Biomedical subjects

Laura Conforti

Publications and source records attributed to Laura Conforti.

At least 19 recordsLinked to original sources

PubMed2006

Identification of a novel signal in the cytoplasmic tail of the Na+:HCO3- cotransporter NBC1 that mediates basolateral targeting.

The Na(+):HCO(3)(-) cotransporter NBC1 (SLC4A4, variant A, kidney specific) is located exclusively on the basolateral membrane of epithelial cells, implying that this molecule has acquired specific signals for targeting to the basolateral membrane. A motif with the sequence QQPFLS (positions 1010-1015) in the cytoplasmic tail of NBC1 was recently demonstrated to mediate targeting of NBC1 to the basolateral membrane. Here, we demonstrate that mutating the amino acid F (phenylalanine) or L (leucine) at positions 1013 or 1014 to alanine, respectively, resulted in the retargeting of NBC1 to the apical membrane. Furthermore, mutation of the FL motif to FF showed similar properties as the wild-type; however, mutation of the FL motif to LL showed significant intracellular retention of NBC1. Mutating the amino acids Q-Q-P and S (positions 1010-1011-1012 and 1015) to A-A-A and A, respectively, did not affect the membrane targeting of NBC1. Functional studies in oocytes with microelectrode demonstrated that the apically targeted mutants, as well as basolaterally targeted mutants, are all functional. We propose that the FL motif in the COOH-terminal tail of NBC1 is essential for the targeting of NBC1 to the basolateral membrane but is distinct from the membrane-targeting di-leucine motif identified in other membrane proteins.

Amino Acid Motifs↗

PubMed2006

The Ca(2+)-activated K(+) channel KCa3.1 compartmentalizes in the immunological synapse of human T lymphocytes.

T cell receptor engagement results in the reorganization of intracellular and membrane proteins at the T cell-antigen presenting cell interface forming the immunological synapse (IS), an event required for Ca(2+) influx. KCa3.1 channels modulate Ca(2+) signaling in activated T cells by regulating the membrane potential. Nothing is known regarding KCa3.1 membrane distribution during T cell activation. Herein, we determined whether KCa3.1 translocates to the IS in human T cells using YFP-tagged KCa3.1 channels. These channels showed electrophysiological and pharmacological properties identical to wild-type channels. IS formation was induced by either anti-CD3/CD28 antibody-coated beads for fixed microscopy experiments or Epstein-Barr virus-infected B cells for fixed and live cell microscopy. In fixed microscopy experiments, T cells were also immunolabeled for F-actin or CD3epsilon, which served as IS formation markers. The distribution of KCa3.1 was determined with confocal and fluorescence microscopy. We found that, upon T cell activation, KCa3.1 channels localize with F-actin and CD3epsilon to the IS but remain evenly distributed on the cell membrane when no stimulus is provided. Detailed imaging experiments indicated that KCa3.1 channels are recruited in the IS shortly after antigen presentation and are maintained there for at least 15-30 min. Interestingly, pretreatment of activated T cells with the specific KCa3.1 blocker TRAM-34 blocked Ca(2+) influx, but channel redistribution to the IS was not prevented. These results indicate that KCa3.1 channels are a part of the signaling complex that forms at the IS upon antigen presentation.

Actins↗

PubMed2006

Signalling during hypoxia in human T lymphocytes--critical role of the src protein tyrosine kinase p56Lck in the O2 sensitivity of Kv1.3 channels.

