Altered K(+) channel gene expression in diabetic rat ventricle: isoform switching between Kv4.2 and Kv1.4

Expression of voltage-gated K(+) channels encoding the K(+) independent transient outward current in the streptozocin-induced diabetic (DM) rat ventricle was studied to determine the basis for slowed cardiac repolarization in diabetes mellitus. Although hypertrophy was not detected in diabetic rats at 12 wk after streptozocin treatment, ventricular Kv4.2 mRNA levels decreased 41% relative to nondiabetic controls. Kv1.4 mRNA levels increased 179% relative to controls, whereas Kv4.3 mRNA levels were unaffected. Immunohistochemistry and Western blot analysis of the diabetic heart showed that the density of the Kv4.2 protein decreased, whereas Kv1.4 protein increased. Thus isoform switching from Kv4.2 to Kv1.4 is most likely the mechanism underlying the slower kinetics of transient outward K(+) current observed in the diabetic ventricle. Brain Kv1.4, Kv4.2, or Kv4.3 mRNA levels were unaffected by diabetes. Myosin heavy chain (MHC) gene expression was altered with a 32% decrease in alpha-MHC mRNA and a 259% increase in beta-MHC mRNA levels in diabetic ventricle. Low-dose insulin-like growth factor-II (IGF-II) treatment during the last 6 of the 12 wk of diabetes (DM + IGF) protected against these changes in MHC mRNAs despite continued hyperglycemia and body weight loss. IGF-II treatment did not change K(+) channel mRNA levels in DM or control rat ventricles. Thus IGF treatment may prevent some, but not all, biochemical abnormalities in the diabetic heart.     

Developmental analysis reveals mismatches in the expression of K+ channel alpha subunits and voltage-gated K+ channel currents in rat ventricular myocytes

In the experiments here, the developmental expression of the functional Ca(2+)-independent, depolarization-activated K+ channel currents, Ito and IK, and of the voltage-gated K+ channel (Kv) alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2 in rat ventricular myocytes were examined quantitatively. Using the whole-cell patch clamp recording method, the properties and the densities of Ito and IK in ventricular myocytes isolated from postnatal day 5 (P5), 10 (P10), 15 (P15), 20 (P20), 25 (P25), 30 (P30), and adult (8-12 wk) rats were characterized and compared. These experiments revealed that mean Ito densities increase fourfold between birth and P30, whereas IK densities vary only slightly. Neither the time- nor the voltage-dependent properties of the currents vary measurably, suggesting that the subunits underlying functional Ito and IK channels are the same throughout postnatal development. In parallel experiments, the developmental expression of each of the voltage-gated K+ channel alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2, was examined quantitatively at the mRNA and protein levels using subunit-specific probes. RNase protection assays revealed that Kv1.4 message levels are high at birth, increase between P0 and P10, and subsequently decrease to very low levels in adult rat ventricles. The decrease in message is accompanied by a marked reduction in Kv1.4 protein, consistent with our previous suggestion that Kv1.4 does not contribute to the formation of functional K+ channels in adult rat ventricular myocytes. In contrast to Kv1.4, the mRNA levels of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 increase (three- to five- fold) between birth and adult. Western analyses, however, revealed that the expression patterns of these subunits proteins vary in distinct ways: Kv1.2 and Kv4.2, for example, increase between P5 and adult, whereas Kv1.5 remains constant and Kv2.1 decreases. Throughout development, therefore, there is a mismatch between the numbers of Kv alpha subunits expressed and the functional voltage-gated K+ channel currents distinguished electrophysiologically in rat ventricular myocytes. Alternative experimental approaches will be required to define directly the Kv alpha subunits that underlie functional voltage- gated K+ channels in these (and other) cells. In addition, the finding that Kv alpha subunit protein expression levels do not necessarily mirror mRNA levels suggests that caution should be exercised in attempting functional interpretations of observed changes in mRNA levels alone.     

Differential association of the auxiliary subunit Kvbeta2 with Kv1.4 and Kv4.3 K+ channels

Kvbeta2 subunits associate with Kv1 and Kv4 K+ channels, but the basis of preferential association is not understood. For example, detergent resistance suggests stronger auxiliary subunit association with Kv4.2 than with Kv1.2, but Kvbeta2 preferentially localizes with the latter channels in brain. Here we examine the interaction of Kvbeta2 with two native binding partners in brain: Kv4.3 and Kv1.4. We show that the auxiliary subunit binds more efficiently to Kv1.4 than to Kv4.3 in mammalian cells. However, preexisting Kvbeta2 complexes with Kv1.4 and Kv4.3 have similar detergent sensitivity. Thus, preferential steady state binding may reflect a difference in initial association rather than stability. We also find that that the cytoplasmic C-terminus of Kv4.3 inhibits Kvbeta2 association. Apparently, a region proximal to the Kv4.3 pore contributes to the inefficient auxiliary subunit interaction that produces preferential binding of Kvbeta2 to Kv1 channels.     

