Expression Of IK Research Articles

IK Channel-Independent Effects of Clotrimazole and Senicapoc on Cancer Cells Viability and Migration.

Many studies highlighted the importance of the IK channel for the proliferation and the migration of different types of cancer cells, showing how IK blockers could slow down cancer growth. Based on these data, we wanted to characterize the effects of IK blockers on melanoma metastatic cells and to understand if such effects were exclusively IK-dependent. For this purpose, we employed two different blockers, namely clotrimazole and senicapoc, and two cell lines: metastatic melanoma WM266-4 and pancreatic cancer Panc-1, which is reported to have little or no IK expression. Clotrimazole and senicapoc induced a decrease in viability and the migration of both WM266-4 and Panc-1 cells irrespective of IK expression levels. Patch-clamp experiments on WM266-4 cells revealed Ca2+-dependent, IK-like, clotrimazole- and senicapoc-sensitive currents, which could not be detected in Panc-1 cells. Neither clotrimazole nor senicapoc altered the intracellular Ca2+ concentration. These results suggest that the effects of IK blockers on cancer cells are not strictly dependent on a robust presence of the channel in the plasma membrane, but they might be due to off-target effects on other cellular targets or to the blockade of IK channels localized in intracellular organelles.

IK: A novel cell mitosis regulator that contributes to carcinogenesis.

Carcinogenesis is characterized by abnormal regulation of cell growth and cell death. IK is a novel cell mitosis regulator that may contribute to carcinogenesis. Previous studies showed that the loss of IK expression resulted in cell mitotic arrest and even cell death. Besides, IK can also inhibit the interferon gamma (IFN-γ)-induced expression of human leukocyte antigen (HLA) class II antigen, which is associated with tumour immune microenvironment. To gain insight into the current research progress regarding IK, we conducted a review and searched the limited literature on IK using PubMed or Web of Science. In this review, we discussed the possible biological functions and mechanisms of IK in cancer and its immune microenvironment. Future perspectives of IK were also mentioned to explore its clinical significance.

Inactivating Mutations of the IK Gene Weaken Ku80/Ku70-Mediated DNA Repair and Sensitize Endometrial Cancer to Chemotherapy.

Simple SummarySome endometrial cancer (EC) patients have different prognoses and responses to treatment, even among those with the same stage and tumor grade as assessed by the International Federation of Gynecology and Obstetrics (FIGO) criteria. Molecular classification may make up for this deficiency to some extent. Our previous investigation of the 271 EC samples from the Cancer Genome Atlas (TCGA) dataset showed that IK somatic mutations were enriched in a cluster of patients with high-grade and high-stage cancers, and this group had longer overall survival. This current study continued to provide insight into how IK somatic mutations contribute to EC pathophysiology. We explored IK gene mutations in depth and used functional studies to characterize an unrecognized function of the IK gene in EC. Our findings may elucidate the molecular mechanism of IK in EC, which might guide future patient stratification and contribute targeted therapy for EC.IK is a mitotic factor that promotes cell cycle progression. Our previous investigation of 271 endometrial cancer (EC) samples from the Cancer Genome Atlas (TCGA) dataset showed IK somatic mutations were enriched in a cluster of patients with high-grade and high-stage cancers, and this group had longer survival. This study provides insight into how IK somatic mutations contribute to EC pathophysiology. We analyzed the somatic mutational landscape of IK gene in 547 EC patients using expanded TCGA dataset. Co-immunoprecipitation and mass spectrometry were used to identify protein interactions. In vitro and in vivo experiments were used to evaluate IK’s role in EC. The patients with IK-inactivating mutations had longer survival during 10-year follow-up. Frameshift and stop-gain were common mutations and were associated with decreased IK expression. IK knockdown led to enrichment of G2/M phase cells, inactivation of DNA repair signaling mediated by heterodimerization of Ku80 and Ku70, and sensitization of EC cells to cisplatin treatment. IK/Ku80 mutations were accompanied by higher mutation rates and associated with significantly better overall survival. Inactivating mutations of IK gene and loss of IK protein expression were associated with weakened Ku80/Ku70-mediated DNA repair, increased mutation burden, and better response to chemotherapy in patients with EC.

