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1.
PLoS One ; 18(9): e0291442, 2023.
Article in English | MEDLINE | ID: mdl-37725617

ABSTRACT

Increasing evidence demonstrate that the electron transfer chain plays a critical role in controlling the effector functions of macrophages. In this work, we have generated a Ndufs4-/- murine macrophage cell lines. The Ndufs4 gene, which encodes a supernumerary subunit of complex I, is a mutational hotspot in Leigh syndrome patients. Ndufs4-/- macrophages showed decreased complex I activity, altered complex I assembly, and lower levels of maximal respiration and ATP production. These mitochondrial respiration alterations were associated with a shift towards a pro-inflammatory cytokine profile after lipopolysaccharide challenge and improved ability to phagocytose Gram-negative bacteria.


Subject(s)
Electron Transport Complex I , Leigh Disease , Humans , Animals , Mice , Electron Transport Complex I/genetics , Macrophages , Phagocytosis , Cell Line
2.
J Cell Physiol ; 238(5): 976-991, 2023 05.
Article in English | MEDLINE | ID: mdl-36852591

ABSTRACT

Voltage-dependent potassium channel Kv1.3 plays a key role on T-cell activation; however, lack of reliable antibodies has prevented its accurate detection under endogenous circumstances. To overcome this limitation, we created a Jurkat T-cell line with endogenous Kv1.3 channel tagged, to determine the expression, location, and changes upon activation of the native Kv1.3 channels. CRISPR-Cas9 technique was used to insert a Flag-Myc peptide at the C terminus of the KCNA3 gene. Basal or activated channel expression was studied using western blot analysis and imaging techniques. We identified two isoforms of Kv1.3 other than the canonical channel (54 KDa) differing on their N terminus: a longer isoform (70 KDa) and a truncated isoform (43 KDa). All three isoforms were upregulated after T-cell activation. We focused on the functional characterization of the truncated isoform (short form, SF), because it has not been previously described and could be present in the available Kv1.3-/- mice models. Overexpression of SF in HEK cells elicited small amplitude Kv1.3-like currents, which, contrary to canonical Kv1.3, did not induce HEK proliferation. To explore the role of endogenous SF isoform in a native system, we generated both a knockout Jurkat clone and a clone expressing only the SF isoform. Although the canonical isoform (long form) localizes mainly at the plasma membrane, SF remains intracellular, accumulating perinuclearly. Accordingly, SF Jurkat cells did not show Kv1.3 currents and exhibited depolarized resting membrane potential (VM ), decreased Ca2+ influx, and a reduction in the [Ca2+ ]i increase upon stimulation. Functional characterization of these Kv1.3 channel isoforms showed their differential contribution to signaling pathways involved in formation of the immunological synapse. We conclude that alternative translation initiation generates at least three endogenous Kv1.3 channel isoforms in T cells that exhibit different functional roles. For some of these functions, Kv1.3 proteins do not need to form functional plasma membrane channels.


Subject(s)
Kv1.3 Potassium Channel , Animals , Humans , Mice , Cell Line , Cell Membrane/metabolism , Jurkat Cells , Protein Isoforms/genetics , Protein Isoforms/metabolism , Kv1.3 Potassium Channel/genetics , Kv1.3 Potassium Channel/metabolism
3.
Mol Metab ; 53: 101306, 2021 11.
Article in English | MEDLINE | ID: mdl-34298200

ABSTRACT

OBJECTIVES: Restenosis after vessel angioplasty due to dedifferentiation of the vascular smooth muscle cells (VSMCs) limits the success of surgical treatment of vascular occlusions. Type 2 diabetes (T2DM) has a major impact on restenosis, with patients exhibiting more aggressive forms of vascular disease and poorer outcomes after surgery. Kv1.3 channels are critical players in VSMC proliferation. Kv1.3 blockers inhibit VSMCs MEK/ERK signalling and prevent vessel restenosis. We hypothesize that dysregulation of microRNAs (miR) play critical roles in adverse remodelling, contributing to Kv1.3 blockers efficacy in T2DM VSMCs. METHODS AND RESULTS: We used clinically relevant in vivo models of vascular risk factors (VRF) and vessels and VSMCs from T2DM patients. RESUKTS: Human T2DM vessels showed increased remodelling, and changes persisted in culture, with augmented VSMCs migration and proliferation. Moreover, there were downregulation of PI3K/AKT/mTOR and upregulation of MEK/ERK pathways, with increased miR-126 expression. The inhibitory effects of Kv1.3 blockers on remodelling were significantly enhanced in T2DM VSMCs and in VRF model. Finally, miR-126 overexpression confered "diabetic" phenotype to non-T2DM VSMCs by downregulating PI3K/AKT axis. CONCLUSIONS: miR-126 plays crucial roles in T2DM VSMC metabolic memory through activation of MEK/ERK pathway, enhancing the efficacy of Kv1.3 blockers in the prevention of restenosis in T2DM patients.


