Expression Of Voltage-gated Potassium Channels Research Articles

Changes underlying arrhythmia in the transgenic heart overexpressing Refsum disease gene-associated protein

Previously, we identified a novel neuron-specific protein (PAHX-AP1) that binds to Refsum disease gene product (PAHX), and we developed transgenic (TG) mice that overexpress heart-targeted PAHX-AP1. These mice have atrial tachycardia and increased susceptibility to aconitine-induced arrhythmia. This study was undertaken to elucidate the possible changes in ion channels underlying the susceptibility to arrhythmia in these mice. RT-PCR analyses revealed that the cardiac expression of adrenergic β 1-receptor ( ADRB1) was markedly lower, whereas voltage-gated potassium channel expression ( Kv2.1) was higher in PAHX-AP1 TG mice compared with non-TG mice. However, the expression of voltage-sensitive sodium and calcium channels, and muscarinic receptor was not significantly different. Propranolol pretreatment, a non-specific β-adrenoceptor antagonist, blocked aconitine-induced arrhythmia in non-TG mice, but not in PAHX-AP1 TG mice. Our results indicate that, in the PAHX-AP1 TG heart, the modulation of voltage-gated potassium channel and ADRB1 expression seem to be important in the electrophysiological changes associated with altered ion channel functions, but ADRB1 is not involved in the greater susceptibility to aconitine-induced arrhythmia.

Analysis of phosphorylation-dependent modulation of Kv1.1 potassium channels

The voltage-gated potassium channel Kv1.1 contains phosphorylation sites for protein kinase A (PKA) and protein kinase C (PKC). To study Kv1.1 protein expression and cellular distribution in regard to its level of phosphorylation, the effects of PKA and PKC activation on Kv1.1 were investigated in HEK 293 cells stably transfected with Kv1.1 (HEK 293/1). Without kinase activation, HEK 293/1 cells carry unphosphorylated Kv1.1 protein in the plasma membranes, whereas large amounts of phosphorylated and unphosphorylated Kv1.1 protein were located intracellularly. Activation of PKA resulted in phosphorylation of intracellular Kv1.1 protein, followed by a rapid translocation of Kv1.1 into the plasma membrane. Patch-clamp analysis revealed an increase in current amplitude upon PKA activation and demonstrated differences in the voltage dependence of current activation between unphosphorylated and phosphorylated Kv1.1 channels. In contrast to PKA, even prolonged activation of PKC did not lead to direct phosphorylation of Kv1.1, but induced Kv1.1 protein synthesis. Thus, protein kinases have direct and indirect effects on the functional expression of voltage-gated potassium channels. Our data suggest that the synergistic action of protein kinases may play an important role in the fine-tuning of Kv channel function.

Voltage-gated potassium ion channels in colon cancer

Voltage-gated potassium channels (VGPCs) have been previously implicated in cellular proliferation. In this study, the expression of VGPCs was examined by immunohistochemistry in seventy-four human colonic carcinoma specimens. Immunostaining for the Kv1.3 type VGPC was absent in two normal human colon specimens. Kv1.3 staining in the 74 colon cancer specimens was low in 9% (7/74), moderate in 61% (45/74) and high in 30% (22/74). Potassium channel (PC) openers, minoxidil and diazoxide (5-50 microg/ml), increased growth of SW1116, LoVo, Colo320DM and LS174t human colon cancer cell lines by 20-40%. PC-blockers, dequalinium and amiodarone, caused marked growth-inhibition of the four cell lines, at concentrations between 1 to 3 microg/ml. PC-blockers such as glibenclamide inhibited cellular proliferation at concentrations above 50 microg/ml while tetraethylammonium and 4-aminopyridine (up to 100 microg/ml) did not have significant growth-suppressive effects. Our results indicate the presence of VGPCs in colon cancer and suggest that PCs could serve as therapeutic targets.

Heterogeneous expression of voltage-gated potassium channels in the heart: roles in normal excitation and arrhythmias.

