertaken to explore the role of Kv1 alpha-subunits in the generation of voltage-gated K(+) currents in SCG neurons. Using the Biolistics Gene Gun, cDNA constructs encoding a Kv1 subfamily-specific dominant negative, Kv1.5W461F, and enhanced green fluorescent protein (EGFP) were introduced into SCG neurons. Whole-cell recordings from EGFP-positive Kv1.5W461F-expressing cells revealed a selective decrease in the percentage of type I cells and an increase in type III cells, indicating that I(Af) is gated by Kv1 alpha-subunits in a subset of type I SCG neurons. I(Af) is eliminated in all SCG cells expressing both Kv1.5W461F and Kv4.2W362F. I(Af) tau(decay) values in Kv1.5W461F-expressing and Kv4.2W362F-expressing type I cells are significantly different, revealing that Kv1 and Kv4 alpha-subunits encode kinetically distinct I(Af) channels. Expression of Kv1.5W461F increases excitability by decreasing action potential current thresholds and converts phasic cells to adapting or tonic firing. Interestingly, the molecular heterogeneity of I(Af) channels has functional significance because Kv1- and Kv4-encoded I(Af) play distinct roles in the regulation of neuronal excitability.

Characterization of mice with a combined suppression of I(to) and I(K,slow).

Cardiac-specific expression of a truncated Kv1.1 polypeptide (Kv1DN) attenuates the slow inactivating outward K(+) current (I(K,slow)), increases action potential duration (APD) and Q-T intervals, and induces spontaneous ventricular arrhythmias. Expression of the pore mutant of Kv4.2 (Kv4DN) eliminates the fast component of the transient outward current (I(to)) and prolongs APDs and Q-T intervals markedly; however, no arrhythmias are seen in Kv4DN mice, suggesting that APD and Q-T prolongation are not per se proarrhythmic. To test this hypothesis, the Kv1DN and Kv4DN lines were crossbred to produce animals (Kv1/Kv4DN) expressing both transgenes in an identical genetic background. Whole cell voltage-clamp recordings from left ventricular apex cells confirmed that in Kv1/Kv4DN left ventricular apex cells, both components (fast and slow) of I(to) and the 4-aminopyridine-sensitive component of I(K,slow) are eliminated, resulting in marked APD prolongation compared with wild-type, Kv1DN, or Kv4DN cells. Telemetric electrocardiogram monitoring (n = 10 mice/group) revealed a significant prolongation of Q-Tc and P-R intervals in Kv1/Kv4DN animals compared with Kv1DN or Kv4DN animals. Spontaneous arrhythmias were observed mainly in Kv1DN mice. Thus the attenuation of fast I(to) in addition to I(K,slow) in Kv1/Kv4DN mice causes significant prolongation of APD and Q-T intervals and attenuation of spontaneous arrhythmias.

Targeted replacement of KV1.5 in the mouse leads to loss of the 4-aminopyridine-sensitive component of I(K,slow) and resistance to drug-induced qt prolongation.

The K(+) channel mKv1.5 is thought to encode a 4-aminopyridine (4-AP)-sensitive component of the current I(K,slow) in the mouse heart. We used gene targeting to replace mKv1.5 with the 4-AP-insensitive channel rKv1.1 (SWAP mice) and directly test the role of Kv1.5 in the mouse ventricle. Kv1.5 RNA and protein were undetectable, rKv1.1 was expressed, and Kv2.1 protein was upregulated in homozygous SWAP hearts. The density of the K(+) current I(K,slow) (depolarizations to +40 mV, pA/pF) was similar in left ventricular myocytes isolated from SWAP homozygotes (17+/-1, n=27) and littermate controls (16+/-2, n=19). The densities and properties of I(peak), I(to,f), I(to,s), and I(ss) were also unchanged. In homozygous SWAP myocytes, the 50-micromol/L 4-AP-sensitive component of IK,slowwas absent (n=6), the density of the 20-mmol/L tetraethylammonium-sensitive component of I(K,slow) was increased (9+/-1 versus 5+/-1, P<0.05), and no 100- to 200-nmol/L alpha-dendrotoxin-sensitive current was found (n=8). APD(90) in SWAP myocytes was similar to controls at baseline but did not prolong in response to 30 micromol/L 4-AP. Similarly, QTc (ms) was not prolonged in anesthetized SWAP mice (64+/-2, homozygotes, n=9; 62+/-2, controls, n=9), and injection with 4-AP prolonged QTc only in controls (63+/-1, homozygotes; 72+/-2, controls; P<0.05). SWAP mice had no increase in arrhythmias during ambulatory telemetry monitoring. Thus, Kv1.5 encodes the 4-AP-sensitive component of I(K,slow) in the mouse ventricle and confers sensitivity to 4-AP-induced prolongation of APD and QTC: Compensatory upregulation of Kv2.1 may explain the phenotypic differences between SWAP mice and the previously described transgenic mice expressing a truncated dominant-negative Kv1.1 construct.