Subject(s)
Coronary Restenosis/metabolism , Diabetes Mellitus, Type 2/metabolism , Epigenesis, Genetic/genetics , Kv1.3 Potassium Channel/metabolism , MicroRNAs/metabolism , Muscle, Smooth, Vascular/metabolism , Aged , Animals , Coronary Restenosis/drug therapy , Diabetes Mellitus, Type 2/drug therapy , Female , Humans , Kv1.3 Potassium Channel/antagonists & inhibitors , Male , Mice , MicroRNAs/genetics , Muscle, Smooth, Vascular/drug effects , Potassium Channel Blockers/pharmacology
4.
Function (Oxf) ; 2(1): zqaa036, 2021.
Article in English | MEDLINE | ID: mdl-35330975

ABSTRACT

Chronic kidney disease (CKD) significantly increases cardiovascular risk. In advanced CKD stages, accumulation of toxic circulating metabolites and mineral metabolism alterations triggers vascular calcification, characterized by vascular smooth muscle cell (VSMC) transdifferentiation and loss of the contractile phenotype. Phenotypic modulation of VSMC occurs with significant changes in gene expression. Even though ion channels are an integral component of VSMC function, the effects of uremia on ion channel remodeling has not been explored. We used an in vitro model of uremia-induced calcification of human aorta smooth muscle cells (HASMCs) to study the expression of 92 ion channel subunit genes. Uremic serum-induced extensive remodeling of ion channel expression consistent with loss of excitability but different from the one previously associated with transition from contractile to proliferative phenotypes. Among the ion channels tested, we found increased abundance and activity of voltage-dependent K+ channel Kv1.3. Enhanced Kv1.3 expression was also detected in aorta from a mouse model of CKD. Pharmacological inhibition or genetic ablation of Kv1.3 decreased the amount of calcium phosphate deposition induced by uremia, supporting an important role for this channel on uremia-induced VSMC calcification.


Subject(s)
Renal Insufficiency, Chronic , Renal Insufficiency , Uremia , Vascular Calcification , Mice , Humans , Animals , Muscle, Smooth, Vascular , Cells, Cultured , Uremia/complications , Vascular Calcification/etiology , Renal Insufficiency/complications , Renal Insufficiency, Chronic/genetics
5.
J Cell Physiol ; 236(6): 4330-4347, 2021 06.
Article in English | MEDLINE | ID: mdl-33230847

ABSTRACT

The voltage-dependent potassium channel Kv1.3 has been implicated in proliferation in many cell types, based on the observation that Kv1.3 blockers inhibited proliferation. By modulating membrane potential, cell volume, and/or Ca2+ influx, K+  channels can influence cell cycle progression. Also, noncanonical channel functions could contribute to modulate cell proliferation independent of K+ efflux. The specificity of the requirement of Kv1.3 channels for proliferation suggests the involvement of molecule-specific interactions, but the underlying mechanisms are poorly identified. Heterologous expression of Kv1.3 channels in HEK cells has been shown to increase proliferation independently of K+ fluxes. Likewise, some of the molecular determinants of Kv1.3-induced proliferation have been located in the C-terminus region, where individual point mutations of putative phosphorylation sites (Y447A and S459A) abolished Kv1.3-induced proliferation. Here, we investigated the mechanisms linking Kv1.3 channels to proliferation exploring the correlation between Kv1.3 voltage-dependent molecular dynamics and cell cycle progression. Using transfected HEK cells, we analyzed both the effect of changes in resting membrane potential on Kv1.3-induced proliferation and the effect of mutated Kv1.3 channels with altered voltage dependence of gating. We conclude that voltage-dependent transitions of Kv1.3 channels enable the activation of proliferative pathways. We also found that Kv1.3 associated with IQGAP3, a scaffold protein involved in proliferation, and that membrane depolarization facilitates their interaction. The functional contribution of Kv1.3-IQGAP3 interplay to cell proliferation was demonstrated both in HEK cells and in vascular smooth muscle cells. Our data indicate that voltage-dependent conformational changes of Kv1.3 are an essential element in Kv1.3-induced proliferation.


Subject(s)
Cell Proliferation , Ion Channel Gating , Kv1.3 Potassium Channel/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/metabolism , HEK293 Cells , Humans , KATP Channels/genetics , KATP Channels/metabolism , Kv1.3 Potassium Channel/chemistry , Kv1.3 Potassium Channel/genetics , Membrane Potentials , Mutation , Protein Conformation , Signal Transduction , Structure-Activity Relationship
6.
Arterioscler Thromb Vasc Biol ; 39(12): e273-e286, 2019 12.
Article in English | MEDLINE | ID: mdl-31597447

ABSTRACT

OBJECTIVE: We have previously described that changes in the expression of Kv channels associate to phenotypic modulation (PM), so that Kv1.3/Kv1.5 ratio is a landmark of vascular smooth muscle cells phenotype. Moreover, we demonstrated that the Kv1.3 functional expression is relevant for PM in several types of vascular lesions. Here, we explore the efficacy of Kv1.3 inhibition for the prevention of remodeling in human vessels, and the mechanisms linking the switch in Kv1.3 /Kv1.5 ratio to PM. Approach and Results: Vascular remodeling was explored using organ culture and primary cultures of vascular smooth muscle cells obtained from human vessels. We studied the effects of Kv1.3 inhibition on serum-induced remodeling, as well as the impact of viral vector-mediated overexpression of Kv channels or myocardin knock-down. Kv1.3 blockade prevented remodeling by inhibiting proliferation, migration, and extracellular matrix secretion. PM activated Kv1.3 via downregulation of Kv1.5. Hence, both Kv1.3 blockers and Kv1.5 overexpression inhibited remodeling in a nonadditive fashion. Finally, myocardin knock-down induced vessel remodeling and Kv1.5 downregulation and myocardin overexpression increased Kv1.5, while Kv1.5 overexpression inhibited PM without changing myocardin expression. CONCLUSIONS: We demonstrate that Kv1.5 channel gene is a myocardin-regulated, vascular smooth muscle cells contractile marker. Kv1.5 downregulation upon PM leaves Kv1.3 as the dominant Kv1 channel expressed in dedifferentiated cells. We demonstrated that the inhibition of Kv1.3 channel function with selective blockers or by preventing Kv1.5 downregulation can represent an effective, novel strategy for the prevention of intimal hyperplasia and restenosis of the human vessels used for coronary angioplasty procedures.