In the mammalian myocardium, there are marked regional differences in action potential waveforms and frequency dependences. This heterogeneity impacts the normal dispersion of ventricular repolarization and appears to reflect the differential expression of voltage-gated K+ channels. Multiple types of voltage-gated K+ currents have been distinguished in mammalian ventricles and, in many cases, the K+ (Kv) channel pore-forming (alpha) and accessory (beta) subunits encoding these channels have been identified. In the diseased myocardium, remodeling of voltage-gated K+ currents occurs, influencing propagation and rhythmicity, effects that can lead to increased dispersion of ventricular repolarization and create substrates for reentrant arrhythmias. Targeting the K+ channels that function to maintain the normal dispersion of ventricular repolarization could be effective in treating cardiac arrhythmias.

Primary pulmonary hypertension: a vascular biology and translational research "Work in progress".

Primary pulmonary hypertension (PPH) is a syndrome of dyspnea, chest pain, and syncope defined by increased pulmonary vascular resistance and the absence of a known cause. It also occurs in a familial form, which is linked to unidentified genes on chromosome 2. This syndrome is characterized by abnormalities of pulmonary vascular biology in each compartment of the blood vessel. The lumen has a prothrombotic diathesis, the endothelium displays an excessive production of vasoconstrictors relative to vasodilators, and the smooth muscle cells are depolarized and calcium-overloaded, which is due in part to reduced expression of voltage-gated potassium channels (Kv). This causes vasoconstriction and may promote cell proliferation. The adventitia displays excessive remodeling, which is associated with exaggerated metalloproteinase and elastase activity. Conceptually, PPH seems to require a permissive genotype, a susceptible phenotype (eg, endothelial dysfunction) and, in many cases, an exogenous trigger (eg, an anorexigen). Although there is not a generally accepted, unifying hypothesis regarding its cause, impaired function and the expression of vascular and platelet Kv channels suggest PPH may be a disease of the ion channels. Abnormal matrix metalloproteinase and elastase activity could also explain the abnormal vascular tone, platelet activation, and remodeling in PPH. Although calcium-channel blockers and prostacyclin, particularly when coadministered with warfarin, improve survival, PPH has a 5-year mortality rate of approximately 50%. Pharmacological and gene therapies aimed at enhancing the activity of prostacyclin, nitric oxide synthases, and Kv channels or at inhibiting endothelin and matrix metalloproteinases are promising areas for future development.

Heterogeneous expression of voltage-gated potassium channels of the shaker family (Kv1) in oligodendrocyte progenitors

Outwardly rectifying K + channels determine the membrane conductance and influence the proliferation rate of glial progenitor cells. To analyze the molecular identity and the functional role of K + channels in glial progenitors of mouse brain, expression of shaker-type Kv1 genes was studied at three levels: (1) presence of Kv1 mRNAs, (2) biosynthesis of channel proteins and (3) electrophysiological and pharmacological properties of K + currents. mRNA expression of Kv1.1 to Kv1.6 genes was studied by single-cell reverse transcription–mediated polymerase chain reaction (RT-PCR) using degenerate primers to amplify the six Kv1 transcripts. Most cells expressed several mRNA combinations simultaneously. In more than half of the cells, messages for Kv1.2, Kv1.5 and Kv1.6 were found, while Kv1.1, Kv1.3 and Kv1.4 were detected in only a minority of cells. In contrast, at the level of protein expression — employing immunocytochemistry with subtype-specific antibodies — Kv1.2 and Kv1.3 were undetectable (<2%), while almost all cells expressed Kv1.4 (85%), Kv1.5 (99%) and Kv1.6 (99%). Kv1.1 was present in a minor cell population (10%). Functional contribution of Kv1 proteins to progenitor membrane conductance was determined by analyzing the voltage-dependence of K + current activation and inactivation as well as their current sensitivities to the subtype-preferring blockers and toxins tetraethylammonium (TEA), 4-aminopyridine (4-AP), charybdotoxin (CTX), α-dendrotoxin (DTX) and mast-cell degranulating peptide (MCDP). From these results, it is concluded: first, glial progenitor cells can express all transcripts of the six Kv1 genes, but do not express all proteins; second, Kv1.4, Kv1.5 and Kv1.6 proteins are most abundant and were found in the majority of cells; and third, K + currents flow predominantly either through heteromeric channel complexes or through homomeric Kv1.5 ion pores, but not through homomeric Kv1.4 or Kv1.6 channels.