Dietary Na+ Depletion Transcriptionally Up‐regulates BK Channel Mediated K+ Secretion in Proximal Colon

BackgroundElectrogenic K+ secretion and H+‐K+‐ATPase‐mediated electroneutral K+ absorption occur in normal rat proximal and distal colon, respectively. Although both high‐K+ diet and dietary‐Na+ depletion induce K+ secretion in distal colon, only the dietary‐Na+ depletion has been shown to stimulate K+ secretion in proximal colon. Dietary‐Na+ depletion induced both intermediate conductance K+ (IK) and large conductance K+ (BK) channels mediated K+ secretion, while high‐K+ diet induced only the BK channel expression and activity in distal colon. Although K+ secretions are present, the molecular identity of K+ channels that mediate K+ secretion in normal and dietary‐Na+ depleted rat proximal colon is not known.HypothesisIt is hypothesized that dietary‐Na+ depletion may induce K+ secretion by stimulating IK and/or BK channel in proximal colon.AimThis study was initiated to identify the molecular identity of the dietary‐Na+ stimulated K+ secretion in rat proximal colon.MethodsNormal rats (Male Sprague‐Dawly; 200–225g) were fed either normal, high‐K+ or Na+‐depleted diet ad libitum for 6–7 days. Mucosal to serosal (m‐s) and serosal to mucosal (s‐m) 86Rb+ (K+ surrogate) fluxes were measured in stripped proximal colon epithelia mounted under voltage clamp condition. Net K+ fluxes were calculated by subtracting s‐m fluxes from m‐s fluxes. Positive and negative net K+ fluxes represent active absorption and secretion, respectively. IK and BK channel mRNA abundance and protein expression were determined by RT‐qPCR and western blot analyses, respectively. Cell and membrane specific IK and BK channel expressions were determined by immunofluorescence studies.ResultsSimilar to earlier demonstration, active K+ secretion is present in normal proximal colon. Dietary‐Na+ depletion, but not high‐K+ diet stimulated K+ secretion in proximal colon (Normal vs Na+‐depletion: 0.42 ± 0.06 vs 2.33 ± 0.16 μEq/cm2.h; p < 0.001). Mucosal iberiotoxin (IbTX; 10 μM; BK channel blocker) completely inhibited the K+ in normal, while it inhibited only 72% in Na+‐depleted rat proximal colon. Mucosal TRAM34 (10 μM; IK channel blocker) did not affect K+ secretion in normal, but it significantly (22%; p < 0.02) inhibited in Na+‐depleted rat colon. RT‐qPCR analyses revealed increased IK (2.2‐fold) and BK (6.2‐fold) specific mRNA abundance, while western blot analyses confirmed increased expression of IK and BK channel expression. Immunofluorescence studies established that in vitro aldosterone (1 μM) incubation increased both IK and BK protein expression in apical membranes of proximal crypt glands.ConclusionWe conclude that high (~1000 ng/ml; Na+‐depletion), but not low (~300 ng/ml; high‐K+ diet) blood aldosterone level stimulates IK/BK expression to increase K+ secretion in proximal colon.Support or Funding InformationThis study was supported by DK112085 and DK104791 from the National Institute of Health Diabetes and Digestive and Kidney Diseases.

P1235Acetylcholine-activated inward-rectifier potassium currents in atrial cardiomyocytes derived from induced pluripotent stem cells and atrial engineered heart muscle preparations