Molecular diversity of the repolarizing voltage-gated K+ currents in mouse atrial cells.

Voltage-clamp studies on atrial myocytes isolated from adult and postnatal day 15 (P15) C57BL6 mice demonstrate the presence of three kinetically distinct Ca2+-independent, depolarization-activated outward K+ currents: a fast, transient outward current (Ito,f), a rapidly activating, slowly inactivating current (IK,s) and a non-inactivating, steady-state current (Iss). The time- and voltage-dependent properties of to,f, IK,s and Iss in adult and P15 atrial cells are indistinguishable. Pharmacological experiments reveal the presence of two components of IK,s: one that is blocked selectively by 50 microM 4-aminopyridine (4-AP), and a 4-AP-insensitive component that is blocked by 25 mM TEA; Iss is also partially attenuated by 25 mM TEA. There are also two components of IK,s recovery from steady-state inactivation. To explore the molecular correlates of mouse atrial IK,s and Iss, whole-cell voltage-clamp recordings were obtained from P15 and adult atrial cells isolated from transgenic mice expressing a mutant Kv2.1 alpha subunit (Kv2.1N216Flag) that functions as a dominant negative, and from P15 atrial myocytes exposed to (1 microM) antisense oligodeoxynucleotides (AsODNs) targeted against Kv1.5 or Kv2.1. Peak outward K+ current densities are attenuated significantly in atrial myocytes isolated from P15 and adult Kv2.1N216Flag-expressing animals and in P15 cells exposed to AsODNs targeted against either Kv1.5 or Kv2.1. Analysis of the decay phases of the outward currents evoked during long (5 s) depolarizing voltage steps revealed that IK, s is selectively attenuated in cells exposed to the Kv1.5 AsODN, whereas both IK,s and Iss are attenuated in the presence of the Kv2. 1 AsODN. In P15 and adult Kv2.1N216Flag-expressing atrial cells, mean +/- s.e.m. IK,s and Iss densities are also significantly lower than in non-transgenic atrial cells. In addition, pharmacological experiments reveal that the TEA-sensitive component IK,s is selectively eliminated in P15 and adult Kv2.1N216Flag-expressing atrial cells. Taken together, the results presented here reveal that both Kv1.5 and Kv2.1 contribute to mouse atrial IK,s, consistent with the presence of two molecularly distinct components of IK,s. In addition, Kv2.1 contributes to mouse atrial Iss.

Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.

We generated transgenic mice in which red, green, yellow, or cyan fluorescent proteins (together termed XFPs) were selectively expressed in neurons. All four XFPs labeled neurons in their entirety, including axons, nerve terminals, dendrites, and dendritic spines. Remarkably, each of 25 independently generated transgenic lines expressed XFP in a unique pattern, even though all incorporated identical regulatory elements (from the thyl gene). For example, all retinal ganglion cells or many cortical neurons were XFP positive in some lines, whereas only a few ganglion cells or only layer 5 cortical pyramids were labeled in others. In some lines, intense labeling of small neuronal subsets provided a Golgi-like vital stain. In double transgenic mice expressing two different XFPs, it was possible to differentially label 3 neuronal subsets in a single animal.

Elimination of the fast transient in superior cervical ganglion neurons with expression of KV4.2W362F: molecular dissection of IA.

