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1.
The Shaker family K(+) channel protein, Kv1.3, is tyrosine phosphorylated by v-Src kinase at Tyr(137) and Tyr(449) to modulate current magnitude and kinetic properties. Despite two proline rich sequences and these phosphotyrosines contained in the carboxyl and amino terminals of the channel, v-Src kinase fails to co-immunoprecipitate with Kv1.3 as expressed in HEK 293 cells, indicating a lack of direct Src homology 3- or Src homology 2-mediated protein-protein interaction between the channel and the kinase. We show that the adaptor proteins, n-Shc and Grb10, are expressed in the olfactory bulb, a region of the brain where Kv1.3 is highly expressed. In HEK 293 cells, co-expression of Kv1.3 plus v-Src with Grb10 causes a decrease in v-Src-induced Kv1.3 tyrosine phosphorylation and a reversal of v-Src-induced Kv1.3 current suppression, increase in inactivation time constant (tau(inact)), and disruption of cumulative inactivation properties. Co-expression of Kv1.3 plus v-Src with n-Shc did not significantly alter v-Src-induced Kv1.3 current suppression but reversed v-Src induced increased tau(inact) and restored the right-shifted voltage at half-activation (V(1/2)) induced by v-Src. The v-Src-induced shift in V(1/2) and increased tau(inact) was retained when Tyr(220), Tyr(221), and Tyr(304) in the CH domain of n-Shc were mutated to Phe (triple Shc mutant) but was reversed back to control values when either wild-type Shc or the family member Sck, which is not a substrate for Src kinase, was substituted for the triple Shc mutant. Thus the portion of the CH domain that includes Tyr(220), Tyr(221), and Tyr(304) may regulate a shift in Kv1.3 voltage dependence and inactivation kinetics produced by n-Shc in the presence of v-Src. Collectively these data indicate that Grb10 and n-Shc adaptor molecules differentially modulate the degree of Kv1.3 tyrosine phosphorylation, the channel's biophysical properties, and the physical complexes associated with Kv1.3 in the presence of Src kinase. 相似文献
2.
Bock J Szabó I Gamper N Adams C Gulbins E 《Biochemical and biophysical research communications》2003,305(4):890-897
Previous studies suggested a central role of sphingomyelin- and cholesterol-enriched membrane rafts in the initiation of signaling via many receptors. Here, we investigated the role of membrane rafts for the function of the voltage-gated potassium channel Kv1.3. We demonstrate that Kv1.3 localizes in the cell membrane to pre-existing small, sphingolipid- and cholesterol-enriched membrane rafts. Transformation of these small rafts to large ceramide-enriched membrane platforms was achieved by stimulation of the endogenous acid sphingomyelinase, addition of exogenous sphingomyelinase or treatment of the cells with C(16)-ceramide and resulted in clustering of Kv1.3 within ceramide-enriched membrane platforms and inhibition of the channel's activity. Likewise, disruption of pre-existing small rafts inhibited Kv1.3 activity. This indicates that intact small membrane rafts are required for Kv1.3 activity and an alteration of the lipid environment of rafts inhibits Kv1.3. These data, thus, may suggest a novel concept for the regulation of ion channels by the cell membrane composition. 相似文献
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4.
Vicente R Escalada A Villalonga N Texidó L Roura-Ferrer M Martín-Satué M López-Iglesias C Soler C Solsona C Tamkun MM Felipe A 《The Journal of biological chemistry》2006,281(49):37675-37685
Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses. 相似文献
5.
Background
Correolide, a nortriterpene isolated from the Costa Rican tree Spachea correa, is a novel immunosuppressant, which blocks Kv1.3 channels in human T lymphocytes. Earlier mutational studies suggest that correolide binds in the channel pore. Correolide has several nucleophilic groups, but the pore-lining helices in Kv1.3 are predominantly hydrophobic raising questions about the nature of correolide-channel interactions. 相似文献6.