Developmental expression of voltage-gated potassium channel β subunits

Expression of potassium channel beta subunits (Kvβ) was determined in the developing mouse CNS using an antiserum against an amino acid sequence present in the C-terminus of Kvβ1, Kvβ2, and Kvβ3. Using the anti-Kvβ antiserum, we determined that Kvβ expression is restricted to the spinal cord and dorsal root ganglia in the embryonic CNS. At birth, Kvβ expression is detected in brainstem and midbrain nuclei, but was not detected in the hippocampus, cerebellum or cerebral cortex. During the first postnatal week, Kvβ expression is present in hippocampal and cortical pyramidal cells and in cerebellar Purkinje cells. Expression of Kvβ subunits reaches adult levels by the third postnatal week in all of the brain regions examined. A rabbit antiserum directed against a unique peptide sequence in the N-terminus of the Kvβ1 protein demonstrates that this subunit displays a novel expression pattern in the developing mouse brain. Kvβ1 expression is high at birth in all brain regions examined and decreases with age. In contrast, Kvβ2 expression is low at birth and increases with age to reach adult levels by the third postnatal week. These findings support the notion that the differential regulation of distinct potassium channel beta subunits, in the developing mouse nervous system, may confer the functional diversity required to mediate both neuronal survival and maturation.

Expression of Kv3.1 and Kv4.2 Genes in Developing Cerebellar Granule Cells

The expression of voltage-gated potassium channels plays an important role in the acquisition of membrane excitability in neurons. We examined the expression pattern of genes in developing cerebellar granule neurons in vivo and in vitro. In situ hybridization of Kv3.1 mRNA demonstrated that the gene was expressed at high levels in the external granule layer (EGL) as well as in the internal granule layer (IGL) at all postnatal stages (P) examined (from P3 to P10). In contrast, Kv4.2 mRNA was detected in the premigratory zone (PMZ) of the EGL, but not in the proliferative zone (PLZ), in addition to the IGL. This indicates that Kv4.2 gene expression initiates in the postmitotic migrating neurons. We also examined the expression of the channel genes in microexplant culture systems. Kv3.1 polypeptide was detected in parallel fibers of granule cells at 2 days in vitro, and the expression continued in later stages. The signal of Kv4.2 protein was very low at 2 days in vitro; however, the number of positive cells and the intensity of the signals were increased at 6 days in vitro. These in vitro observations matched those in vivo and our previous electrophysiological studies in which we demonstrated that delayed- rectifier-type current was predominant in the immature granule cells followed by the later appearance of A-type current. The patterns of K⁺ channel expression suggest that sequential expression of these channel genes primarily determines the membrane excitability.

Changes in expression of voltage-gated potassium channels in dorsal root ganglion neurons following axotomy.