Electrophysiological and molecular studies have revealed considerable heterogeneity in voltage-gated K(+) currents and in the subunits that underlie these channels in mammalian neurons. At present, however, the relationship between native K(+) currents and cloned subunits is poorly understood. In the experiments here, a molecular genetic approach was exploited to define the molecular correlate of the fast transient outward K(+) current, I(Af), in sympathetic neurons and to explore the functional role of I(Af) in shaping action potential waveforms and controlling repetitive firing patterns. Using the biolistic gene gun, cDNAs encoding a dominant negative mutant Kv4.2 alpha-subunit (Kv4.2W362F) and enhanced green fluorescent protein (EGFP) were introduced into rat sympathetic neurons in vitro. Whole-cell voltage-clamp recordings obtained from EGFP-positive cells revealed that I(Af) is selectively eliminated in cells expressing Kv4.2W362F, demonstrating that Kv4 alpha-subunits underlie I(Af) in sympathetic neurons. In addition, I(Af) density is increased significantly in cells overexpressing wild-type Kv4.2. In cells expressing Kv4.2W362F, input resistances are increased and (current) thresholds for action potential generation are decreased, demonstrating that I(Af) plays a pivotal role in regulating excitability. Expression of Kv4.2W362F and elimination of I(Af) also alters the distribution of repetitive firing patterns observed in response to a prolonged injection of depolarizing current. The wild-type superior cervical ganglion is composed of phasic, adapting, and tonic firing neurons. Elimination of I(Af) increases the percentage of adapting cells by shifting phasic cells to the adapting firing pattern, and increased I(Af) density reduces the number of adapting cells.

Functional consequences of elimination of i(to,f) and i(to,s): early afterdepolarizations, atrioventricular block, and ventricular arrhythmias in mice lacking Kv1.4 and expressing a dominant-negative Kv4 alpha subunit.

It was recently reported that the slow transient outward K(+) current, I(to, s), that is evident in mouse left ventricular septal cells is eliminated in mice with a targeted deletion of the Kv1.4 gene (Kv1.4(-/-)). The rapidly inactivating transient outward K(+) current, I(to, f), in contrast, is selectively eliminated in ventricular myocytes isolated from transgenic mice expressing a dominant-negative Kv4 alpha subunit, Kv4.2W362F. Expression of Kv4. 2W362F results in marked prolongation of action potentials and QT intervals. In addition, a slow transient outward K(+) current, that is similar to I(to,s) in wild-type mouse left ventricular septal cells, is evident in all Kv4.2W362F-expressing (left and right) ventricular cells. To test directly the hypothesis that upregulation of Kv1.4 alpha subunit underlies the appearance of this slow transient outward K(+) current in Kv4.2W362F-expressing ventricular cells and to explore the functional consequences of elimination of I(to,f) and I(to,s), mice expressing Kv4.2W362F in the Kv1.4(-/-) background (Kv4.2W362FxKv1.4(-/-)) were generated. Histological and echocardiographic studies revealed no evidence of structural abnormalities or contractile dysfunction in Kv4.2W362FxKv1.4(-/-) mouse hearts. Electrophysiological recordings from the majority (approximately 80%) of cells isolated from the right ventricle and left ventricular apex of Kv4.2W362FxKv1.4(-/-) animals demonstrated that both I(to, f) and I(to,s) are eliminated; action potentials are prolonged significantly; and, in some cells, early afterdepolarizations were observed. In addition, in vivo telemetric ECG recordings from Kv4.2W362FxKv1.4(-/-) animals revealed marked QT prolongation, atrioventricular block, and ventricular tachycardia. These observations demonstrate that upregulation of Kv1.4 contributes to the electrical remodeling evident in the ventricles of Kv4.2W362F-expressing mice and that elimination of both I(to,f) and I(to,s) has dramatic functional consequences.

Molecular basis of electrical remodeling in atrial fibrillation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia, and is often associated with other cardiovascular disorders and diseases. AF can lead to thromboembolism, reduced left ventricular function and stroke, and, importantly, it is independently associated with increased mortality. AF is a progressive disease; numerous lines of evidence suggest that disease progression results from cumulative electrophysiological and structural remodeling of the atria. There is considerable interest in delineating the molecular mechanisms involved in the remodeling that occurs in the atria of patients with AF. Cellular electrophysiological studies have revealed marked reductions in the densities of the L-type voltage-gated Ca2+ current, I(Ca,L), the transient outward K+ current, I(TO), and the ultrarapid delayed rectifier K+ current, I(Kur), in atrial myocytes from patients in chronic AF. Similar (but not identical) changes in currents are evident in myocytes isolated from a canine model of AF and, in this case, the changes in currents are correlated with reduced expression of the underlying channel forming subunits. In both human and canine AF, the reduction in I(Ca,L) appears to be sufficient to explain the observed decreases in action potential duration and effective refractory period that are characteristic features of the remodeled atria. In addition, expression of the sarcoplasmic reticulum Ca2+ ATPase is reduced, suggesting that calcium cycling is affected in AF. These recent studies suggest that calcium overload and perturbations in calcium handling play prominent roles in AF-induced atrial remodeling. Although considerable progress has been made, further studies focused on defining the detailed structural, cellular and molecular changes that accompany the different stages of AF in humans, as well as in animal models of AF, are clearly warranted. It is anticipated that molecular insights gleaned from these studies will facilitate the development of improved therapeutic approaches to treat AF and to prevent the progression of the arrhythmia.

Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium.

In the mammalian heart, Ca2+-independent, depolarization-activated potassium (K+) currents contribute importantly to shaping the waveforms of action potentials, and several distinct types of voltage-gated K+ currents that subserve this role have been characterized. In most cardiac cells, transient outward currents, Ito,f and/or Ito,s, and several components of delayed reactivation, including IKr, IKs, IKur and IK,slow, are expressed. Nevertheless, there are species, as well as cell-type and regional, differences in the expression patterns of these currents, and these differences are manifested as variations in action potential waveforms. A large number of voltage-gated K+ channel pore-forming (alpha) and accessory (beta, minK, MiRP) subunits have been cloned from or shown to be expressed in heart, and a variety of experimental approaches are being exploited in vitro and in vivo to define the relationship(s) between these subunits and functional voltage-gated cardiac K+ channels. Considerable progress has been made in defining these relationships recently, and it is now clear that distinct molecular entities underlie the various electrophysiologically distinct repolarizing K+ currents (i.e. Ito,f, Ito,s, IKr, IKs, IKur, IK,slow, etc.) in myocyardial cells.

Rapid ventricular repolarization in rodents: electrocardiographic manifestations, molecular mechanisms, and clinical insights.

This article examines specific electrocardiographic (ECG) and electrophysiological features of ventricular repolarization in rats and mice, and the role of depolarization-activated potassium currents in mediating the unique features of ECG recordings in these rodents. This article describes the currents that underlie ventricular repolarization in these rodents, identifies terminology that appropriately describes the unique features of murine ECG recordings, and correlates these unique findings with selected human ECG ventricular repolarization abnormalities. The absence of a distinct isoelectric interval between the QRS complex and the T wave, accompanied by a relatively short QT interval, are common features of ECG recordings in mice and rats, but not in ECGs in guinea pigs. The murine ECG morphology is apparently attributable to the presence of large outward K+ currents that dominate the early phase of ventricular repolarization. In rats and mice, the predominant current underlying the early phase of repolarization appears to be the rapidly activating and inactivating 4-aminopyridine-sensitive transient outward current (ie, I(to)). Importantly, the density of I(to) in rats and mice is high, whereas this current is not evident in the ventricular myocytes of guinea pigs. The high density of I(to) appears to underlie the prominent J wave or downsloping ST-segment elevation seen in rats and mice, whereas the ST-segment is isoelectric in guinea pigs. The unusual J wave and ST-segment pattern in murine ECGs, however, does bear some resemblance to ECG features observed in humans with Brugada syndrome, and with hypothermia and ischemia. These patterns in rats and mice might, therefore, serve as an experimental model for the idiopathic J wave.

Do glia have heart? Expression and functional role for ether-a-go-go currents in hippocampal astrocytes.

Potassium homeostasis plays an important role in the control of neuronal excitability, and diminished buffering of extracellular K results in neuronal Hyperexcitability and abnormal synchronization. Astrocytes are the cellular elements primarily involved in this process. Potassium uptake into astrocytes occurs, at least in part, through voltage-dependent channels, but the exact mechanisms involved are not fully understood. Although most glial recordings reveal expression of inward rectifier currents (K(IR)), it is not clear how spatial buffering consisting of accumulation and release of potassium may be mediated by exclusively inward potassium fluxes. We hypothesized that a combination of inward and outward rectifiers cooperate in the process of spatial buffering. Given the pharmacological properties of potassium homeostasis (sensitivity to Cs(+)), members of the ether-a-go-go (ERG) channel family widely expressed in the nervous system could underlie part of the process. We used electrophysiological recordings and pharmacological manipulations to demonstrate the expression of ERG-type currents in cultured and in situ hippocampal astrocytes. Specific ERG blockers (dofetilide and E 4031) inhibited hyperpolarization- and depolarization-activated glial currents, and ERG blockade impaired clearance of extracellular potassium with little direct effect on hippocampal neuron excitability. Immunocytochemical analysis revealed ERG protein mostly confined to astrocytes; ERG immunoreactivity was absent in presynaptic and postsynaptic elements, but pronounced in glia surrounding the synaptic cleft. Oligodendroglia did not reveal ERG immunoreactivity. Intense immunoreactivity was also found in perivascular astrocytic end feet at the blood-brain barrier. cDNA amplification showed that cortical astrocytes selectively express HERG1, but not HERG2-3 genes. This study provides insight into a possible physiological role of hippocampal ERG channels and links activation of ERG to control of potassium homeostasis.