Abstract Background Abnormal activity of muscarinic receptor (M-receptor) activated potassium current (IK,ACh) has been suggested as a potential atrial-specific drug target for antiarrhythmic therapy in patients with atrial fibrillation (AF). Recent work suggests that atrial myocytes derived from human induced pluripotent stem cells (iPSC-CM) hold great potential for in vitro disease modeling and patient-specific drug testing. Here we aim to investigate functional expression of IK,ACh channels in these cells. Methods Atrial differentiation was initiated by temporal WNT and retinoic acid signaling modulation. Action potentials (APs) were recorded using sharp electrode technique in engineered heart muscle preparations (EHM). Currents were measured with voltage-clamp technique in atrial and ventricular iPSC-CM. IK,ACh was activated with a saturating concentration of the non-selective M-receptor agonist carbachol (CCh, 2 μM). Results APs measured in atrial EHMs were shorter compared to their ventricular counterparts (AP duration at 90% repolarization [APD90]: 163.2±3.7 ms, n=6 [EHMs] vs. 210.2±3.1 ms, n=3, p<0.001). In addition, application of CCh resulted in APD90 reduction by ∼18% and hyperpolarisation of the resting membrane potential in atrial but not in ventricular EHMs, pointing to functional IK,ACh expression in atrial preparations. Basal inward-rectifier current, (at −100 mV) in the absence of muscarinic receptor agonists was larger in ventricular iPSC-CM compared to atrial differentiation (−26.1±3.8 pA/pF, n=82/5 [n = cells/line] vs. −15.6±1.6 pA/pF, n=71/5, p<0.02.). CCh-induced current increase, attributed to IK,ACh, was detectable in all myocytes from atrial preparations (−25.2±3.6 pA/pF, n=71/5). The time course of activation with a slow decrease in current amplitude during the continuous presence of CCh resembled measurements in freshly isolated human atrial myocytes. In ventricular iPSC-CM preparations only 13 out of 73 myocytes showed an increase in inward-rectifier current in response to CCh, suggesting that ventricular preparations may contain a minor fraction of atrial myocytes. Of note, in the presence of the selective IK,ACh blocker tertiapin (100 nM), CCh had no effect on basal inward-rectifier current, providing further evidence for the molecular identity of the CCh-induced current increase as IK,ACh. Interestingly, TTP reduced basal current amplitude in CM isolated from atrial EHMs by ∼54% suggesting that agonist-independent, constitutively active IK,ACh contributes to the basal inward-rectifier current amplitude in these myocytes. Conclusion Our data demonstrate that atrial iPSC-CMs express functional IK,ACh-channels, which can be activated by M-receptor agonists. These findings further outline the potential of atrial iPSC-CMs and EHMs as a promising tool for the investigation of channel-associated pathologies such as AF. Acknowledgement/Funding grants from the DFG (VO 1568/3-1, IRTG1816, and SFB1002 project A13), the Else-Kröner-Fresenius Foundation (EKFS 2016_A20) and the DZHK (DZHK GOE MD3)

Ikaros deficiency in host hematopoietic cells separates GVL from GVHD after experimental allogeneic hematopoietic cell transplantation

The graft-versus-leukemia (GVL) effect following allogeneic hematopoietic stem cell transplantation (allo-HCT) is critical for its curative potential. Hwever, GVL is tightly linked to graft-versus-host disease (GVHD). Among hematological malignancies, acute lymphoblastic leukemia (ALL) is the most resistant to GVL, although the reasons for this remain poorly understood. Clinical studies have identified alterations in Ikaros (Ik) transcription factor as the major marker associated with poor outcomes in ALL. We have shown that the absence of Ik in professional host-derived hematopoietic antigen-presenting cells (APCs) exacerbates GVHD. However, whether Ik expression plays a role in resistance to GVL is not known. In this study we used multiple clinically relevant murine models of allo-HCT to explore whether Ik expression in hematopoietic APCs and/or leukemic cells is critical for increasing resistance to GVL and thus inducing relapse. We found that Ik deficiency in host APCs failed to enhance GVL despite increased GVHD severity. Mechanistic studies with bone marrow (BM) chimeras and tetramer analyses demonstrated reduced tumor-specific immunodominant (gag+) antigen responses in the [B6Ik−/−→B6] group. Loss of GVL was observed when both the leukemia cells and the host APCs were deficient in Ik. We found that calreticulin (CRT) expression in host antigen-presenting dendritic cells (DCs) of Ik−/− animals was significantly lower than in wild-type animals. Rescuing CRT expression in Ik−/− DCs improved leukemic-specific cytotoxic T cell function. Together, our data demonstrate that the absence of Ikaros in host hematopoietic cells promotes resistance to GVL despite increasing GVHD and thus provides a potential mechanism for the poor outcome of Ik−/− ALL patients.

