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Michael J Welsh

Publications and source records attributed to Michael J Welsh.

At least 55 records · Page 3Linked to original sources
PubMed2003

cAMP-dependent protein kinase phosphorylation of the acid-sensing ion channel-1 regulates its binding to the protein interacting with C-kinase-1.

The acid-sensing ion channel-1 (ASIC1) contributes to synaptic plasticity and may influence the response to cerebral ischemia and acidosis. We found that cAMP-dependent protein kinase phosphorylated heterologously expressed ASIC1 and endogenous ASIC1 in brain slices. ASIC1 also showed significant phosphorylation under basal conditions. Previous studies showed that the extreme C-terminal residues of ASIC1 bind the PDZ domain of the protein interacting with C-kinase-1 (PICK1). We found that protein kinase A phosphorylation of Ser-479 in the ASIC1 C terminus interfered with PICK1 binding. In contrast, minimizing phosphorylation or mutating Ser-479 to Ala enhanced PICK1 binding. Phosphorylation-dependent disruption of PICK1 binding reduced the cellular colocalization of ASIC1 and PICK1. Thus, the ASIC1 C terminus contains two sites that influence its binding to PICK1. Regulation of this interaction by phosphorylation provides a mechanism to control the cellular localization of ASIC1.

Acid Sensing Ion Channels↗
PubMed2003

Effects of C-terminal deletions on cystic fibrosis transmembrane conductance regulator function in cystic fibrosis airway epithelia.

To better understand the function of the conserved C terminus of the cystic fibrosis (CF) transmembrane conductance regulator, we studied constructs containing deletions in the C-terminal tail. When expressed in well differentiated CF airway epithelia, each construct localized predominantly to the apical membrane and generated transepithelial Cl(-) current. The results suggested that neither the C-terminal PSD-95/Discs-large/ZO-1 (PDZ)-interacting motif nor other C-terminal sequences were absolutely required for apical expression in airway epithelia. Surprisingly, deleting an acidic cluster near the C terminus reduced both channel opening rate and transepithelial Cl(-) transport, indicating that it influences channel gating. These results may help explain the relative paucity of CF-associated mutations in the C terminus.

Amino Acid Sequence↗
PubMed2003

Drosophila DEG/ENaC pickpocket genes are expressed in the tracheal system, where they may be involved in liquid clearance.

The Drosophila tracheal system and mammalian airways are branching networks of tubular epithelia that deliver oxygen to the organism. In mammals, the epithelial Na(+) channel (ENaC) helps clear liquid from airways at the time of birth and removes liquid from the airspaces in adults. We tested the hypothesis that related Drosophila degenerin (DEG)/ENaC family members might play a similar role in the fly. Among 16 Drosophila DEG/ENaC genes, called pickpocket (PPK) genes, we found 9 expressed in the tracheal system. By in situ hybridization, expression appeared in late-stage embryos after tracheal tube formation, with individual PPK genes showing distinct temporal and spatial expression patterns as development progressed. Promoters for several PPK genes drove reporter gene expression in the larval and adult tracheal systems. Adding the DEG/ENaC channel blocker amiloride to the medium inhibited liquid clearance from the trachea of first instar larvae. Moreover, when RNA interference was used to silence PPK4 and PPK11, larvae failed to clear tracheal liquid. These data suggest substantial molecular diversity of DEG/ENaC channel expression in the Drosophila tracheal system where the PPK proteins likely play a role in Na(+) absorption. Extensive similarities between Drosophila and mammalian airways offer opportunities for genetic studies that may decipher further the structure and function of DEG/ENaC proteins and development of the airways.

Amiloride↗
PubMed2003

ASIC3 and ASIC1 mediate FMRFamide-related peptide enhancement of H+-gated currents in cultured dorsal root ganglion neurons.