Subject(s)
Coronary Artery Disease/genetics , Coronary Vessels/pathology , Gene Expression Regulation , Kv1.3 Potassium Channel/genetics , Kv1.5 Potassium Channel/genetics , Muscle, Smooth, Vascular/metabolism , Nuclear Proteins/genetics , Trans-Activators/genetics , Cells, Cultured , Coronary Artery Disease/metabolism , Coronary Artery Disease/pathology , Coronary Vessels/metabolism , Coronary Vessels/physiopathology , Humans , Immunohistochemistry , Kv1.3 Potassium Channel/antagonists & inhibitors , Kv1.3 Potassium Channel/biosynthesis , Kv1.5 Potassium Channel/biosynthesis , Muscle, Smooth, Vascular/pathology , Nuclear Proteins/biosynthesis , Organ Culture Techniques , Phenotype , RNA/genetics , Trans-Activators/biosynthesis , Vascular Remodeling
7.
Front Physiol ; 9: 89, 2018.
Article in English | MEDLINE | ID: mdl-29483881

ABSTRACT

Patients with chronic kidney disease (CKD) have a markedly increased incidence of cardiovascular disease (CVD). The high concentration of circulating uremic toxins and alterations in mineral metabolism and hormone levels produce vascular wall remodeling and significant vascular damage. Medial calcification is an early vascular event in CKD patients and is associated to apoptosis or necrosis and trans-differentiation of vascular smooth muscle cells (VSMC) to an osteogenic phenotype. VSMC obtained from bovine or rat aorta and cultured in the presence of increased inorganic phosphate (Pi) have been extensively used to study these processes. In this study we used human aortic VSMC primary cultures to compare the effects of increased Pi to treatment with serum obtained from uremic patients. Uremic serum induced calcification, trans-differentiation and phenotypic remodeling even with normal Pi levels. In spite of similar calcification kinetics, there were fundamental differences in osteochondrogenic marker expression and alkaline phosphatase induction between Pi and uremic serum-treated cells. Moreover, high Pi induced a dramatic decrease in cell viability, while uremic serum preserved it. In summary, our data suggests that primary cultures of human VSMC treated with serum from uremic patients provides a more informative model for the study of vascular calcification secondary to CKD.

8.
Am J Physiol Cell Physiol ; 314(1): C27-C42, 2018 01 01.
Article in English | MEDLINE | ID: mdl-28931540

ABSTRACT

Kv1.3 channels are involved in the switch to proliferation of normally quiescent cells, being implicated in the control of cell cycle in many different cell types and in many different ways. They modulate membrane potential controlling K+ fluxes, sense changes in potential, and interact with many signaling molecules through their intracellular domains. From a mechanistic point of view, we can describe the role of Kv1.3 channels in proliferation with at least three different models. In the "membrane potential model," membrane hyperpolarization resulting from Kv1.3 activation provides the driving force for Ca2+ influx required to activate Ca2+-dependent transcription. This model explains most of the data obtained from several cells from the immune system. In the "voltage sensor model," Kv1.3 channels serve mainly as sensors that transduce electrical signals into biochemical cascades, independently of their effect on membrane potential. Kv1.3-dependent proliferation of vascular smooth muscle cells (VSMCs) could fit this model. Finally, in the "channelosome balance model," the master switch determining proliferation may be related to the control of the Kv1.3 to Kv1.5 ratio, as described in glial cells and also in VSMCs. Since the three mechanisms cannot function independently, these models are obviously not exclusive. Nevertheless, they could be exploited differentially in different cells and tissues. This large functional flexibility of Kv1.3 channels surely gives a new perspective on their functions beyond their elementary role as ion channels, although a conclusive picture of the mechanisms involved in Kv1.3 signaling to proliferation is yet to be reached.