IK Induced by Coxsackievirus B3 Infection Transiently Downregulates Expression of MHC Class II Through Increasing cAMP

Major histocompatibility complex (MHC) class II expression is critical for the presentation of antigens in the immune response to viral infection. Consequently, some viruses regulate the MHC class II-mediated presentation of viral antigens as a mechanism of immune escape. In this study, we found that Coxsackievirus B3 (CVB3) infection transiently increased IK expression, which reduced the expression of MHC class II (I-A/I-E) on splenic B cells. Interestingly, CVB3-induced IK elevated cAMP, a downstream molecule of the G protein-coupled receptors, which inhibited MHC class II presentation on B cells. Transgenic mice expressing truncated IK showed lower expression of MHC class II on B cells than did wild-type mice after CVB3 infection. Taken together, these results imply that IK plays a role in downregulating MHC class II expression on B cells during CVB3 infection through the induction of cAMP.

Density functional study of the pressure tensor for inhomogeneous Lennard—Jones fluids

Based on classical density functional theory, an expression of the pressure tensor for inhomogeneous fluids is presented. This takes into account greater correlation between particles, especially for systems that are geometrically confined or involve an interface. The density and pressure components of Lennard-Jones fluids confined in hard and softened nano-cavities are calculated. A comparison between the results of this work and IK expression suggests that the agreement depends on temperature. The interfacial tension for hard sphere fluids agrees well with the Monte Carlo result when the bulk density is not too large. The results of the solid-fluid interfacial tension for Lennard—Jones fluids demonstrate that different types of external potentials modulate the interfacial tension in different manners.

The GPR55 agonist lysophosphatidylinositol directly activates intermediate-conductance Ca2+-activated K+ channels

Lysophosphatidylinositol (LPI) was recently shown to act both as an extracellular mediator binding to G protein-coupled receptor 55 (GPR55) and as an intracellular messenger directly affecting a number of ion channels including large-conductance Ca2+ and voltage-gated potassium (BKCa) channels. Here, we explored the effect of LPI on intermediate-conductance Ca2+-activated K+ (IKCa) channels using excised inside-out patches from endothelial cells. The functional expression of IKCa was confirmed by the charybdotoxin- and TRAM-34-sensitive hyperpolarization to histamine and ATP. Moreover, the presence of single IKCa channels with a slope conductance of 39 pS in symmetric K+ gradient was directly confirmed in inside-out patches. When cytosolically applied in the range of concentrations of 0.3–10 μM, which are well below the herein determined critical micelle concentration of approximately 30 μM, LPI potentiated the IKCa single-channel activity in a concentration-dependent manner, while single-channel current amplitude was not affected. In the whole-cell configuration, LPI in the pipette was found to facilitate membrane hyperpolarization in response to low (0.5 μM) histamine concentrations in a TRAM-34-sensitive manner. These results demonstrate a so far not-described receptor-independent effect of LPI on the IKCa single-channel activity of endothelial cells, thus, highlighting LPI as a potent intracellular messenger capable of modulating electrical responses in the vasculature.

Distinct Cellular and Molecular Mechanisms Underlie Functional Remodeling of Repolarizing K + Currents With Left Ventricular Hypertrophy

Left ventricular hypertrophy (LVH) is associated with electric remodeling and increased arrhythmia risk, although the underlying mechanisms are poorly understood. In the experiments here, functional voltage-gated (Kv) and inwardly rectifying (Kir) K(+) channel remodeling was examined in a mouse model of pressure overload-induced LVH, produced by transverse aortic constriction (TAC). Action potential durations (APDs) at 90% repolarization in TAC LV myocytes and QT(c) intervals in TAC mice were prolonged. Mean whole-cell membrane capacitance (C(m)) was higher, and I(to,f), I(K,slow), I(ss), and I(K1) densities were lower in TAC, than in sham, LV myocytes. Although the primary determinant of the reduced current densities is the increase in C(m), I(K,slow) amplitudes were decreased and I(ss) amplitudes were increased in TAC LV cells. Further experiments revealed regional differences in the effects of LVH. Cellular hypertrophy and increased I(ss) amplitudes were more pronounced in TAC endocardial LV cells, whereas I(K,slow) amplitudes were selectively reduced in TAC epicardial LV cells. Consistent with the similarities in I(to,f) and I(K1) amplitudes, Kv4.2, Kv4.3, and KChIP2 (I(to,f)), as well as Kir2.1 and Kir2.2 (I(K1)), transcript and protein expression levels were similar in TAC and sham LV. Unexpectedly, expression of I(K,slow) channel subunits Kv1.5 and Kv2.1 was increased in TAC LV. Biochemical experiments also demonstrated that, although total protein was unaltered, cell surface expression of TASK1 was increased in TAC LV. Functional changes in repolarizing K(+) currents with LVH, therefore, result from distinct cellular (cardiomyocyte enlargement) and molecular (alterations in the numbers of functional channels) mechanisms.