The acid-sensing ion channels (ASICs) form cation channels that are transiently activated by extracellular protons. They are expressed in dorsal root ganglia (DRG) neurons and in the periphery where they play a function in nociception and mechanosensation. Previous studies showed that FMRFamide and related peptides potentiate H(+)-gated currents. To better understand this potentiation, we examined the effect of FMRFamide-related peptides on DRG neurons from wild-type mice and animals missing individual ASIC subunits. We found that FMRFamide and FRRFamide potentiated H(+)-gated currents of wild-type DRG in a dose-dependent manner. They increased current amplitude and slowed desensitization following a proton stimulus. Deletion of ASIC3 attenuated the response to FMRFamide-related peptides, whereas the loss of ASIC1 increased the response. The loss of ASIC2 had no effect on FMRFamide-dependent enhancement of H(+)-gated currents. These data suggest that FMRFamide-related peptides modulate DRG H(+)-gated currents through an effect on both ASIC1 and ASIC3 and that ASIC3 plays the major role. The recent discovery of RFamide-related peptides (RFRP) in mammals suggested that they might also modulate H(+)-gated current. We found that RFRP-1 slowed desensitization of H(+)-gated DRG currents, whereas RFRP-2 increased the peak amplitude. COS-7 cells heterologously expressing ASIC1 or ASIC3 showed similar effects. These results suggest that FMRFamide-related peptides, including the newly identified RFRPs, modulate H(+)-gated DRG currents through ASIC1 and ASIC3. The presence of several ASIC subunits, the diversity of FMRFamide-related peptides, and the distinct effects on H(+)-gated currents suggest the possibility of substantial complexity in modulation of current in DRG sensory neurons.

Acid Sensing Ion Channels↗
PubMed2003

Chronic hyperalgesia induced by repeated acid injections in muscle is abolished by the loss of ASIC3, but not ASIC1.

Clinically, chronic pain and hyperalgesia induced by muscle injury are disabling and difficult to treat. Cellular and molecular mechanisms underlying chronic muscle-induced hyperalgesia are not well understood. For this reason, we developed an animal model where repeated injections of acidic saline into one gastrocnemius muscle produce bilateral, long-lasting mechanical hypersensitivity of the paw (i.e. hyperalgesia) without associated tissue damage. Since acid sensing ion channels (ASICs) are found on primary afferent fibers and respond to decreases in pH, we tested the hypothesis that ASICs on primary afferent fibers innervating muscle are critical to development of hyperalgesia and central sensitization in response to repeated intramuscular acid. Dorsal root ganglion neurons innervating muscle express ASIC3 and respond to acidic pH with fast, transient inward and sustained currents that resemble those of ASICs. Mechanical hyperalgesia produced by repeated intramuscular acid injections is prevented by prior treatment of the muscle with the non-selective ASIC antagonist, amiloride, suggesting ASICs might be involved. ASIC3 knockouts do not develop mechanical hyperalgesia to repeated intramuscular acid injection when compared to wildtype littermates. In contrast, ASIC1 knockouts develop hyperalgesia similar to their wildtype littermates. Extracellular recordings of spinal wide dynamic range (WDR) neurons from wildtype mice show an expansion of the receptive field to include the contralateral paw, an increased response to von Frey filaments applied to the paw both ipsilaterally and contralaterally, and increased response to noxious pinch contralaterally after the second intramuscular acid injection. These changes in WDR neurons do not occur in ASIC3 knockouts. Thus, activation of ASIC3s on muscle afferents is required for development of mechanical hyperalgesia and central sensitization that normally occurs in response to repeated intramuscular acid. Therefore, interfering with ASIC3 might be of benefit in treatment or prevention of chronic hyperalgesia.

Acid Sensing Ion Channels↗
PubMed2003

Identification and function of thermosensory neurons in Drosophila larvae.

Although the ability to sense temperature is critical for many organisms, the underlying mechanisms are poorly understood. Using the calcium reporter yellow cameleon 2.1 and electrophysiological recordings, we identified thermosensitive neurons and examined their physiologic response in Drosophila melanogaster larvae. In the head, terminal sensory organ neurons showed increased activity in response to cooling by < or =1 degrees C, heating reduced their basal activity, and different units showed distinct response patterns. Neither cooling nor heating affected dorsal organ neurons. Body wall neurons showed a variety of distinct response patterns to both heating and cooling; the diverse thermal responses were strikingly similar to those described in mammals. These data establish a functional map of thermoresponsive neurons in Drosophila larvae and provide a foundation for understanding mechanisms of thermoreception in both insects and mammals.

Animals↗
PubMed2003

The sperm outer dense fiber protein is the 10th member of the superfamily of mammalian small stress proteins.