Subject(s)
Cell Proliferation , Kv1.3 Potassium Channel/metabolism , Potassium/metabolism , Animals , Calcium Signaling , Cell Proliferation/drug effects , Humans , Ion Channel Gating , Kv1.3 Potassium Channel/antagonists & inhibitors , Kv1.3 Potassium Channel/chemistry , Kv1.3 Potassium Channel/genetics , Membrane Potentials , Models, Biological , Potassium Channel Blockers/pharmacology , Protein Conformation , Structure-Activity Relationship
9.
Microcirculation ; 25(1)2018 01.
Article in English | MEDLINE | ID: mdl-29110368

ABSTRACT

Kv channels are present in virtually all VSMCs and strongly influence contractile responses. However, they are also instrumental in the proliferative, migratory, and secretory functions of synthetic, dedifferentiated VSMCs upon PM. In fact, Kv channels not only contribute to all these processes but also are active players in the phenotypic switch itself. This review is focused on the role(s) of Kv channels in VSMC proliferation, which is one of the best characterized functions of dedifferentiated VSMCs. VSMC proliferation is a complex process requiring specific Kv channels at specific time and locations. Their identification is further complicated by their large diversity and the differences in expression across vascular beds. Of interest, both conserved changes in some Kv channels and vascular bed-specific regulation of others seem to coexist and participate in VSMC proliferation through complementary mechanisms. Such a system will add flexibility to the process while providing the required robustness to preserve this fundamental cellular response.


Subject(s)
Cell Proliferation , Muscle, Smooth, Vascular/cytology , Potassium Channels, Voltage-Gated/physiology , Animals , Humans , Vascular Remodeling
10.
Front Physiol ; 8: 500, 2017.
Article in English | MEDLINE | ID: mdl-28747891

ABSTRACT

K+ channels encoded by the ether-a-go-go related gene (ERG1 or KCNH2) are important determinants of the cardiac action potential. Expression of both cardiac isoforms (ERG1a and ERG1b) were identified in murine portal vein and distinctive voltage-gated K+ currents were recorded from single myocytes. The aim of the present study was to ascertain the expression and functional impact of ERG channels in murine arteries. Methods: Quantitative RT-PCR was undertaken on RNA extracted from a number of murine arteries. Immunofluorescence was performed on single vascular smooth muscle cells using antibodies against the ERG1 expression product (Kv11.1). Single cell electrophysiology was performed on myocytes from portal vein and several different arteries, complimented by isometric tension recordings. Proliferation assays were undertaken on smooth muscle cells isolated from femoral arteries. Results: ERG1 transcripts were detected in all murine blood vessels, and Kv11.1 immunofluorescence was observed in all smooth muscle cells. However, K+ currents with properties consistent with ERG channels were only recorded in portal vein myocytes. Moreover, ERG channel blockers (E4031 or dofetilide, 1 µM) failed to depolarize carotid arteries or produce contraction. Proliferation of arterial smooth muscle cells was associated with a marked increase in ERG1 expression and ERG blockers suppressed proliferation significantly. Conclusions: These data reveal that arterial blood vessels express ERG channels that appear to be functional silent in contractile smooth muscle but contribute to proliferative response.

11.
PLoS One ; 10(2): e0118148, 2015.
Article in English | MEDLINE | ID: mdl-25659150

ABSTRACT

Despite the substantial knowledge on the antidiabetic, antiobesity and antihypertensive actions of tungstate, information on its primary target/s is scarce. Tungstate activates both the ERK1/2 pathway and the vascular voltage- and Ca2+-dependent large-conductance BKαß1 potassium channel, which modulates vascular smooth muscle cell (VSMC) proliferation and function, respectively. Here, we have assessed the possible involvement of BKαß1 channels in the tungstate-induced ERK phosphorylation and its relevance for VSMC proliferation. Western blot analysis in HEK cell lines showed that expression of vascular BKαß1 channels potentiates the tungstate-induced ERK1/2 phosphorylation in a Gi/o protein-dependent manner. Tungstate activated BKαß1 channels upstream of G proteins as channel activation was not altered by the inhibition of G proteins with GDPßS or pertussis toxin. Moreover, analysis of Gi/o protein activation measuring the FRET among heterologously expressed Gi protein subunits suggested that tungstate-targeting of BKαß1 channels promotes G protein activation. Single channel recordings on VSMCs from wild-type and ß1-knockout mice indicated that the presence of the regulatory ß1 subunit was essential for the tungstate-mediated activation of BK channels in VSMCs. Moreover, the specific BK channel blocker iberiotoxin lowered tungstate-induced ERK phosphorylation by 55% and partially reverted (by 51%) the tungstate-produced reduction of platelet-derived growth factor (PDGF)-induced proliferation in human VSMCs. Our observations indicate that tungstate-targeting of BKαß1 channels promotes activation of PTX-sensitive Gi proteins to enhance the tungstate-induced phosphorylation of ERK, and inhibits PDGF-stimulated cell proliferation in human vascular smooth muscle.


Subject(s)
Calcium Signaling/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Large-Conductance Calcium-Activated Potassium Channels/metabolism , MAP Kinase Signaling System/drug effects , Muscle, Smooth, Vascular/metabolism , Tungsten Compounds/pharmacology , Animals , Calcium/metabolism , Calcium Signaling/genetics , Extracellular Signal-Regulated MAP Kinases/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , HEK293 Cells , Humans , Large-Conductance Calcium-Activated Potassium Channels/genetics , MAP Kinase Signaling System/genetics , Mice , Mice, Knockout , Muscle, Smooth, Vascular/cytology , Phosphorylation/drug effects , Platelet-Derived Growth Factor/pharmacology
12.
J Physiol ; 591(24): 6175-91, 2013 Dec 15.
Article in English | MEDLINE | ID: mdl-24167226