Expression of distinct ERG proteins in rat, mouse, and human heart. Relation to functional I(Kr) channels.

One form of inherited long QT syndrome, LQT2, results from mutations in HERG1, the human ether-a-go-go-related gene, which encodes a voltage-gated K(+) channel alpha subunit. Heterologous expression of HERG1 gives rise to K(+) currents that are similar (but not identical) to the rapid component of delayed rectification, I(Kr), in cardiac myocytes. In addition, N-terminal splice variants of HERG1 and MERG1 (mouse ERG1) referred to as HERG1b and MERG1b have been cloned and suggested to play roles in the generation of functional I(Kr) channels. In the experiments here, antibodies generated against HERG1 were used to examine ERG1 protein expression in heart and in brain. In Western blots of extracts of QT-6 cells expressing HERG1, MERG1, or RERG1 (rat ERG1) probed with antibodies targeted against the C terminus of HERG1, a single 155-kDa protein is identified, whereas a 95-kDa band is evident in blots of extracts from cells expressing MERG1b or HERG1b. In immunoblots of fractionated rat (and mouse) brain and heart membrane proteins, however, two prominent high molecular mass proteins of 165 and 205 kDa were detected. Following treatment with glycopeptidase F, the 165- and 205-kDa proteins were replaced by two new bands at 175 and 130 kDa, suggesting that ERG1 is differentially glycosylated in rat/mouse brain and heart. In human heart, a single HERG1 protein with an apparent molecular mass of 145 kDa is evident. In rats, ERG1 protein (and I(Kr)) expression is higher in atria than ventricles, whereas in humans, HERG1 expression is higher in ventricular, than atrial, tissue. Taken together, these results suggest that the N-terminal alternatively spliced variants of ERG1 (i.e. ERG1b) are not expressed at the protein level in rat, mouse, or human heart and that these variants do not, therefore, play roles in the generation of functional cardiac I(Kr) channels.

Double-blind, placebo-controlled study to evaluate two miconazole conditioners for the treatment of Malassezia dermatitis in dogs.

Eighteen dogs with Malassezia dermatitis participated in a clinical trial to evaluate the efficacy of miconazole conditioners. Dogs were randomly assigned to receive vehicle only, miconazole 1%, or miconazole 2% conditioner. Conditioners were used three times weekly for 2 weeks and then twice weekly for 2 weeks. Investigators evaluated erythema, pruritus, and yeast counts weekly. Owners scored pruritus daily. Yeast number decreased in all treatment groups. Yeast number in the vehicle group was higher than in both the miconazole treatment groups but was not different between the two miconazole groups. Clinical scores decreased but no difference was detected among groups.

Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes.

1. Two kinetically and pharmacologically distinct transient outward K+ currents, referred to as Ito,f and Ito,s, have been distinguished in mouse left ventricular myocytes. Ito,f is present in all left ventricular apex cells and in most left ventricular septum cells, whereas Ito,s is identified exclusively in left ventricular septum cells. 2. Electrophysiological recordings from ventricular myocytes isolated from animals with a targeted deletion of the Kv1.4 gene (Kv1.4-/- mice) reveal that Ito,s is undetectable in cells isolated from the left ventricular septum (n = 26). Ito,f density in both apex and septum cells, in contrast, is not affected by deletion of Kv1.4. 3. Neither the 4-AP-sensitive, slowly inactivating K+ current, IK,slow, nor the steady-state non-inactivating K+ current, ISS, is affected in Kv1.4-/- mouse left ventricular cells. 4. In myocytes isolated from transgenic mice expressing a dominant negative Kv4.2 alpha subunit, Kv4.2W362F, Ito,f is eliminated in both left ventricular apex and septum cells. In addition, a slowly inactivating transient outward K+ current similar to Ito,s in wild-type septum cells is evident in myocytes isolated from left ventricular apex of Kv4.2W362F-expressing transgenics. The density of Ito,s in septum cells, however, is unaffected by Kv4.2W362F expression. 5. Western blots of fractionated mouse ventricular membrane proteins reveal a significant increase in Kv1.4 protein level in Kv4.2W362F-expressing transgenic mice. The protein levels of other Kv alpha subunits, Kv1.2 and Kv2.1, in contrast, are not affected by the expression of the Kv4.2W362F transgene. 6. The results presented here demonstrate that the molecular correlates of Ito,f and Ito,s in adult mouse ventricle are distinct. Kv1.4 underlies mouse ventricular septum Ito,s, whereas Kv alpha subunits of the Kv4 subfamily underlie mouse ventricular apex and septum Ito, f. The appearance of the slow transient outward K+ current in Kv4. 2W362F-expressing left ventricular apex cells with properties indistinguishable from Ito,s in wild-type cells is accompanied by an increase in Kv1.4 protein expression, suggesting that the upregulation of Kv1.4 underlies the observed electrical remodeling in Kv4.2W362F-expressing transgenics.

Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant-negative Kv2 alpha subunit.

An in vivo experimental strategy, involving cardiac-specific expression of a mutant Kv 2.1 subunit that functions as a dominant negative, was exploited in studies focused on exploring the role of members of the Kv2 subfamily of pore-forming (alpha) subunits in the generation of functional voltage-gated K(+) channels in the mammalian heart. A mutant Kv2.1 alpha subunit (Kv2.1N216) was designed to produce a truncated protein containing the intracellular N terminus, the S1 membrane-spanning domain, and a portion of the S1/S2 loop. The truncated Kv2.1N216 was epitope tagged at the C terminus with the 8-amino acid FLAG peptide to generate Kv2. 1N216FLAG. No ionic currents are detected on expression of Kv2. 1N216FLAG in HEK-293 cells, although coexpression of this construct with wild-type Kv2.1 markedly reduced the amplitudes of Kv2. 1-induced currents. Using the alpha-myosin heavy chain promoter to direct cardiac specific expression of the transgene, 2 lines of Kv2. 1N216FLAG-expressing transgenic mice were generated. Electrophysiological recordings from ventricular myocytes isolated from these animals revealed that I(K, slow) is selectively reduced. The attenuation of I(K, slow) is accompanied by marked action potential prolongation, and, occasionally, spontaneous triggered activity (apparently induced by early afterdepolarizations) is observed. The time constant of inactivation of I(K, slow) in Kv2. 1N216FLAG-expressing cells (mean+/-SEM=830+/-103 ms; n=17) is accelerated compared with the time constant of I(K, slow) inactivation (mean+/-SEM=1147+/-57 ms; n=25) in nontransgenic cells. In addition, unlike I(K, slow) in wild-type cells, the component of I(K, slow) remaining in the Kv2.1N216FLAG-expressing cells is insensitive to 25 mmol/L tetraethylammonium. Taken together, these observations suggest that there are 2 distinct components of I(K, slow) in mouse ventricular myocytes and that Kv2 alpha subunits underlie the more slowly inactivating, tetraethylammonium-sensitive component of I(K, slow). In vivo telemetric recordings also reveal marked QT prolongation, consistent with a defect in ventricular repolarization, in Kv2.1N216FLAG-expressing transgenic mice.

Atrial L-type Ca2+ currents and human atrial fibrillation.

Chronic atrial fibrillation (AF) is characterized by decreased atrial contractility, shortened action potential duration, and decreased accommodation of action potential duration to changes in activation rate. Studies on experimental animal models of AF implicate a reduction in L-type Ca2+ current (I(Ca)) density in these changes. To evaluate the effect of AF on human I(Ca), we compared I(Ca) in atrial myocytes isolated from 42 patients in normal sinus rhythm at the time of cardiac surgery with that of 11 chronic AF patients. I(Ca) was significantly reduced in the myocytes of patients with chronic AF (mean -3.35+/-0.5 pA/pF versus -9.13+/-1. 0 pA/pF in the controls), with no difference between groups in the voltage dependence of activation or steady-state inactivation. Although I(Ca) was lower in myocytes from the chronic AF patients, their response to maximal beta-adrenergic stimulation was not impaired. Postoperative AF frequently follows cardiac surgery. Half of the patients in the control group (19/38) of this study experienced postoperative AF. Whereas chronic AF is characterized by reduced atrial I(Ca), the patients with the greatest I(Ca) had an increased incidence of postoperative AF, independent of patient age or diagnosis. This observation is consistent with the concept that calcium overload may be an important factor in the initiation of AF. The reduction in functional I(Ca) density in myocytes from the atria of chronic AF patients may thus be an adaptive response to the arrhythmia-induced calcium overload.