Mice lacking the transcription factor Ikaros display behavioral alterations of an anti-depressive phenotype

The Ikaros (Ik) family of transcription factors has critical functions in immune regulation, lymphohematopoiesis and the hypothalamic-pituitary axis. Ik influences cell fate decisions through transcriptional activation of target genes and its interaction with chromatin remodeling complexes. While Ik is well-described in the lymphoid system and pituitary, its presence and function in the brain has received limited attention to date. This study describes the transient spatio-temporal expression of Ik in striatal medium spiny neurons of the developing murine CNS. To determine the impact of Ik deficiency, standardized behavioral tests were performed. In the elevated plus-maze and contextual fear conditioning tests, homozygous Ik-deficient mice performed similarly to wild-type or heterozygote mice. However, significant differences were observed in Ik-null mice in several behavioral tests. Pinch-induced catalepsy was markedly extended. In the Porsolt forced swim test, Ik-null mice showed reduced immobility, consistent with an anti-depressive effect. The acoustic startle response of Ik-null mice was also markedly diminished. Our findings extend the role of the Ikaros zinc-finger protein to the maturation and differentiation of striatal medium spiny neurons and indicate important actions for Ik in the development of neurocognitive functions and affecting depressive behaviors.

Catheter Angiography, MR Angiography, and MR Perfusion in Posterior Reversible Encephalopathy Syndrome

The cause of posterior reversible encephalopathy syndrome (PRES) is unknown. Two primary hypotheses exist: 1) hypertension exceeding auto-regulatory limits leading to forced hyper-perfusion and 2) vasoconstriction and hypo-perfusion leading to ischemia with resultant edema. The purpose of this study was to evaluate the catheter angiography (CA), MR angiography (MRA), and MR perfusion (MRP) features in PRES in order to render further insight into its mechanism of origin. In 47 patients with PRES, 9 CAs and 43 MRAs were evaluated for evidence of vasculopathy (vasoconstriction and vasodilation), and 15 MRP studies were evaluated for altered relative cerebral blood volume (rCBV) in PRES lesions and regions. Visualization of vessels on MRA and toxicity blood pressures were compared with the extent of hemispheric vasogenic edema. Vasculopathy was present in 8 of 9 patients on CA (direct correlation to MRA in 3/6 patients). At MRA, moderate to severe vessel irregularity consistent with vasoconstriction and vasodilation was present in 30 of 43 patients and vessel pruning or irregularity in 7 patients, with follow-up MRA demonstrating reversal of vasoconstriction or vasodilation in 9 of 11 patients. Vasogenic edema was less in patients with hypertension compared with patients who were normotensive. Preserved normal length of the posterior cerebral artery (PCA) was commonly seen in patients with severe hypertension despite diffuse or focal vasoconstriction or vasodilation. In these patients, lengthier visualization of the distal PCA correlated with a lower grade of hemispheric edema (P = .002). Cortical rCBV was significantly reduced in 51 of 59 PRES lesions and regions compared with a healthy reference cortex (average 61% of reference cortex) with mild decrease in the remainder. Vasculopathy was a common finding on CA and MRA in our patients with PRES, and MRP demonstrated reduced cortical rCBV in PRES lesions. Vasogenic edema was reduced in patients with hypertension, and superior distal PCA visualization correlated with reduced hemispheric edema in patients with PRES and severe hypertension.