T lymphocytes encounter hypoxia when they migrate to pathological sites such as tumours and wounds. The inability of T cells to provide an efficient defence at these sites can in part be explained by the hypoxic environment. Kv 1.3 channels, important components of the T cell activation process are inhibited by hypoxia and their inhibition accounts for a hypoxia-induced decrease in T cell proliferation. Although Kv 1.3 channels play a key role in T cell O(2) sensing, the signalling mechanisms mediating their response to hypoxia are still not understood. In this study, we show that the src-protein tyrosine kinase p56Lck (Lck) is required for Kv 1.3 channel response to hypoxia. Pre-exposure to the src inhibitor PP2 abolished the hypoxia-induced inhibition of Kv 1.3 channels in primary human T lymphocytes. Moreover, Kv 1.3 channel sensitivity to hypoxia was lost in Lck-deficient Jurkat T cells. Further studies with recombinant Kv 1.3 channels showed that Kv 1.3 channels lack intrinsic O(2) sensitivity, but delivery of Lck into the cells and transfection of a constitutively active Lck (Y505FLck) restored their sensitivity to hypoxia. Although Lck is necessary for the Kv 1.3 channel response to hypoxia, it does not directly inhibit Kv 1.3 channels. Indeed, under normal oxygen tension, delivery of active Lck into L929 cells and overexpression of Y505FLck did not decrease recombinant Kv 1.3 currents. On the contrary, activation of endogenous src kinases increased wild-type Kv 1.3 currents in T lymphocytes. Our findings indicate that Lck is required for the acute response to hypoxia of human T lymphocytes as it is necessary to confer O(2) sensitivity on Kv 1.3 channels.

Cell Hypoxia↗

PubMed2005

The slow Wallerian degeneration protein, WldS, binds directly to VCP/p97 and partially redistributes it within the nucleus.

Slow Wallerian degeneration (Wld(S)) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD(+) synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld(S) targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld(S) lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD(+) synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld(S) protein influence the intranuclear location of both ubiquitin proteasome and NAD(+) synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.

Adenosine Triphosphatases↗

PubMed2005

Tetracycline-regulated gene expression in the NSC-34-tTA cell line for investigation of motor neuron diseases.

The motor neuron-like cell line NSC-34 has become a widely used in vitro model for motor neuron biology and pathology. We established a tetracycline-regulated gene expression system in this cell line by stably transfecting pTet-Off, which codifies for the tetracycline transactivator, the regulatory protein tTA. The monoclonal cell lines (NSC-34-tTA) were evaluated for the presence of functional tTA after transient transfection with pBI-EGFP, analyzing the expression of the reporter gene enhanced green fluorescent protein. We evaluated the regulation of tTA function with doxycycline using fluorescence microscopy and quantitative cytofluorimetric analysis on viable transfected cells. The best-regulated cell line (NSC-34-tTA40) had a 66.4-fold induction for the reporter gene fluorescence in comparison to NSC-34. Alpha-tubulin, GAP-43 and phosphorylated medium and heavy neurofilaments, proteins of importance for the motor neuronal phenotype, were evident in NSC-34-tTA40 by Western blot and immunocytochemistry; they were expressed similarly in NSC-34-tTA40 and in NSC-34. The cDNA of human Cu/Zn superoxide dismutase, a gene of interest for amyotrophic lateral sclerosis, was cloned into pBI-EGFP, downstream of the tetracycline-responsive bidirectional promoter. This plasmid was transiently transfected into NSC-34-tTA40, and the functionality of bidirectional transcription was verified by determining the expression of enhanced green fluorescent protein and of human Cu/Zn superoxide dismutase. Both proteins were regulated by doxycycline. This novel cell line, NSC-34 tTA40, that permits tetracycline-regulated gene expression may prove useful to unravel the mechanisms of motor neuron degeneration.

Animals↗

PubMed2005

Hypoxia-activated metabolic pathway stimulates phosphorylation of p300 and CBP in oxygen-sensitive cells.