Molecular correlates of altered expression of potassium currents in failing rabbit myocardium

Action potential (AP) prolongation is a hallmark of failing myocardium. Functional downregulation of K currents is a prominent feature of cells isolated from failing ventricles. The detailed changes in K current expression differ depending on the species, the region of the heart, and the mechanism of induction of heart failure. We used complementary approaches to study K current downregulation in pacing tachycardia-induced heart failure in the rabbit. The AP duration (APD) at 90% repolarization was significantly longer in cells isolated from failing hearts compared with controls (539 +/- 162 failing vs. 394 +/- 114 control, P < 0.05). The major K currents in the rabbit heart, inward rectifier potassium current (I(K1)), transient outward (I(to)), and delayed rectifier current (I(K)) were functionally downregulated in cells isolated from failing ventricles. The mRNA levels of Kv4.2, Kv1.4, KChIP2, and Kir2.1 were significantly downregulated, whereas the Kv4.3, Erg, KvLQT1, and minK were unaltered in the failing ventricles compared with the control left ventricles. Significant downregulation in the long splice variant of Kv4.3, but not in the total Kv4.3, Kv4.2, and KChIP2 immunoreactive protein, was observed in cells isolated from the failing ventricle with no change in Kv1.4, KvLQT1, and in Kir2.1 immunoreactive protein levels. Multiple cellular and molecular mechanisms underlie the downregulation of K currents in the failing rabbit ventricle.     

Differential expression of Kv4 pore-forming and KChIP auxiliary subunits in rat uterus during pregnancy

Regulation of voltage-gated K(+) (K(v)) channel expression may be involved in controlling contractility of uterine smooth muscle cells during pregnancy. Functional expression of these channels is not only controlled by the levels of pore-forming subunits, but requires their association with auxiliary subunits. Specifically, rapidly inactivating K(v) current is prominent in myometrial cells and may be carried by complexes consisting of Kv4 pore-forming and KChIP auxiliary subunits. To determine the molecular identity of the channel complexes and their changes during pregnancy, we examined the expression and localization of these subunits in rat uterus. RT-PCR analysis revealed that rat uterus expressed all three Kv4 pore-forming subunits and KChIP2 and -4 auxiliary subunits. The expression of mRNAs for these subunits was dynamically and region selectively regulated during pregnancy. In the corpus, Kv4.2 mRNA level increased before parturition, whereas the expression of Kv4.1 and Kv4.3 mRNAs decreased during pregnancy. A marked increase in KChIP2 mRNA level was also seen at late gestation. In the cervix, the expression of all three pore-forming and two auxiliary subunit mRNAs increased at late gestation. Immunoprecipitation followed by immunoblot analysis indicated that Kv4.2-KChIP2 complexes were significant in uterus at late pregnancy. Kv4.2- and KChIP2-immunoreactive proteins were present in both circular and longitudinal myometrial cells. Finally, Kv4.2 and KChIP2 mRNA levels were similarly elevated in pregnant and nonpregnant corpora of one side-conceived rats. These results suggest that diffusible factors coordinate the pregnancy-associated changes in molecular compositions of myometrial Kv4-KChIP channel complexes.     

Voltage-gated potassium channel (Kv) subunits expressed in the rat cochlear nucleus.

Because the neuronal membrane properties and firing characteristics are crucially affected by the depolarization-activated K(+) channel (Kv) subunits, data about the Kv distribution may provide useful information regarding the functionality of the neurons situated in the cochlear nucleus (CN). Using immunohistochemistry in free-floating slices, the distribution of seven Kv subunits was described in the rat CN. Positive labeling was observed for Kv1.1, 1.2, 1.6, 3.1, 3.4, 4.2, and 4.3 subunits. Giant and octopus neurons showed particularly strong immunopositivity for Kv3.1; octopus neurons showed intense Kv1.1- and 1.2-specific reactions also. In the latter case, an age-dependent change of the expression pattern was also documented; although both young and older animals produced definite labeling for Kv1.2, the intensity of the reaction increased in older animals and was accompanied with the translocation of the Kv1.2 subunits to the cell surface membrane. The granule cell layer exhibited strong Kv4.2-specific immunopositivity, and markedly Kv4.2-positive glomerular synapses were also seen. It was found that neither giant nor pyramidal cells were uniform in terms of their Kv expression patterns. Our data provide new information about the Kv expression of the CN and also suggest potential functional heterogeneity of the giant and pyramidal cells.     