Binding of R(+)-bupivacaine to open-state homology models of the mammalian K(v)1.5 membrane ion channel is studied using automated docking and molecular dynamics (MD) methods. Homology models of K(v)1.5 are built using the 3D structures of the KcsA and MthK channels as a template. The packing of transmembrane (TM) alpha-helices in the KcsA structure corresponds to a closed channel state. Opening of the channel may be reached by a conformational transition yielding a bent structure of the internal S6 helices. Our first model of the K(v) open state involves a PVP-type of bending hinge in the internal helices, while the second model corresponds to a Gly-type of bending hinge as found in the MthK channel. Ligand binding to these models is probed using the common local anaesthetic bupivacaine, where blocker binding from the intracellular side of the channel is considered. Conformational properties and partial atomic charges of bupivacaine are determined from quantum mechanical HF/6-31G* calculations with inclusion of solvent effects. The automated docking and MD calculations for the PVP-bend model predict that bupivacaine could bind either in the central cavity or in the PVP region of the channel pore. Linear interaction energy (LIE) estimates of the binding free energies for bupivacaine predict strongest binding to the PVP region. Surprisingly, no binding is predicted for the Gly-bend model. These results are discussed in light of electrophysiological data which show that the K(v)1.5 channel is unable to close when bupivacaine is bound. 相似文献
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8.
Yin SJ Jiang L Yi H Han S Yang DW Liu ML Liu H Cao ZJ Wu YL Li WX 《Journal of proteome research》2008,7(11):4890-4897
The low selectivity of Kv1 peptide inhibitors for specific isoforms makes them poor candidates for the development of theraputics. Using combined approaches, we showed that the Kv1 turret is the critical determinant for ADWX-1 peptide inhibitor selectivity of Kv1.3 over Kv1.1. Mutation of Kv1.1 turret residues to match the sequence of Kv1.3 lead to increased inhibition of Kv1.1 activity. These studies may lead to improvements in peptide inhibitor drug development. 相似文献
9.
Cianci J Baell JB Flynn BL Gable RW Mould JA Paul D Harvey AJ 《Bioorganic & medicinal chemistry letters》2008,18(6):2055-2061
Chalcone derivatives of the natural product khellinone were synthesised and screened for bioactivity against the voltage-gated potassium channel Kv1.3. X-ray crystallography was employed to investigate relationships between the structure and function of a selection of the reported chalcones. 相似文献
10.
The mitral and short axon cells of the olfactory bulb 总被引:5,自引:0,他引:5
11.
In the olfactory bulb, input from olfactory receptor neurons is processed by neuronal networks before it is relayed to higher
brain regions. In many neurons, hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels generate and control
oscillations of the membrane potential. Oscillations also appear crucial for information processing in the olfactory bulb.
Four channel isoforms exist (HCN1–HCN4) that can form homo- or heteromers. Here, we describe the expression pattern of HCN
isoforms in the olfactory bulb of mice by using a novel and comprehensive set of antibodies against all four isoforms. HCN
isoforms are abundantly expressed in the olfactory bulb. HCN channels can be detected in most cell populations identified
by commonly used marker antibodies. The combination of staining with marker and HCN antibodies has revealed at least 17 different
staining patterns in juxtaglomerular cells. Furthermore, HCN isoforms give rise to an unexpected wealth of co-expression patterns
but are rarely expressed in the same combination and at the same level in two given cell populations. Therefore, heteromeric
HCN channels may exist in several cell populations in vivo. Our results suggest that HCN channels play an important role in
olfactory information processing. The staining patterns are consistent with the possibility that both homomeric and heteromeric
HCN channels are involved in oscillations of the membrane potential of juxtaglomerular cells. 相似文献
12.
Inhibition of the olfactory cyclic nucleotide gated ion channel by intracellular calcium. 总被引:10,自引:0,他引:10
F Zufall G M Shepherd S Firestein 《Proceedings. Biological sciences / The Royal Society》1991,246(1317):225-230
When olfactory receptor neurons are exposed to sustained application of odours, the elicited ionic current is transient. This adaptation-like effect appears to require the influx of Ca2+ through the odour-sensitive conductance; in the absence of extracellular Ca2+ the current remains sustained. Odour transduction proceeds through a G-protein-based second messenger system, resulting finally in the direct activation of an ion channel by cyclic AMP. This channel is one possible site for a negative feedback loop using Ca2+ as a messenger. In recordings of single cyclic AMP gated channels from olfactory receptor neurons, the open probability of the channel in saturating cAMP concentrations was dependent on the concentration of intracellular Ca2+. It could be reduced from 0.6 in 100 nm Ca2+ to 0.09 in 3 microM Ca2+. However, as neither the single channel conductance nor the mean open time were affected by Ca+ concentration, this does not appear to be a mechanism of simple channel block. Rather, these results suggest that intracellular Ca2+ acts allosterically to stabilize a closed state of the channel. 相似文献
13.