Transcription co-activators and histone acetyltransferases, p300 and cyclic AMP responsive element-binding protein-binding protein (CBP), participate in hypoxic activation of hypoxia-inducible genes. Here, we show that exposure of PC12 and cells to 1-10% oxygen results in hyperphosphorylation of p300/CBP. This response is fast, long lasting and specific for hypoxia, but not for hypoxia-mimicking agents such as desferioxamine or Co2+ ions. It is also cell-type specific and occurs in pheochromocytoma PC12 cells and the carotid body of rats but not in hepatoblastoma cells. The p300 hyperphosphorylation specifically depends on the release of intracellular calcium from inositol 1,4,5-triphosphate (IP3)-sensitive stores. However, it is not inhibited by pharmacological inhibitors of any of the kinases traditionally known to be directly or indirectly calcium regulated. On the other hand, p300 hyperphosphorylation is inhibited by several different inhibitors of the glucose metabolic pathway from generation of NADH by glyceraldehyde 3-phosphate dehydrogenase, through the transfer of NADH through the glycerol phosphate shuttle to ubiquinone and complex III of the mitochondrial respiratory chain. Inhibition of IP3-sensitive calcium stores decreases generation of ATP, and this inhibition is significantly stronger in hypoxia than in normoxia. We propose that the NADH glycerol phosphate shuttle participates in generating a pool of ATP that serves either as a co-factor or a modulator of the kinases involved in the phosphorylation of p300/CBP during hypoxia.

Animals↗

PubMed2005

Missense mutations in Na+:HCO3- cotransporter NBC1 show abnormal trafficking in polarized kidney cells: a basis of proximal renal tubular acidosis.

The kidney Na(+):HCO(3)(-) cotransporter NBC1 is located exclusively on the basolateral membrane of kidney proximal tubule cells and is responsible for the reabsorption of majority of filtered bicarbonate. Two well-described missense mutations in NBC1, R510H and S427L, are associated with renal tubular acidosis (RTA). However, the exact relationship between these mutations and NBC1 dysregulation remains largely unknown. To address this question, cDNAs for wild-type kidney NBC1 and its mutants R510H and S427L were generated, fused in frame with NH(2) terminally tagged GFP, and transiently expressed in Madin-Darby canine kidney cells. In parallel studies, oocytes were injected with the wild-type and mutant NBC1 cRNAs and studied for membrane expression and activity. In monolayer cells grown to polarity, the wild-type GFP-NBC1 was exclusively localized on the basolateral membrane domain. However, GFP-NBC1 mutant R510H was detected predominantly in the cytoplasm. GFP-NBC1 mutant S427L, on the other hand, was detected predominantly on the apical membrane with residual cytoplasmic retention and basolateral membrane labeling. In oocytes injected with the wild-type or mutant GFP-NBC1 cRNAs, Western blot analysis showed that wild-type NBC1 is predominantly localized in the membrane fraction, whereas NBC1-R510H mutant was predominantly expressed in the cytoplasm. NBC1-S427L mutant was mostly expressed in the membrane fraction. Functional analysis of NBC1 activity in oocytes by membrane potential recording demonstrated that compared with wild-type GFP-NBC1, the GFP-NBC1 mutants H510R and S427L exhibited significant reduction in activity. These findings suggest that the permanent isolated proximal RTA in patients with H510R or S427L mutation resulted from a combination of inactivation and mistargeting of kidney NBC1, with H510R mutant predominantly retained in the cytoplasm, whereas S427L mutant is mistargeted to the apical membrane.

Acidosis, Renal Tubular↗

PubMed2005

Hypoxia modulates early events in T cell receptor-mediated activation in human T lymphocytes via Kv1.3 channels.