ABSTRACT

Hypertension is a clinical syndrome characterized by increased arterial tone. Although the mechanisms are varied, the generally accepted view is that increased CaV1.2 channel function is a common feature of this pathological condition. Here, we investigated the mechanisms underlying vascular dysfunction in a mouse model of genetic hypertension. Contrary to expectation, we found that whole-cell CaV1.2 currents (ICa) were lower in hypertensive (BPH line) than normotensive (BPN line) myocytes. However, local CaV1.2 sparklet activity was higher in BPH cells, suggesting that the relatively low ICa in these cells was produced by a few hyperactive CaV1.2 channels. Furthermore, our data suggest that while the lower expression of the pore-forming α1c subunit of CaV1.2 currents underlies the lower ICa in BPH myocytes, the increased sparklet activity was due to a different composition in the auxiliary subunits of the CaV1.2 complexes. ICa currents in BPN cells were produced by channels composed of α1c/α2δ/ß3 subunits, while in BPH myocytes currents were probably generated by the opening of channels formed by α1c/α2δ/ß2 subunits. In addition, Ca(2+) sparks evoked large conductance, Ca(2+)-activated K(+) (BK) currents of lower magnitude in BPH than in BPN myocytes, because BK channels were less sensitive to Ca(2+). Our data are consistent with a model in which a decrease in the global number of CaV1.2 currents coexist with the existence of a subpopulation of highly active channels that dominate the resting Ca(2+) influx. The decrease in BK channel activity makes the hyperpolarizing brake ineffective and leads BPH myocytes to a more contracted resting state.


Subject(s)
Calcium Channels, L-Type/metabolism , Calcium/metabolism , Down-Regulation , Hypertension/metabolism , Myocytes, Smooth Muscle/metabolism , Action Potentials , Animals , Calcium Channels, L-Type/genetics , Calcium Signaling , Large-Conductance Calcium-Activated Potassium Channels/metabolism , Mice , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/physiology , Myocytes, Smooth Muscle/physiology , Protein Subunits/metabolism
13.
J Physiol ; 590(23): 6075-91, 2012 Dec 01.
Article in English | MEDLINE | ID: mdl-22966162

ABSTRACT

The increased vascular tone that defines essential hypertension is associated with depolarization of vascular smooth muscle cells (VSMCs) and involves a change in the expression profile of ion channels promoting arterial contraction. As a major regulator of VSMC resting membrane potential (V(M)), K(+) channel activity is an important determinant of vascular tone and vessel diameter. However, hypertension-associated changes in the expression and/or modulation of K(+) channels are poorly defined, due to their large molecular diversity and their bed-specific pattern of expression. Moreover, the impact of these changes on the integrated vessel function and their contribution to the development of altered vascular tone under physiological conditions need to be confirmed. Hypertensive (BPH) and normotensive (BPN) mice strains obtained by phenotypic selection were used to explore whether changes in the functional expression of VSMC inward rectifier K(+) channels contribute to the more depolarized resting V(M) and the increased vascular reactivity of hypertensive arteries. We determined the expression levels of inward rectifier K(+) channel mRNA in several vascular beds from BPN and BPH animals, and their functional contribution to VSMC excitability and vascular tone in mesenteric arteries. We found a decrease in the expression of Kir2.1, Kir4.1, Kir6.x and SUR2 mRNA in BPH VSMCs, and a decreased functional contribution of both K(IR) and K(ATP) channels in isolated BPH VSMCs. However, only the effect of K(ATP) channel modulators was impaired when exploring vascular tone, suggesting that decreased functional expression of K(ATP) channels may be an important element in the remodelling of VSMCs in essential hypertension.


Subject(s)
Hypertension/physiopathology , Mesenteric Arteries/physiology , Muscle, Smooth, Vascular/physiology , Myocytes, Smooth Muscle/physiology , Potassium Channels, Inwardly Rectifying/physiology , ATP-Binding Cassette Transporters/physiology , Animals , Mice , Protein Subunits/physiology , Receptors, Drug/physiology , Sulfonylurea Receptors
14.
Arterioscler Thromb Vasc Biol ; 32(5): 1299-307, 2012 May.
Article in English | MEDLINE | ID: mdl-22383699

ABSTRACT

OBJECTIVE: Phenotypic modulation of vascular smooth muscle cells has been associated with a decreased expression of all voltage-dependent potassium channel (Kv)1 channel encoding genes but Kcna3 (which encodes Kv1.3 channels). In fact, upregulation of Kv1.3 currents seems to be important to modulate proliferation of mice femoral vascular smooth muscle cells in culture. This study was designed to explore if these changes in Kv1 expression pattern constituted a landmark of phenotypic modulation across vascular beds and to investigate the mechanisms involved in the proproliferative function of Kv1.3 channels. METHODS AND RESULTS: Changes in Kv1.3 and Kv1.5 channel expression were reproduced in mesenteric and aortic vascular smooth muscle cells, and their correlate with protein expression was electrophysiologicaly confirmed using selective blockers. Heterologous expression of Kv1.3 and Kv1.5 channels in HEK cells has opposite effects on the proliferation rate. The proproliferative effect of Kv1.3 channels was reproduced by "poreless" mutants but disappeared when voltage-dependence of gating was suppressed. CONCLUSIONS: These findings suggest that the signaling cascade linking Kv1.3 functional expression to cell proliferation is activated by the voltage-dependent conformational change of the channels without needing ion conduction. Additionally, the conserved upregulation of Kv1.3 on phenotypic modulation in several vascular beds makes this channel a good target to control unwanted vascular remodeling.