Cell cycle‐dependent expression of potassium channels and cell proliferation in rat mesenchymal stem cells from bone marrow

Recently, our team has demonstrated that voltage-gated delayed rectifier K(+) current (IK(DR)) and Ca(2+)-activated K(+) current (I(KCa)) are present in rat bone marrow-derived mesenchymal stem cells; however, little is known of their physiological roles. The present study was designed to investigate whether functional expression of IK(DR) and I(KCa) would change with cell cycle progression, and whether they could regulate proliferation in undifferentiated rat mesenchymal stem cells (MSCs). Membrane potentials and ionic currents were recorded using whole-cell patch clamp technique, cell cycling was analysed by flow cytometry, cell proliferation was assayed with DNA incorporation method and the related genes were down-regulated by RNA interference (RNAi) and examined using RT-PCR. It was found that membrane potential hyperpolarized, and cell size increased during the cell cycle. In addition, IK(DR) decreased, while I(KCa) increased during progress from G(1) to S phase. RT-PCR revealed that the mRNA levels of Kv1.2 and Kv2.1 (likely responsible for IK(DR)) reduced, whereas the mRNA level of KCa3.1 (responsible for intermediate-conductance I(KCa)) increased with the cell cycle progression. Down-regulation of Kv1.2, Kv2.1 or KCa3.1 with the specific RNAi, targeted to corresponding gene inhibited proliferation of rat MSCs. These results demonstrate that membrane potential, IK(DR) and I(KCa) channels change with cell cycle progression and corresponding alteration of gene expression. IK(DR) and intermediate-conductance I(KCa) play an important role in maintaining membrane potential and they participate in modulation of proliferation in rat MSCs.

Ikaros Is Regulated through Multiple Histone Modifications and Deoxyribonucleic Acid Methylation in the Pituitary

The transcription factor Ikaros (Ik) is at the center of a functionally diverse chromatin-remodeling network that is critical for the development and regulation of both the immune and endocrine systems. Dominant negative forms of Ik result in neoplastic growth in mouse genetic studies and have been identified in human tumors. Ik modulates chromatin accessibility through associations with members of the NURD complex including histone deacetylase complexes. We show here that Ik expression in mouse pituitary corticotroph cells is itself regulated through histone modifications as well as DNA methylation. Examination of primary human pituitary specimens also identified a correlation of loss of Ik expression with the presence of DNA methylation in the untranslated exon 1 CpG island. These findings have important implications for the understanding of Ikaros' role in epigenetic functions and suggest a potential role for demethylating agents in the treatment of related disorders.

Properties of hyposmotically activated K channels in human lens epithelial cells (FHL124 LECs)

FHL24 LECs (a gift of Dr. John Reddan, Oakland University), when exposed to hyposmotic media (150mOsM) exhibit regulatory volume decrease (RVD) mediated primarily by a K-selective channel (Lauf et al., ARVO 2006) which was further characterized by Rb influx/K efflux measurements ± inhibitors, and by RT-PCR, Western blot analysis and immunochemistry. In the presence of 0.1 mM ouabain and 5 μM bumetanide, the rate constant of cellular K loss, okK, from FHL124 LECs increased from 0.0015 min−1 at 300 mOsM to 0.005 min−1 at 150 mOsM, equivalent to a permeability change of >3 fold causing a 40% K and 26% water loss within 15 min. The permeability sequence of this Na-independent channel appears to be K>Rb>Cs. Of some 20 inhibitors tested, chlotrimazole (CLT) completely inhibited K loss at 50 μM, whereas TEA and apamin failed, suggesting Ca-dependent intermediate (IK) rather than big (BK) or small (SK) conductance channels mediate RVD in FHL124 LECs. RT-PCR showed the presence of mRNA for IK channels, and for all BK channel subunits (KCNβA1, βA2, βA3, βA4, SLACK), as well as for the SK channel isoforms 1, 2 and 3. Western blots using commercial antibodies revealed the presence of high and low molecular weight protein bands consistent with membrane expression of IK, SK2 and 3, and probably BK channels. Verification by immunofluorescence is in progress. Results suggest that FHL124 LEC express three classes of K channels. However, only a CLT-sensitive Ca-activated IK channel may mediate RVD response to hyposmotic challenge.