Nana‐Maria Wagner Gunnar Brandhorst Frauke Czepluch Mareike Lankeit Christoph Eberle Sebastian Herzberg Vivien Faustin Joachim Riggert Michael Oellerich Gerd Hasenfuss Stavros Konstantinides Katrin Schäfer 《Obesity (Silver Spring, Md.)》2013,21(3):461-468
Objective:
Reduced numbers of regulatory T (Treg) cells have been observed in visceral adipose tissue of obese mice and humans. However, it is unknown whether human obesity affects circulating Treg cells and whether their number is associated with markers of systemic inflammation or glucose intolerance.Design and Methods:
Peripheral blood mononuclear cells were isolated from venous blood of obese (BMI ≥ 27 kg/m2; n = 30) and nonobese (BMI ≥ 27 kg/m2; n = 13) individuals and analyzed using flow cytometry for the expression of CD4, CD25, and Foxp3.Results:
Reduced circulating Treg‐cell numbers were detected in obese compared with nonobese study participants (P = 0.038). Circulating CD4+CD25+CD127?Foxp3 Treg cells inversely correlated with body weight (P = 0.009), BMI (P = 0.004) and plasma leptin levels (P = 0.004) and were reduced in subjects with hsCRP ≥ 3.0 mg/L (P = 0.034) or HbA1c ≥ 5.5% (P < 0.005). Receiver operating characteristic curve analysis revealed a cutoff of circulating Treg cells < 1.06% to be predictive for hsCRP levels ≥ 3.0 mg/L, and logistic regression showed that the risk of having hsCRP levels ≥ 3.0 mg/L was increased 9.6‐fold (P = 0.008), if Treg cells were below this threshold. The Treg cutoff for HbA1c levels ≥ 5.5% was 0.73%, and this cutoff also predicted an increased risk of having elevated levels of both hsCRP and HbA1c, if only obese subjects were examined.Conclusion:
Our findings thus reveal an association between circulating Treg cells and measures of adiposity, inflammation, and glucose intolerance. Although further prospective studies are needed, we present data suggesting that the determination of Treg cells might be useful to identify obese subjects at increased risk of developing cardiovascular and/or metabolic complications.14.
《Seminars in cell biology》1994,5(1):65-74
The olfactory system shares many principles of functional organization with other sensory systems, but differs in that the sensory input is in the form of molecular information carried in odor molecules. Current studies are providing new insights into how this information is processed. In analogy with the spatial receptive fields of visual neurons, the molecular receptive range of olfactory cells is defined as the range of odor molecules that will affect the firing of that cell. Olfactory receptor molecules belong to a large gene family; it is hypothesized that individual receptor molecule may have relatively broad molecular receptive ranges, and that an individual receptor cell need therefore express only one or a few different types of receptors to cover a broad range. Mitral/tufted cells have narrower molecular receptive ranges, comprising molecules with related structures (odotopes). This is believed to reflect processing through the olfactory glomeruli, each glomerulus acting as a convergence center for related inputs. Varying overlapping specificities of receptor cells, glomeruli and mitral/tufted cells appear to provide the basis for discrimination of odor molecules, in analogy with discrimination of color in the visual systems. 相似文献
15.
Insulin found within the brain is derived from the blood and can affect various central nervous system (CNS) functions. The olfactory bulb contains one of the highest concentrations of insulin and insulin receptors within the CNS. To determine the mechanism underlying this high concentration of insulin, we used radioactively iodinated insulin to compare the blood to tissue transport rates and tissue degradation rates for the olfactory bulb, whole brain and spinal cord. We found that the olfactory bulb had both the highest transport rate across the blood-brain barrier (BBB) and the highest rate of degradation. Because a higher degradation rate would decrease, not increase, tissue concentrations of insulin, BBB transport may be the primary mechanism by which high concentrations of insulin are maintained within the olfactory bulb. This illustrates an adaptive aspect of the BBB in its regulation of the exchange of information molecules between the blood and the CNS. 相似文献
16.