Subject(s)
Gene Expression Regulation , Kv1.3 Potassium Channel/genetics , Muscle, Smooth, Vascular/physiology , RNA, Messenger/genetics , Vasoconstriction/physiology , Animals , Blotting, Western , Cell Proliferation , Cells, Cultured , Kv1.3 Potassium Channel/biosynthesis , Mice , Muscle, Smooth, Vascular/cytology , Phenotype , Polymerase Chain Reaction
15.
Arterioscler Thromb Vasc Biol ; 30(6): 1203-11, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20299686

ABSTRACT

OBJECTIVE: Vascular smooth muscle cells (VSMCs) contribute significantly to occlusive vascular diseases by virtue of their ability to switch to a noncontractile, migratory, and proliferating phenotype. Although the participation of ion channels in this phenotypic modulation (PM) has been described previously, changes in their expression are poorly defined because of their large molecular diversity. We obtained a global portrait of ion channel expression in contractile versus proliferating mouse femoral artery VSMCs, and explored the functional contribution to the PM of the most relevant changes that we observed. METHODS AND RESULTS: High-throughput real-time polymerase chain reaction of 87 ion channel genes was performed in 2 experimental paradigms: an in vivo model of endoluminal lesion and an in vitro model of cultured VSMCs obtained from explants. mRNA expression changes showed a good correlation between the 2 proliferative models, with only 2 genes, Kv1.3 and Kvbeta2, increasing their expression on proliferation. The functional characterization demonstrates that Kv1.3 currents increased in proliferating VSMC and that their selective blockade inhibits migration and proliferation. CONCLUSIONS: These findings establish the involvement of Kv1.3 channels in the PM of VSMCs, providing a new therapeutical target for the treatment of intimal hyperplasia.


Subject(s)
Cell Proliferation , Kv1.3 Potassium Channel/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Animals , Cell Movement , Cell Proliferation/drug effects , Cells, Cultured , Cluster Analysis , Disease Models, Animal , Femoral Artery/metabolism , Femoral Artery/pathology , Gene Expression Profiling , Genotype , Hyperplasia , Kv1.3 Potassium Channel/antagonists & inhibitors , Kv1.3 Potassium Channel/genetics , Membrane Potentials , Mice , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/injuries , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/pathology , Phenotype , Potassium Channel Blockers/pharmacology , RNA, Messenger/metabolism , Shaker Superfamily of Potassium Channels/genetics , Shaker Superfamily of Potassium Channels/metabolism , Up-Regulation , Vasoconstriction
16.
Cardiovasc Res ; 86(3): 383-91, 2010 Jun 01.
Article in English | MEDLINE | ID: mdl-20093253

ABSTRACT

AIMS: Vascular smooth muscle cell (VSMC) proliferation is involved in cardiovascular pathologies associated with unwanted arterial wall remodelling. Coordinated changes in the expression of several K+ channels have been found to be important elements in the phenotypic switch of VSMCs towards proliferation. We have previously demonstrated the association of functional expression of Kv3.4 channels with proliferation of human uterine VSMCs. Here, we sought to gain deeper insight on the relationship between Kv3.4 channels and cell cycle progression in this preparation. METHODS AND RESULTS: Expression and function of Kv3.4 channels along the cell cycle was explored in uterine VSMCs synchronized at different checkpoints, combining real-time PCR, western blotting, and electrophysiological techniques. Flow cytometry, Ki67 expression and BrdU incorporation techniques allowed us to explore the effects of Kv3.4 channels blockade on cell cycle distribution. We found cyclic changes in Kv3.4 and MiRP2 mRNA and protein expression along the cell cycle. Functional studies showed that Kv3.4 current amplitude and Kv3.4 channels contribution to cell excitability increased in proliferating cells. Finally, both Kv3.4 blockers and Kv3.4 knockdown with siRNA reduced the proportion of proliferating VSMCs. CONCLUSION: Our data indicate that Kv3.4 channels exert a permissive role in the cell cycle progression of proliferating uterine VSMCs, as their blockade induces cell cycle arrest after G2/M phase completion. The modulation of resting membrane potential (V(M)) by Kv3.4 channels in proliferating VSMCs suggests that their role in cell cycle progression could be at least in part mediated by their contribution to the hyperpolarizing signal needed to progress through the G1 phase.