Mitogenic modulation of Ca2+‐activated K+ channels in proliferating A7r5 vascular smooth muscle cells

Modulation of Ca(2+)-activated K(+) channels (K(Ca)) has been implicated in the control of proliferation in vascular smooth muscle cells (VSMC) and other cell types. In the present study, we investigated the underlying signal transduction mechanisms leading to mitogen-induced alterations in the expression pattern of intermediate-conductance K(Ca) in VSMC. Regulation of expression of IK(Ca)/rK(Ca)3.1 and BK(Ca)/rK(Ca)1.1 in A7r5 cells, a cell line derived from rat aortic VSMC, was investigated by patch-clamp technique, quantitative RT-PCR, immunoblotting procedures, and siRNA strategy.PDGF stimulation for 2 and 48 h induced an 11- and 3.5-fold increase in rK(Ca)3.1 transcript levels resulting in a four- and seven-fold increase in IK(Ca) currents after 4 and 48 h, respectively. Upregulation of rK(Ca)3.1 transcript levels and channel function required phosphorylation of extracellular signal-regulated kinases (ERK1/2) and Ca(2+) mobilization, but not activation of p38-MAP kinase, c-Jun NH(2)-terminal kinase, protein kinase C, calcium-calmodulin kinase II and Src kinases. In contrast to rK(Ca)3.1, mRNA expression and functions of BK(Ca)/rK(Ca)1.1 were decreased by half following mitogenic stimulation. Downregulation of rK(Ca)1.1 did not require ERK1/2 phosphorylation or Ca(2+) mobilization. In an in vitro-proliferation assay, knockdown of rK(Ca)3.1 expression by siRNA completely abolished functional IK(Ca) channels and mitogenesis. Mitogen-induced upregulation of rK(Ca)3.1 expression is mediated via activation of the Raf/MEK- and ERK-signaling cascade in a Ca(2+)-dependent manner. Upregulation of rK(Ca)3.1 promotes VSMC proliferation and may thus represent a pharmacological target in cardiovascular disease states characterized by abnormal cell proliferation.

Ca2+-Activated K+-Current Density Is Correlated With Soma Size in Rat Vestibular-Afferent Neurons in Culture

Vestibular-afferent neurons (VANs) transmit information about linear and angular accelerations during head movements from vestibular end organs to vestibular nuclei. In situ, these neurons show heterogeneous discharge patterns that may be produced by differences in their intrinsic properties. However, little is known about the ionic currents underlying their different firing patterns. Using the whole cell patch-clamp technique, we analyzed the expression of Ca(2+) and Ca(2+)-activated K(+) currents (I(KCa)) in primary cultured neurons isolated from young rats (p7-p10). We found two overlapping subpopulations of VANs classified according to low-threshold Ca(2+)-current [low-voltage-activated (LVA)] expression; LVA (-) neurons, formed by small cells, and LVA (+) neurons composed of medium to large cells. The I(KCa) in both cell-groups was carried through channels of high (BK), intermediate (IK), and low conductance (SK), besides a resistant channel to classical blockers (IR). BK was expressed preferentially in LVA (+) cells, whereas IR expression was preferentially in LVA (-) cells. No correlation between SK and IK expression with the soma size was found. Current-clamp experiments showed that BK participates in the adaptation of discharge and in the duration of the action potential, whereas SK and IK did not show a significant contribution to electrical discharge of cultured VANs. However, because of the low number of VANs in culture with repetitive firing it is difficult to interpret our results in terms of discharge patterns. Our results demonstrate that vestibular-afferent neurons possess different Ca(2+)-activated K(+) (K(Ca)) channels and that their expression, heterogeneous among the cells, would contribute to explain some of the differences in the electrical-firing properties of these neurons.