T lymphocytes are exposed to hypoxia during their development and when they migrate to hypoxic pathological sites. Although it has been shown that hypoxia inhibits Kv1.3 channels and proliferation in human T cells, the mechanisms by which hypoxia regulates T cell activation are not fully understood. Herein we test the hypothesis that hypoxic inhibition of Kv1.3 channels induces membrane depolarization, thus modulating the increase in cytoplasmic Ca2+ that occurs during activation. Hypoxia causes membrane depolarization in human CD3+ T cells, as measured by fluorescence-activated cell sorting (FACS) with the voltage-sensitive dye DiBAC4(3). Similar depolarization is produced by the selective Kv1.3 channel blockers ShK-Dap22 and margatoxin. Furthermore, pre-exposure to such blockers prevents any further depolarization by hypoxia. Since membrane depolarization is unfavourable to the influx of Ca2+ through the CRAC channels (necessary to drive many events in T cell activation such as cytokine production and proliferation), the effect of hypoxia on T cell receptor-mediated increase in cytoplasmic Ca2+ was determined using fura-2. Hypoxia depresses the increase in Ca2+ induced by anti-CD3/CD28 antibodies in approximately 50% of lymphocytes. In the remaining cells, hypoxia either did not elicit any change or produced a small increase in cytoplasmic Ca2+. Similar effects were observed in resting and pre-activated CD3+ cells and were mimicked by ShK-Dap22. These effects appear to be mediated solely by Kv1.3 channels, as we find no influence of hypoxia on IKCa1 and CRAC channels. Our findings indicate that hypoxia modulates Ca2+ homeostasis in T cells via Kv1.3 channel inhibition and membrane depolarization.

Cell Hypoxia↗

PubMed2005

The slow Wallerian degeneration gene, WldS, inhibits axonal spheroid pathology in gracile axonal dystrophy mice.

Axonal dystrophy is the hallmark of axon pathology in many neurodegenerative disorders of the CNS, including Alzheimer's disease, Parkinson's disease and stroke. Axons can also form larger swellings, or spheroids, as in multiple sclerosis and traumatic brain injury. Some spheroids are terminal endbulbs of axon stumps, but swellings may also occur on unbroken axons and their role in axon loss remains uncertain. Similarly, it is not known whether spheroids and axonal dystrophy in so many different CNS disorders arise by a common mechanism. These surprising gaps in current knowledge result largely from the lack of experimental methods to manipulate axon pathology. The slow Wallerian degeneration gene, Wld(S), delays Wallerian degeneration after injury, and also delays 'dying-back' in peripheral nervous system disorders, revealing a mechanistic link between two forms of axon degeneration traditionally considered distinct. We now report that Wld(S) also inhibits axonal spheroid pathology in gracile axonal dystrophy (gad) mice. Both gracile nucleus (P < 0.001) and cervical gracile fascicle (P = 0.001) contained significantly fewer spheroids in gad/Wld(S) mice, and secondary signs of axon pathology such as myelin loss were also reduced. Motor nerve terminals at neuromuscular junctions continued to degenerate in gad/Wld(S) mice, consistent with previous observations that Wld(S) has a weaker effect on synapses than on axons, and probably contributing to the fact that Wld(S) did not alleviate gad symptoms. Wld(S) acts downstream of the initial pathogenic events to block gad pathology, suggesting that its effect on axonal swelling need not be specific to this disease. We conclude that axon degeneration mechanisms are more closely related than previously thought and that a link exists in gad between spheroid pathology and Wallerian degeneration that could hold for other disorders.

Animals↗

PubMed2004

Increased internalisation and degradation of GLT-1 glial glutamate transporter in a cell model for familial amyotrophic lateral sclerosis (ALS).