Several families of voltage-gated potassium channels (Kv), including a spectrum of subtypes, are involved in regulating and modifying the integration and transmission of electrical signals in the nervous system. However, the specific patterns of Kv expression in normal or injured dorsal root ganglion (DRG) neurons have not been studied. Previous studies have examined the expression of voltage-gated sodium channels in DRG neurons, and also the selective up- and downregulation of several of these channels following axonal injury to the DRG neurons. In the present study, we used immunocytochemical methods to investigate the expression of Kv channels (Kv1.1, 1.2, 1.3, 1.4, 1.5, 1.6, and 2.1) in DRG cells cultured from control and axotomized adult rats. Kv1.2 and 2.1 immunoreactivity in DRG neurons showed large decreases following axotomy, whereas Kv1.1 and 1.3 showed smaller decreases. Kv1.4 and 1.6 immunostaining were not altered by axotomy, and Kv1.5 immunoreactivity was low in both control and axotomized DRG neurons. These results provide molecular correlates for the expression of multiple K+ currents in normal DRG neurons and indicate that, in relation to changes in sodium channel expression, there are decreases in specific potassium channels following axotomy in these cells. The alterations in K+ and Na+ channel expression following axonal injury may lead to changes in electrical excitability of the DRG neurons, and might contribute to chronic pain syndromes.

Effects of thyroid status on expression of voltage-gated potassium channels in rat left ventricle.

Thyroid hormone modifies cardiac action potentials and outward potassium currents directly and indirectly e.g. through beta-adrenergic signaling pathway. We thus examined the expression of six voltage-gated potassium channel alpha-subunits in the rat left ventricle under hypo- and hyperthyroid status, and tested roles of beta-adrenergic signaling pathway in their expressions under both status. Hypothyroidism and hyperthyroidism were induced by administration of methimazole (MMI) for 4 weeks and by injection of L-thyroxine (T4) to the MMI-treated rats for the last 7 days, respectively. To distinguish the effects of T4 and the beta-adrenergic system, propranolol (Pro) was administered to the MMI-treated rats together with T4, and isoproterenol (Iso) was injected to MMI-treated rats for the last 7 days. The mRNA levels of Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2 and Kv4.3 in the left ventricles were determined by ribonuclease protection assay. MMI treatment induced hypothyroidism and resulted in a significant decrease in the mRNA levels of Kv1.5, Kv2.1 and Kv4.2 (19%, 77% and 61% of control value, respectively; n = 6, p < 0.05). T4 administration induced hyperthyroidism and cardiac hypertrophy, and it increased the Kv1.5 and Kv2.1 mRNA levels over the control value (212% and 140%, respectively; n = 6, p < 0.05). Kv4.2 mRNA level was restored to the control level by T4. In contrast, the Kv1.2 and Kv1.4 mRNA levels increased in hypothyroid rats (161% and 186% of control value, respectively; n = 6, p < 0.01) and decreased in hyperthyroid rats (14% and 33% of control value, respectively; n = 6, p < 0.01). The Kv4.3 mRNA level was not altered by thyroid status. Pro did not inhibit the T4-induced hypertrophy. Iso induced cardiac hypertrophy. Pro or Iso by itself did not alter Kv mRNA levels except for Kv1.2, the message of which was decreased by Iso. Thyroid hormone differentially regulates the expression of Kv1.4, Kv1.5, Kv2.1 and Kv4.2 mRNA levels in the rat left ventricle. This effect is not mediated through beta-adrenergic signaling pathway. On the other hand, the reduction in Kv1.2 mRNA level was associated with cardiac hypertrophy induced by T4 or Iso.

Expression of Voltage-Gated Potassium Channels Decreases Cellular Protein Tyrosine Phosphorylation

Protein tyrosine phosphorylation by endogenous and expressed tyrosine kinases is reduced markedly by the expression of functional voltage-gated potassium (Kv) channels. The levels of tyrosine kinase protein and cellular protein substrates are unaffected, consistent with a reduction in tyrosine phosphorylation that results from inhibition of protein tyrosine kinase activity. The attenuation of protein tyrosine phosphorylation is correlated with the gating properties of expressed wild-type and mutant Kv channels. Furthermore, cellular protein tyrosine phosphorylation is reduced within minutes by acute treatment with the electrogenic potassium ionophore valinomycin. Because tyrosine phosphorylation in turn influences Kv channel activity, these results suggest that reciprocal modulatory interactions occur between Kv channel and protein tyrosine phosphorylation signaling pathways.