Subject(s)
Cell Cycle , Cell Proliferation , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Shaw Potassium Channels/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Proliferation/drug effects , Cells, Cultured , Female , Humans , Membrane Potentials , Muscle, Smooth, Vascular/drug effects , Myocytes, Smooth Muscle/drug effects , Phenotype , Potassium Channel Blockers/pharmacology , Potassium Channels, Voltage-Gated/genetics , Potassium Channels, Voltage-Gated/metabolism , RNA Interference , RNA, Messenger/metabolism , Shaw Potassium Channels/antagonists & inhibitors , Shaw Potassium Channels/genetics , Signal Transduction , Uterine Artery/metabolism
17.
J Physiol ; 587(3): 625-40, 2009 Feb 01.
Article in English | MEDLINE | ID: mdl-19074965

ABSTRACT

Essential hypertension involves a gradual and sustained increase in total peripheral resistance, reflecting an increased vascular tone. This change associates with a depolarization of vascular myocytes, and relies on a change in the expression profile of voltage-dependent ion channels (mainly Ca(2+) and K(+) channels) that promotes arterial contraction. However, changes in expression and/or modulation of voltage-dependent K(+) channels (Kv channels) are poorly defined, due to their large molecular diversity and their vascular bed-specific expression. Here we endeavor to characterize the molecular and functional expression of Kv channels in vascular smooth muscle cells (VSMCs) and their regulation in essential hypertension, by using VSMCs from resistance (mesenteric) or conduit (aortic) arteries obtained from a hypertensive inbred mice strain, BPH, and the corresponding normotensive strain, BPN. Real-time PCR reveals a differential distribution of Kv channel subunits in the different vascular beds as well as arterial bed-specific changes under hypertension. In mesenteric arteries, the most conspicuous change was the de novo expression of Kv6.3 (Kcng3) mRNA in hypertensive animals. The functional relevance of this change was studied by using patch-clamp techniques. VSMCs from BPH arteries were more depolarized than BPN ones, and showed significantly larger capacitance values. Moreover, Kv current density in BPH VSMCs is decreased mainly due to the diminished contribution of the Kv2 component. The kinetic and pharmacological profile of Kv2 currents suggests that the expression of Kv6.3 could contribute to the natural development of hypertension.


Subject(s)
Hypertension/genetics , Hypertension/physiopathology , Muscle, Smooth, Vascular/physiopathology , Potassium Channels, Voltage-Gated/metabolism , Animals , Aorta/metabolism , Cell Line , Gene Expression Profiling , Hypertension/metabolism , Indoles/pharmacology , Ion Channel Gating/drug effects , Mesenteric Arteries/drug effects , Mesenteric Arteries/metabolism , Mice , Muscle, Smooth, Vascular/drug effects , Patch-Clamp Techniques , Potassium Channel Blockers/pharmacology , Potassium Channels, Voltage-Gated/antagonists & inhibitors , Shab Potassium Channels/antagonists & inhibitors , Shab Potassium Channels/metabolism , Shaker Superfamily of Potassium Channels/antagonists & inhibitors , Shaker Superfamily of Potassium Channels/metabolism , Triterpenes/pharmacology
18.
J Gen Physiol ; 131(5): 455-71, 2008 May.
Article in English | MEDLINE | ID: mdl-18411327

ABSTRACT

Shal-type (Kv4) channels are expressed in a large variety of tissues, where they contribute to transient voltage-dependent K+ currents. Kv4 are the molecular correlate of the A-type current of neurons (I(SA)), the fast component of I(TO) current in the heart, and also of the oxygen-sensitive K+ current (K(O2)) in rabbit carotid body (CB) chemoreceptor cells. The enormous degree of variability in the physiological properties of Kv4-mediated currents can be attributable to the complexity of their regulation together with the large number of ancillary subunits and scaffolding proteins that associate with Kv4 proteins to modify their trafficking and their kinetic properties. Among those, KChIPs and DPPX proteins have been demonstrated to be integral components of I(SA) and I(TO) currents, as their coexpression with Kv4 subunits recapitulates the kinetics of native currents. Here, we explore the presence and functional contribution of DPPX to K(O2) currents in rabbit CB chemoreceptor cells by using DPPX functional knockdown with siRNA. Additionally, we investigate if the presence of DPPX endows Kv4 channels with new pharmacological properties, as we have observed anomalous tetraethylammonium (TEA) sensitivity in the native K(O2) currents. DPPX association with Kv4 channels induced an increased TEA sensitivity both in heterologous expression systems and in CB chemoreceptor cells. Moreover, TEA application to Kv4-DPPX heteromultimers leads to marked kinetic effects that could be explained by an augmented closed-state inactivation. Our data suggest that DPPX proteins are integral components of K(O2) currents, and that their association with Kv4 subunits modulate the pharmacological profile of the heteromultimers.


Subject(s)
Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Ion Channel Gating/drug effects , Shal Potassium Channels/drug effects , Shal Potassium Channels/metabolism , Tetraethylammonium/pharmacology , Animals , Carotid Body/metabolism , Cells, Cultured , Chemoreceptor Cells/physiology , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics , Gene Expression , Gene Silencing , Humans , Ion Channel Gating/genetics , Ion Transport/drug effects , Kinetics , Multiprotein Complexes/drug effects , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Oxygen/pharmacology , Patch-Clamp Techniques , Potassium Channel Blockers/pharmacology , Protein Subunits/genetics , Protein Subunits/metabolism , Rabbits , Shal Potassium Channels/genetics
19.
Rev Esp Cardiol ; 61(1): 66-75, 2008 Jan.
Article in Spanish | MEDLINE | ID: mdl-18221693