Expression of the Ikaros gene family in childhood acute lymphoblastic leukaemia

The Ikaros (Ik) gene family, which includes Ik, Aiolos (Ai), and Helios (He), is a primary regulator of lymphocyte differentiation, and is involved in the development of acute lymphoblastic leukaemia (ALL). We analysed the expression of the Ik gene family isoforms in 97 ALL cases, consisting of 64 childhood and 33 infant ALL cases, using reverse transcription-polymerase chain reaction (RT-PCR). Expression of Ik was detected in all cases, 87 of which expressed either Ik1 or Ik2, or both, five of which expressed Ik1/Ik2 and Ik6, and another five of which expressed only Ik6. Therefore, the dominant negative isoform of Ik6 was expressed in 10 of the 38 cases of childhood precursor B ALL, but was absent in other types of childhood ALL (26.3%, chi2-test, P = 0.0001). In terms of Aiolos and Helios expression, 49 (65.3%) out of the 75 and 40 (50%) out of the 80 ALL cases tested showed non-spliced Ai1 and He1 respectively. Only one case of T lineage ALL expressed a small-sized isoform of Helios (designated as He6). It was also found that the expression of Ai1 and He1 was low in Ik6-positive patients (Fisher's exact test; Ai1 P = 0.005, Hel P = 0.035).

Fibroblast growth factor receptor 4 is a target for the zinc-finger transcription factor Ikaros in the pituitary.

Fibroblast growth factor receptors (FGFRs) have been implicated in a multitude of endocrine cell hormonal and proliferative properties, and FGFR4 is differentially expressed in normal and neoplastic pituitary. We therefore examined the functionally important cis-DNA elements and multiprotein complexes implicated in the cooperative control of expression of the human FGFR4 gene in pituitary cells. Using deletional mapping, we defined a 214-bp (-115/+99) promoter that was functional in pituitary GH4 and PRL 235 cells. Overlapping 40- to 50-bp fragments of this minimal promoter were examined by EMSA. Interestingly, fragment C (-64/-26) included potential binding sites for the hematopoietic zinc finger-containing transcription factor Ikaros (Ik) flanked by binding sites for Sp and Ets-type factors. DNA binding by Ik, Sp, and Ets-like factors was confirmed by oligonucleotide competition and supershifting with specific antibodies. Transcriptional regulation of FGFR4 by Ik was demonstrated by cotransfection of Ik1 with or without Sp1 or Ets overexpression and by disruption of the Ik binding site. Although both Ets-1 and Sp1 overexpression stimulated promoter activity, mutation of the Ik-binding site completely eliminated the Ik1 effect. Specific Ik expression was identified by Western blotting of pituitary GH4 and PRL235 cells and localized in primary mouse hormone-producing anterior pituitary cells by immunocytochemistry. Our findings point to a new role for Ik outside the hematopoietic system and suggest a novel transcriptional contribution with Ets and Sp1 in regulation of FGFR4 in the pituitary.

Developmental expression of the potassium current IK,n contributes to maturation of mouse outer hair cells.

1. The expression of K+ currents in mouse outer hair cells (OHCs) was investigated as a function of developmental age between postnatal day (P) 0 and P26, using whole-cell patch clamp. 2. During the first postnatal week, a slow outward K+ current (IK,neo) was expressed by all OHCs from the apical coil of the cochlea. The amplitude of this current increased greatly between P0 and P6. Then, at the beginning of the second postnatal week, IK,neo decreased. At the same time, from P8 onwards, IK,n, a K+ current characteristic of mature OHCs, was rapidly expressed. 3. The expression of IK,n coincided with the onset of electromotility of the cell body of the OHCs, which could also be detected from P8 onwards and increased substantially in size thereafter. 4. IK,n was reversibly blocked by linopirdine, an inhibitor of members of the KCNQ family of K+ channels, with a KD of 0.7 microM. In the cochlea, KCNQ4 is only expressed in OHCs and is responsible for a form of non-syndromic autosomal dominant deafness. Linopirdine had no effect on other OHC K+ currents at concentrations up to 200 microM. We conclude that ion channels underlying IK,n contain the KCNQ4 subunit. 5. In current clamp, depolarizing current injections from the resting potential triggered action potentials in OHCs during the first postnatal week. Thereafter, more rapid and graded voltage responses occurred from more negative resting potentials. Thus, OHCs mature rapidly from P8 onwards, and IK,n contributes to this maturation.