It has been suggested that glutamate-induced excitotoxicity plays a central role in the development of motor neuron diseases such as amyotrophic lateral sclerosis (ALS). The GLT-1 isoform of the glutamate transporter gene family is the most important transporter involved in keeping extracellular glutamate concentration below neurotoxic levels. Its loss and an increase in extracellular glutamate has been documented in cases of sporadic and familial ALS, as well as in animal models expressing ALS-linked Cu2+-Zn2+ superoxide dismutase (SOD1) mutations, but the underlying molecular mechanisms are still unclear. We developed and characterised a cell model consisting of polarised epithelial Madin-Darby Canine Kidney (MDCK) cell lines stably expressing wild-type SOD1 or the ALS-linked SOD1 G93A mutant, and analysed the expression of glutamate transporters after transient transfection of the corresponding cDNAs. Like ALS patients and animal models of ALS, the G93A-expressing MDCK cell system showed reduced total glial GLT-1 expression, with no change in the expression of the neuronal EAAC1 glutamate transporter isoform. Morphological analysis revealed the intracellular redistribution of GLT-1 to acidic compartments, whereas the surface distribution of other glutamate transporters (neuronal EAAC1 and glial GLAST) was not affected. Moreover, mutant SOD1 affected the cytosolic tail of GLT-1 because reduced protein expression of EAAC-GLT but not GLT-EAAC chimeras was found in G93A-expressing cell lines. GLT-1 downregulation was greatly induced by inhibition of protein synthesis, and prevented by treatment with chloroquine aimed at inhibiting the activity of acidic degradative compartments. Negligible effect on the protein level or distribution of GLT-1 was observed in cells overexpressing wild-type SOD1. The specific decrease in the GLT-1 isoform of glutamate transporters is therefore recapitulated in G93A-expressing MDCK cell lines, thus suggesting an autonomous cell mechanism underlying the loss of GLT-1 in ALS. Our data indicate that the continuous expression of mutant SOD1 causes the downregulation of GLT-1 by increasing the internalisation and degradation of the surface transporter, and suggest that the cytosolic tail of GLT-1 is required to target the transporter to degradation.

Amyotrophic Lateral Sclerosis↗

PubMed2004

Identification of a carboxyl-terminal motif essential for the targeting of Na+-HCO-3 cotransporter NBC1 to the basolateral membrane.

The Na+-HCO3- cotransporter NBC1 is located exclusively on the basolateral membrane and mediates vectorial transport of bicarbonate in a number of epithelia, including kidney and pancreas. To identify the motifs that direct the targeting of kidney NBC1 to basolateral membrane, wild type and various carboxyl-terminally truncated kidney NBC1 mutants were generated, fused translationally in-frame to GFP, and transiently expressed in kidney epithelial cells. GFP was linked to the NH2 terminus of NBC1, and labeling was examined by confocal microscopy. Full-length (1035 aa) and mutants with the deletion of 3 or 20 amino acids from the COOH-terminal end of NBC1 (lengths 1032 and 1015 aa, respectively) showed strong and exclusive targeting on the basolateral membrane. However, the deletion of 26 amino acid residues from the COOH-terminal end (length 1010 aa) resulted in retargeting of NBC1 to the apical membrane. Expression studies in oocytes demonstrated that the NBC1 mutant with the deletion of 26 amino acid residues from the COOH-terminal end is functional. Additionally, the deletion of the last 23 amino acids or mutation in the conserved residue Phe at position 1013 on the COOH-terminal end demonstrated retargeting to the apical membrane. We propose that a carboxyl-terminal motif with the sequence QQPFLS, which spans amino acid residues 1010-1015, and specifically the amino acid residue Phe (position 1013) are essential for the exclusive targeting of NBC1 to the basolateral membrane.

Amino Acid Motifs↗

PubMed2004

Regulation of the spontaneous contractile activity of the portal vein by the sarcoplasmic reticulum: evidence from the phospholamban gene-ablated mouse.

The rapid contraction/relaxation cycles of phasic smooth muscles necessitates intracellular calcium cycling at a more rapid rate than that of tonic smooth muscles. Recent studies suggest that sarcoplasmic reticulum calcium handling is an important determinant of portal vein phasic contractions. We evaluated the importance and role of phospholamban, a protein which inhibits the sarcoplasmic reticulum (SR) calcium ATPase (SERCA), in regulating the contractility of the phasic mouse portal vein. PLB gene ablation significantly reduced the basal frequency of spontaneous mechanical activity and increased force development of the portal vein. Cyclopiazonic acid (CPA), an inhibitor of SERCA, did not significantly affect the spontaneous activity of the wild-type (WT) portal vein. CPA (1 microM) eliminated the differences in frequency and force between the PLB-KO and WT, localizing the effects to the SR. The PLB-KO portal vein had a lower resting membrane potential than WT controls. There were no significant differences between WT and KO responses to charybdotoxin (250 nM), indicating that calcium-activated potassium channels do not contribute to altered KO portal vein contractility. While contractile sensitivity to acetylcholine was not different between WT and PLB-KO portal veins, force generated in response to a given concentration of acetylcholine was significantly greater in the PLB-KO portal vein, both in the absence and presence of CPA. Our results confirm that SR activity can play a major role in modulating the frequency of the spontaneous mechanical activity of portal veins and removal of PLB inhibition of the SR calcium ATPase has significant effects on the spontaneous activity of the portal vein.