The thyroid hormone analog DITPA restores I(to) in rats after myocardial infarction

Previous studies have established that reductions in repolarizing currents occur in heart disease and can contribute to life-threatening arrhythmias in myocardium. In this study, we investigated whether the thyroid hormone analog 3, 5-diiodothyropropionic acid (DITPA) could restore repolarizing transient outward K(+) current (I(to)) density and gene expression in rat myocardium after myocardial infarction (MI). Our findings show that I(to) density was reduced after MI (14.0 +/- 1.0 vs. 10.2 +/- 0.9 pA/pF, sham vs. post-MI at +40 mV). mRNA levels of Kv4.2 and Kv4.3 genes were decreased but Kv1.4 mRNA levels were increased post-MI. Corresponding changes in Kv4.2 and Kv1.4 protein were also observed. Chronic treatment of post-MI rats with 10 mg/kg DITPA restored I(to) density (to 15.2 +/- 1.1 pA/pF at +40 mV) as well as Kv4.2 and Kv1.4 expression to levels observed in sham-operated controls. Other membrane currents (Na(+), L-type Ca(2+), sustained, and inward rectifier K(+) currents) were unaffected by DITPA treatment. Associated with the changes in I(to) expression, action potential durations (current-clamp recordings in isolated single right ventricular myocytes and monophasic action potential recordings from the right free wall in situ) were prolonged after MI and restored with DITPA treatment. Our results demonstrate that DITPA restores I(to) density in the setting of MI, which may be useful in preventing complications associated with I(to) downregulation.     

Position-dependent attenuation by Kv1.6 of N-type inactivation of Kv1.4-containing channels

Assembly of distinct α subunits of Kv1 (voltage-gated K(+) channels) into tetramers underlies the diversity of their outward currents in neurons. Kv1.4-containing channels normally exhibit N-type rapid inactivation, mediated through an NIB (N-terminal inactivation ball); this can be over-ridden if associated with a Kv1.6 α subunit, via its NIP (N-type inactivation prevention) domain. Herein, NIP function was shown to require positioning of Kv1.6 adjacent to the Kv1.4 subunit. Using a recently devised gene concatenation, heterotetrameric Kv1 channels were expressed as single-chain proteins on the plasmalemma of HEK (human embryonic kidney)-293 cells, so their constituents could be arranged in different positions. Placing the Kv1.4 and 1.6 genes together, followed by two copies of Kv1.2, yielded a K(+) current devoid of fast inactivation. Mutation of critical glutamates within the NIP endowed rapid inactivation. Moreover, separating Kv1.4 and 1.6 with a copy of Kv1.2 gave a fast-inactivating K(+) current with steady-state inactivation shifted to more negative potentials and exhibiting slower recovery, correlating with similar inactivation kinetics seen for Kv1.4-(1.2)(3). Alternatively, separating Kv1.4 and 1.6 with two copies of Kv1.2 yielded slow-inactivating currents, because in this concatamer Kv1.4 and 1.6 should be together. These findings also confirm that the gene concatenation can generate K(+) channels with α subunits in pre-determined positions.     

Two N-terminal domains of Kv4 K(+) channels regulate binding to and modulation by KChIP1

The family of calcium binding proteins called KChIPs associates with Kv4 family K(+) channels and modulates their biophysical properties. Here, using mutagenesis and X-ray crystallography, we explore the interaction between Kv4 subunits and KChIP1. Two regions in the Kv4.2 N terminus, residues 7-11 and 71-90, are necessary for KChIP1 modulation and interaction with Kv4.2. When inserted into the Kv1.2 N terminus, residues 71-90 of Kv4.2 are also sufficient to confer association with KChIP1. To provide a structural framework for these data, we solved the crystal structures of Kv4.3N and KChIP1 individually. Taken together with the mutagenesis data, the individual structures suggest that that the Kv4 N terminus is required for stable association with KChIP1, perhaps through a hydrophobic surface interaction, and that residues 71-90 in Kv4 subunits form a contact loop that mediates the specific association of KChIPs with Kv4 subunits.     