ABSTRACT

In 2002, a group of researchers in the fields of cell electrophysiology, cardiology, population genetics, epidemiology, proteomics, molecular biology, bioinformatics and statistics decided to take up the challenge of investigating the mechanisms and genetics of arterial hypertension (AH). Mechanisms related to ion channel regulation of arterial smooth muscle function were identified. The HERACLES (Hipertensión Esencial: Red de Análisis de Canales iónicos de la musculatura Lisa arterial y su Explotación terapéutica Sistemática) network was honored with a distinguished mention in the 2005 evaluation, and was strengthened by the incorporation of new research groups in 2007. The work of the HERACLES network is characterized as much by the transfer of knowledge "from bedside to bench" as by its converse: "from bench to bedside". The current objectives of the HERACLES network are: a) to study the Ca2+-dependent K+ channels, the transient receptor potential (TRP) cation channels, and the Ca2+-dependent Cl- channels that are involved in vascular physiology; b) to study protein expression maps in plasma and cardiovascular tissue and their significance for drug treatment; c) to study the effect of flavonoids on ion transport and responses to oxidative stress, and d) to identify biomarkers of risk, prognosis, and treatment responses in extreme AH phenotypes. Our project includes a number of lines of research coordinated within cross-sectional programs based on centralized facilities, which are used by them. The HERACLES network has published more than 60 articles (available from: http://www.redheracles.net), funding has been received for more than 90 projects in competitive submissions to Spanish and six international bodies, and there is a DNA Biobank.


Subject(s)
Biomedical Research/organization & administration , Cardiovascular Diseases , Hypertension , Cardiovascular Diseases/physiopathology , Cardiovascular Diseases/therapy , Humans , Hypertension/physiopathology , Hypertension/therapy , Ion Channels , Muscle, Smooth, Vascular/physiopathology , Spain
20.
Rev. esp. cardiol. (Ed. impr.) ; 61(1): 66-75, ene. 2008. ilus, tab
Article in Es | IBECS | ID: ibc-058566

ABSTRACT

En 2002, un grupo de investigadores en electrofisiología celular, cardiología, genética de poblaciones, epidemiología, proteómica, biología molecular, bioinformática y estadística decidió afrontar el reto de analizar los mecanismos y la genética de la hipertensión arterial (HTA). Se identificaron mecanismos relacionados con la regulación de la función de la musculatura lisa arterial por canales iónicos. La Red HERACLES (Hipertensión Esencial: Red de Análisis de Canales iónicos de la musculatura Lisa arterial y su Explotación terapéutica Sistemática) fue calificada con mención Excelente en la evaluación de 2005, y se ha consolidado con la incorporación de nuevos grupos en la convocatoria 2007. Las actividades de la red se enmarcan en la transferencia de conocimiento del bedside-to-bench, así como su inversa, bench-to-bedside. Los objetivos actuales de la red son: a) estudio de canales de K+ dependientes de Ca2+, canales catiónicos TRP y canales de Cl- dependientes de Ca2+ que participan en la fisiología vascular; b) estudio de los mapas de expresión proteínica en plasma y tejido cardiovascular y su relevancia en el tratamiento farmacológico; c) estudios del efecto de los flavonoides en el transporte iónico y la respuesta al estrés oxidativo, y d) identificación de marcadores biológicos de riesgo, pronóstico y respuesta al tratamiento en fenotipos de HTA extremos. Nuestro proyecto incluye un conjunto de líneas de investigación coordinadas con programas horizontales basados en plataformas centrales, que las sirven. La red ha publicado más de 60 manuscritos (disponibles en: http://www.redheracles.net) y ha recibido financiación para más de 90 proyectos en convocatorias competitivas nacionales y 6 internacionales, y un Biobanco ADN (AU)


In 2002, a group of researchers in the fields of cell electrophysiology, cardiology, population genetics, epidemiology, proteomics, molecular biology, bioinformatics and statistics decided to take up the challenge of investigating the mechanisms and genetics of arterial hypertension (AH). Mechanisms related to ion channel regulation of arterial smooth muscle function were identified. The HERACLES (Hipertensión Esencial: Red de Análisis de Canales iónicos de la musculatura Lisa arterial y su Explotación terapéutica Sistemática) network was honored with a distinguished mention in the 2005 evaluation, and was strengthened by the incorporation of new research groups in 2007. The work of the HERACLES network is characterized as much by the transfer of knowledge «from bedside to bench» as by its converse: «from bench to bedside». The current objectives of the HERACLES network are: a) to study the Ca2+-dependent K+ channels, the transient receptor potential (TRP) cation channels, and the Ca2+-dependent Cl- channels that are involved in vascular physiology; b) to study protein expression maps in plasma and cardiovascular tissue and their significance for drug treatment; c) to study the effect of flavonoids onion transport and responses to oxidative stress, and d) to identify biomarkers of risk, prognosis, and treatment responses in extreme AH phenotypes. Our project includes a number of lines of research coordinated within cross-sectional programs based on centralized facilities, which are used by them. The HERACLES network has published more than 60 articles (available from: http://www.redheracles.net), funding has been received for more than 90 projects in competitive submissions to Spanish and six international bodies, and there is a DNA Biobank (AU)


Subject(s)
Humans , Electrophysiologic Techniques, Cardiac/trends , Biomedical Research/trends , Ion Channels , Hypertension/physiopathology , Research Support as Topic , Cardiovascular Diseases/diagnosis , Cooperative Behavior
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