Animals↗

PubMed2004

Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress.

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1wt) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1wt in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1wt expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [3H]d-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1wt. The hSOD1-induced decline in GLT-1 protein and [3H]d-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.

Amyotrophic Lateral Sclerosis↗

PubMed2003

SLC26A7: a basolateral Cl-/HCO3- exchanger specific to intercalated cells of the outer medullary collecting duct.

The outer medullary collecting duct (OMCD) plays an important role in bicarbonate reabsorption and acid-base regulation. An apical V-type H+-ATPase and a basolateral Cl-/HCO3- exchanger, located in intercalated cells of OMCD, mediate the bicarbonate reabsorption. Here we report the identification of a new basolateral Cl-/HCO3- exchanger in OMCD intercalated cells in rat kidney. Northern hybridizations demonstrated the predominant expression of this transporter, also known as SLC26A7, in the outer medulla, with lower expression levels in the inner medulla. SLC26A7 was recognized as a approximately 90-kDa band in the outer medulla by immunoblot analysis and was localized on the basolateral membrane of a subset of OMCD cells by immunocytochemical staining. No labeling was detected in the cortex. Double-immunofluorescence labeling with the aquaporin-2 and SLC26A7 antibodies or anion exchanger-1 and SLC26A7 antibodies identified the SLC26A7-expressing cells as alpha-intercalated cells. Functional studies in oocytes demonstrated that increasing the osmolality of the media (to simulate the physiological milieu in the medulla) increased the Cl-/HCO3- exchanger activity mediated via SLC26A7 by about threefold (P < 0.02 vs. normal condition). We propose that SLC26A7 is a basolateral Cl-/HCO3- exchanger in intercalated cells of the OMCD and may play an important role in bicarbonate reabsorption in medullary collecting duct.

Animals↗

PubMed2003

The pore region of the Kv1.2alpha subunit is an important component of recombinant Kv1.2 channel oxygen sensitivity.

Oxygen-sensitive K(+) channels are important elements in the cellular response to hypoxia. Although much progress has been made in identifying their molecular composition, the structural components associated to their O(2)-sensitivity are not yet understood. Recombinant Kv1.2 currents expressed in Xenopus oocytes are inhibited by a decrease in O(2) availability. On the contrary, heterologous Kv2.1 channels are O(2)-insensitive. To elucidate the protein segment responsible for the O(2)-sensitivity of Kv1.2 channels, we analyzed the response to anoxia of Kv1.2/Kv2.1 chimeric channels. Expression of chimeric Kv2.1 channels each containing the S4, the S1-S3 or the S6-COOH segments of Kv1.2 polypeptide resulted in a K(+) current insensitive to anoxia. In contrast, transferring the S5-S6 segment of Kv1.2 into Kv2.1 produced an O(2)-sensitive K(+) current. Finally, mutating a redox-sensitive methionine residue (M380) of Kv1.2 polypeptide did not affect O(2)-sensitivity. Thus, the pore and its surrounding regions of Kv1.2 polypeptide confer its hypoxic inhibition. This response is independent on the redox modulation of methionine residues in this protein segment.

Amino Acid Sequence↗

PubMed2003

Kif1Bbeta isoform is enriched in motor neurons but does not change in a mouse model of amyotrophic lateral sclerosis.