Distinct transient outward potassium current (Ito) phenotypes and distribution of fast-inactivating potassium channel alpha subunits in ferret left ventricular myocytes

The biophysical characteristics and alpha subunits underlying calcium-independent transient outward potassium current (Ito) phenotypes expressed in ferret left ventricular epicardial (LV epi) and endocardial (LV endo) myocytes were analyzed using patch clamp, fluorescent in situ hybridization (FISH), and immunofluorescent (IF) techniques. Two distinct Ito phenotypes were measured (21-22 degrees C) in the majority of LV epi and LV endo myocytes studied. The two Ito phenotypes displayed marked differences in peak current densities, activation thresholds, inactivation characteristics, and recovery kinetics. Ito,epi recovered rapidly [taurec, -70 mV = 51 +/- 3 ms] with minimal cumulative inactivation, while Ito,endo recovered slowly [taurec, -70 mV = 3,002 +/- 447 ms] with marked cumulative inactivation. Heteropoda toxin 2 (150 nM) blocked Ito,epi in a voltage-dependent manner, but had no effect on Ito,endo. Parallel FISH and IF measurements conducted on isolated LV epi and LV endo myocytes demonstrated that Kv1.4, Kv4.2, and Kv4.3 alpha subunit expression in LV myocyte types was quite heterogenous: (a) Kv4.2 and Kv4.3 were more predominantly expressed in LV epi than LV endo myocytes, and (b) Kv1.4 was expressed in the majority of LV endo myocytes but was essentially absent in LV epi myocytes. In combination with previous measurements on recovery kinetics (Kv1.4, slow; Kv4.2/4.3, relatively rapid) and Heteropoda toxin block (Kv1.4, insensitive; Kv4.2, sensitive), our results strongly support the hypothesis that, in ferret heart, Kv4.2/Kv4.3 and Kv1.4 alpha subunits, respectively, are the molecular substrates underlying the Ito,epi and Ito,endo phenotypes. FISH and IF measurements were also conducted on ferret ventricular tissue sections. The three Ito alpha subunits again showed distinct patterns of distribution: (a) Kv1.4 was localized primarily to the apical portion of the LV septum, LV endocardium, and approximate inner 75% of the LV free wall; (b) Kv4. 2 was localized primarily to the right ventricular free wall, epicardial layers of the LV, and base of the heart; and (c) Kv4.3 was localized primarily to epicardial layers of the LV apex and diffusely distributed in the LV free wall and septum. Therefore, in intact ventricular tissue, a heterogeneous distribution of candidate Ito alpha subunits not only exists from LV epicardium to endocardium but also from apex to base.     

KChAP/Kvbeta1.2 interactions and their effects on cardiac Kv channel expression

KChAP and voltage-dependent K+ (Kv) beta-subunits are two different types of cytoplasmic proteins that interact with Kv channels. KChAP acts as a chaperone for Kv2.1 and Kv4.3 channels. It also binds to Kv1.x channels but, with the exception of Kv1.3, does not increase Kv1.x currents. Kvbeta-subunits are assembled with Kv1.x channels; they exhibit "chaperone-like" behavior and change gating properties. In addition, KChAP and Kvbeta-subunits interact with each other. Here we examine the consequences of this interaction on Kv currents in Xenopus oocytes injected with different combinations of cRNAs, including Kvbeta1.2, KChAP, and either Kv1.4, Kv1.5, Kv2.1, or Kv4.3. We found that KChAP attenuated the depression of Kv1.5 currents produced by Kvbeta1.2, and Kvbeta1.2 eliminated the increase of Kv2.1 and Kv4.3 currents produced by KChAP. Both KChAP and Kvbeta1.2 are expressed in cardiomyocytes, where Kv1.5 and Kv2.1 produce sustained outward currents and Kv4.3 and Kv1.4 generate transient outward currents. Because they interact, either KChAP or Kvbeta1.2 may alter both sustained and transient cardiac Kv currents. The interaction of these two different classes of modulatory proteins may constitute a novel mechanism for regulating cardiac K+ currents.     

Differential expression of K+ channel mRNAs in the rat brain and down-regulation in the hippocampus following seizures.