Felix JP Bugianesi RM Schmalhofer WA Borris R Goetz MA Hensens OD Bao JM Kayser F Parsons WH Rupprecht K Garcia ML Kaczorowski GJ Slaughter RS 《Biochemistry》1999,38(16):4922-4930
A novel nortriterpene, termed correolide, purified from the tree Spachea correae, inhibits Kv1.3, a Shaker-type delayed rectifier potassium channel present in human T lymphocytes. Correolide inhibits 86Rb+ efflux through Kv1.3 channels expressed in CHO cells (IC50 86 nM; Hill coefficient 1) and displays a defined structure-activity relationship. Potency in this assay increases with preincubation time and with time after channel opening. Correolide displays marked selectivity against numerous receptors and voltage- and ligand-gated ion channels. Although correolide is most potent as a Kv1.3 inhibitor, it blocks all other members of the Kv1 family with 4-14-fold lower potency. C20-29-[3H]dihydrocorreolide (diTC) was prepared and shown to bind in a specific, saturable, and reversible fashion (Kd = 11 nM) to a single class of sites in membranes prepared from CHO/Kv1.3 cells. The molecular pharmacology and stoichiometry of this binding reaction suggest that one diTC site is present per Kv1.3 channel tetramer. This site is allosterically coupled to peptide and potassium binding sites in the pore of the channel. DiTC binding to human brain synaptic membranes identifies channels composed of other Kv1 family members. Correolide depolarizes human T cells to the same extent as peptidyl inhibitors of Kv1.3, suggesting that it is a candidate for development as an immunosuppressant. Correolide is the first potent, small molecule inhibitor of Kv1 series channels to be identified from a natural product source and will be useful as a probe for studying potassium channel structure and the physiological role of such channels in target tissues of interest. 相似文献
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18.
Sokolova OS Shaĭtan KV Grizel' AV Popinako AV Karlova MG Kirpichnikov MP 《Bioorganicheskaia khimiia》2012,38(2):177-184
Here we present a three-dimensional structure of human voltage gated Kv10.2 ion channel solved at 2.5 nm resolution. We demonstrated that Kv10.2 channel structure is subdivided into two layers. For interpretation of the structure we used the homology modeling, using the transmembrane regions of MlotiK1 channel (C subunit), and cytoplasmic PAS-PAC and cNBD domains of the N-terminal tail of hERG (A subunit) and the bacterial cyclic nucleotide-activated K+ channel binding domain as the templates. The homologous transmembrane part can be fitted into the upper part of the reconstruction. The cytoplasmic domains form the structure, similar to a "hanging gondola", which is connected to the membrane-embedded domain with linkers. The length of linkers allow contacts between C-terminal cNBD domains and N-terminal PAS domains. 相似文献
19.
Heidi De Wet Marcus Allen Chris Holmes Michelle Stobbart Jonathan D. Lippiat Heidi De Wet 《Molecular membrane biology》2013,30(5):420-429
BK channels regulate vascular tone by hyperpolarizing smooth muscle in response to fluctuating calcium concentrations. Oestrogen has been reported to lower blood pressure by increasing BK channel open probability through direct binding to the regulatory β1-subunit(s) associated with the channel. The present investigation demonstrates that 17β-oestradiol activates the BK channel complex by increasing the burst duration of channel openings. A subconductance state was observed in 25% of recordings following the addition of 17β-oestradiol and could reflect uncoupling between the pore forming α1-subunit and the regulatory β1-subunit. We also present evidence that more than one β1-subunit is required to facilitate binding of 17β-oestradiol to the channel complex. 相似文献
20.
De Wet H Allen M Holmes C Stobbart M Lippiat JD Callaghan R 《Molecular membrane biology》2006,23(5):420-429
BK channels regulate vascular tone by hyperpolarizing smooth muscle in response to fluctuating calcium concentrations. Oestrogen has been reported to lower blood pressure by increasing BK channel open probability through direct binding to the regulatory beta1-subunit(s) associated with the channel. The present investigation demonstrates that 17beta-oestradiol activates the BK channel complex by increasing the burst duration of channel openings. A subconductance state was observed in 25% of recordings following the addition of 17beta-oestradiol and could reflect uncoupling between the pore forming alpha1-subunit and the regulatory beta1-subunit. We also present evidence that more than one beta1-subunit is required to facilitate binding of 17beta-oestradiol to the channel complex. 相似文献