The kinesin superfamily motor protein Kif1B is expressed in two isoforms, Kif1Balpha and Kif1Bbeta, with distinct cargo-binding domains. We examined the mRNA distribution of the two isoforms in adjacent sections of brain and spinal cord of adult mice using in situ hybridization analysis. Kif1Bbeta mRNA is enriched in several regions of brain and spinal cord. Its levels are four to five times higher than that of the alpha isoform, which was barely detectable. The highest mRNA levels of Kif1Bbeta were found in the cortex, hippocampus, cerebellum and the grey matter of the spinal cord. At the cellular level the highest signal was found in motor neurons in the motor nuclei of medulla oblongata and the ventral horn of spinal cord. Because expression of other Kif genes is altered in amyotrophic lateral sclerosis (ALS) models, we examined the expression level of Kif1Bbeta mRNA in the spinal cord of transgenic mice carrying the SOD1G93A mutation, a model of familial ALS, at presymptomatic and early stages of the disease. No changes were observed in Kif1Bbeta mRNA in motor neurons or in other regions of the spinal cord. These findings indicate that Kif1Balpha, which modulates the transport of mitochondria, may play a major role in tissues other than the central nervous system. Instead Kif1Bbeta, responsible for the transport of synaptic vesicle precursors, seems to play an important role in the nervous system, particularly in the lower motor neurons. The absence of changes of Kif1Bbeta mRNA in transgenic SOD1G93A mice suggests that other molecular mechanisms may play a role in the disruption of axonal transport occurring in the motor neurons of these mice.

Amyotrophic Lateral Sclerosis↗

PubMed2003

Hypoxia regulates expression and activity of Kv1.3 channels in T lymphocytes: a possible role in T cell proliferation.

T lymphocytes are exposed to hypoxia during their development and also when they migrate to hypoxic pathological sites such as tumors and wounds. Although hypoxia can affect T cell development and function, the mechanisms by which immune cells sense and respond to changes in O(2)-availability are poorly understood. K(+) channels encoded by the Kv1.3 subtype of the voltage-dependent Kv1 gene family are highly expressed in lymphocytes and are involved in the control of membrane potential and cell function. In this study, we investigate the sensitivity of Kv1.3 channels to hypoxia in freshly isolated human T lymphocytes and leukemic Jurkat T cells. Acute exposure to hypoxia (20 mmHg, 2 min) inhibits Kv1.3 currents in both cell types by 20%. Prolonged exposure to hypoxia (1% O(2) for 24 h) selectively decreases Kv1.3 protein levels in Jurkat T cells by 47%, but not Kvbeta2 and SK2 Ca-activated K(+) channel subunit levels. The decrease in Kv1.3 protein levels occurs with no change in Kv1.3 mRNA expression and is associated with a significant decrease in K(+) current density. A decrease in Kv1.3 polypeptide levels similar to that obtained during hypoxia is produced by Kv1.3 channel blockage. Our results indicate that hypoxia produces acute and long-term inhibition of Kv1.3 channels in T lymphocytes. This effect could account for the inhibition of lymphocyte proliferation during hypoxia. Indeed, we herein present evidence showing that hypoxia selectively inhibits TCR-mediated proliferation and that this inhibition is associated with a decrease in Kv1.3 proteins.

Adult↗

PubMed2003

Neurites undergoing Wallerian degeneration show an apoptotic-like process with Annexin V positive staining and loss of mitochondrial membrane potential.

Wallerian degeneration, the disintegration of the distal part of an injured axon, is an important event in many neurodegenerative diseases. We studied Wallerian degeneration in dorsal root ganglion (DRG) explants in culture by separating neurites from their cell bodies with a scalpel. The severed neurites showed Annexin V positive staining, that spreads distally with a rate comparable to that of slow axonal transport in intact neurons in vivo. Moreover, the injured neurites showed loss of mitochondrial membrane potential. These features resemble those seen when cells undergo apoptosis. These data contribute to a new understanding of the mechanism of axonal degeneration, have implications for the response of stromal cells in central nervous system (CNS) and raise the prospect of new pharmacological treatments for those neurodegenerative pathologies where the protection of the cell body alone does not alleviate the disease.

Animals↗