K+ channels are major determinants of membrane excitability. Differences in neuronal excitability within the nervous system may arise from differential expression of K+ channel genes, regulated spatially in a cell type-specific manner, or temporally in response to neuronal activity. We have compared the distribution of mRNAs of three K+ channel genes, Kv1.1, Kv1.2, and Kv4.2 in rat brain, and examined activity-dependent changes following treatment with the convulsant drug pentylenetetrazole. Both regional and cell type-specific differences of K+ channel gene expression were found. In addition, seizure activity caused a reduction of Kv1.2 and Kv4.2 mRNAs in the dentate granule cells of the hippocampus, raising the possibility that K+ channel gene regulation may play a role in long-term neuronal plasticity.     

Subunit composition determines Kv1 potassium channel surface expression

Shaker-related or Kv1 voltage-gated K(+) channels play critical roles in regulating the excitability of mammalian neurons. Native Kv1 channel complexes are octamers of four integral membrane alpha subunits and four cytoplasmic beta subunits, such that a tremendous diversity of channel complexes can be assembled from the array of alpha and beta subunits expressed in the brain. However, biochemical and immunohistochemical studies have demonstrated that only certain complexes predominate in the mammalian brain, suggesting that regulatory mechanisms exist that ensure plasma membrane targeting of only physiologically appropriate channel complexes. Here we show that Kv1 channels assembled as homo- or heterotetrameric complexes had distinct surface expression characteristics in both transfected mammalian cells and hippocampal neurons. Homotetrameric Kv1.1 channels were localized to endoplasmic reticulum, Kv1.4 channels to the cell surface, and Kv1.2 channels to both endoplasmic reticulum and the cell surface. Heteromeric assembly with Kv1.4 resulted in dose-dependent increases in cell surface expression of coassembled Kv1.1 and Kv1.2, while coassembly with Kv1.1 had a dominant-negative effect on Kv1.2 and Kv1.4 surface expression. Coassembly with Kv beta subunits promoted cell surface expression of each Kv1 heteromeric complex. These data suggest that subunit composition and stoichiometry determine surface expression characteristics of Kv1 channels in excitable cells.     

Augmentation of Kv4.2-encoded currents by accessory dipeptidyl peptidase 6 and 10 subunits reflects selective cell surface Kv4.2 protein stabilization

Rapidly activating and inactivating somatodendritic voltage-gated K(+) (Kv) currents, I(A), play critical roles in the regulation of neuronal excitability. Considerable evidence suggests that native neuronal I(A) channels function in macromolecular protein complexes comprising pore-forming (α) subunits of the Kv4 subfamily together with cytosolic, K(+) channel interacting proteins (KChIPs) and transmembrane, dipeptidyl peptidase 6 and 10 (DPP6/10) accessory subunits, as well as other accessory and regulatory proteins. Several recent studies have demonstrated a critical role for the KChIP subunits in the generation of native Kv4.2-encoded channels and that Kv4.2-KChIP complex formation results in mutual (Kv4.2-KChIP) protein stabilization. The results of the experiments here, however, demonstrate that expression of DPP6 in the mouse cortex is unaffected by the targeted deletion of Kv4.2 and/or Kv4.3. Further experiments revealed that heterologously expressed DPP6 and DPP10 localize to the cell surface in the absence of Kv4.2, and that co-expression with Kv4.2 does not affect total or cell surface DPP6 or DPP10 protein levels. In the presence of DPP6 or DPP10, however, cell surface Kv4.2 protein expression is selectively increased. Further addition of KChIP3 in the presence of DPP10 markedly increases total and cell surface Kv4.2 protein levels, compared with cells expressing only Kv4.2 and DPP10. Taken together, the results presented here demonstrate that the expression and localization of the DPP accessory subunits are independent of Kv4 α subunits and further that the DPP6/10 and KChIP accessory subunits independently stabilize the surface expression of Kv4.2.     

Subcellular segregation of two A-type K+ channel proteins in rat central neurons.

In the mammalian nervous system, K+ channels regulate diverse aspects of neuronal function and are encoded by a large set of K+ channel genes. The roles of different K+ channel proteins could be dictated by their localization to specific subcellular domains. We report that two K+ channel polypeptides, Kv1.4 and Kv4.2, which form transient (A-type) K+ channels when expressed in Xenopus oocytes, are segregated in rat central neurons. Kv1.4 protein is targeted to axons and possibly terminals, while Kv4.2 is concentrated in dendrites and somata. This differential distribution implies distinct roles for these channel proteins in vivo. Their localizations suggest that Kv1.4 and Kv4.2 may regulate synaptic transmission via presynaptic, or postsynaptic mechanisms, respectively.