Nine proteins have been assigned to date to the superfamily of mammalian small heat shock proteins (sHsps): Hsp27 (HspB1, Hsp25), myotonic dystrophy protein kinase-binding protein (MKBP) (HspB2), HspB3, alphaA-crystallin (HspB4), alphaB-crystallin (HspB5), Hsp20 (p20, HspB6), cardiovascular heat shock protein (cvHsp [HspB7]), Hsp22 (HspB8), and HspB9. The most pronounced structural feature of sHsps is the alpha-crystallin domain, a conserved stretch of approximately 80 amino acid residues in the C-terminal half of the molecule. Using the alpha-crystallin domain of human Hsp27 as query in a BLAST search, we found sequence similarity with another mammalian protein, the sperm outer dense fiber protein (ODFP). ODFP occurs exclusively in the axoneme of sperm cells. Multiple alignment of human ODFP with the other human sHsps reveals that the primary structure of ODFP fits into the sequence pattern that is typical for this protein superfamily: alpha-crystallin domain (conserved), N-terminal domain (less conserved), central region (variable), and C-terminal tails (variable). In a phylogenetic analysis of 167 proteins of the sHsp superfamily, using Bayesian inference, mammalian ODFPs form a clade and are nested within previously identified sHsps, some of which have been implicated in cytoskeletal functions. Both the multiple alignment and the phylogeny suggest that ODFP is the 10th member of the superfamily of mammalian sHsps, and we propose to name it HspB10 in analogy with the other sHsps. The C-terminal tail of HspB10 has a remarkable low-complexity structure consisting of 10 repeats of the motif C-X-P. A BLAST search using the C-terminal tail as query revealed similarity with sequence elements in a number of Drosophila male sperm proteins, and mammalian type I keratins and cornifin-alpha. Taken together, the following findings suggest a specialized role of HspB10 in cytoskeleton: (1) the exclusive location in sperm cell tails, (2) the phylogenetic relationship with sHsps implicated in cytoskeletal functions, and (3) the partial similarity with cytoskeletal proteins.

Amino Acid Sequence↗
PubMed2002

Adenovirus fiber disrupts CAR-mediated intercellular adhesion allowing virus escape.

Adenovirus binds its receptor (CAR), enters cells, and replicates. It must then escape to the environment to infect a new host. We found that following infection, human airway epithelia first released adenovirus to the basolateral surface. Virus then traveled between epithelial cells to emerge on the apical surface. Adenovirus fiber protein, which is produced during viral replication, facilitated apical escape. Fiber binds CAR, which sits on the basolateral membrane where it maintains tight junction integrity. When fiber bound CAR, it disrupted junctional integrity, allowing virus to filter between the cells and emerge apically. Thus, adenovirus exploits its receptor for two important but distinct steps in its life cycle: entry into host cells and escape across epithelial barriers to the environment.

Adenoviridae↗
PubMed2002

A component of innate immunity prevents bacterial biofilm development.

Antimicrobial factors form one arm of the innate immune system, which protects mucosal surfaces from bacterial infection. These factors can rapidly kill bacteria deposited on mucosal surfaces and prevent acute invasive infections. In many chronic infections, however, bacteria live in biofilms, which are distinct, matrix-encased communities specialized for surface persistence. The transition from a free-living, independent existence to a biofilm lifestyle can be devastating, because biofilms notoriously resist killing by host defence mechanisms and antibiotics. We hypothesized that the innate immune system possesses specific activity to protect against biofilm infections. Here we show that lactoferrin, a ubiquitous and abundant constituent of human external secretions, blocks biofilm development by the opportunistic pathogen Pseudomonas aeruginosa. This occurs at lactoferrin concentrations below those that kill or prevent growth. By chelating iron, lactoferrin stimulates twitching, a specialized form of surface motility, causing the bacteria to wander across the surface instead of forming cell clusters and biofilms. These findings reveal a specific anti-biofilm defence mechanism acting at a critical juncture in biofilm development, the time bacteria stop roaming as individuals and aggregate into durable communities.

Biofilms↗
PubMed2002

The acid-activated ion channel ASIC contributes to synaptic plasticity, learning, and memory.

Many central neurons possess large acid-activated currents, yet their molecular identity is unknown. We found that eliminating the acid sensing ion channel (ASIC) abolished H(+)-gated currents in hippocampal neurons. Neuronal H(+)-gated currents and transient acidification are proposed to play a role in synaptic transmission. Investigating this possibility, we found ASIC in hippocampus, in synaptosomes, and in dendrites localized at synapses. Moreover, loss of ASIC impaired hippocampal long-term potentiation. ASIC null mice had reduced excitatory postsynaptic potentials and NMDA receptor activation during high-frequency stimulation. Consistent with these findings, null mice displayed defective spatial learning and eyeblink conditioning. These results identify ASIC as a key component of acid-activated currents and implicate these currents in processes underlying synaptic plasticity, learning, and memory.

Acid Sensing Ion Channels↗
PubMed2002

Heteromultimers of DEG/ENaC subunits form H+-gated channels in mouse sensory neurons.

Acidic extracellular solution activates transient H(+)-gated currents in dorsal root ganglion (DRG) neurons. The biophysical properties of three degenerin/epithelial sodium (DEG/ENaC) channel subunits (BNC1, ASIC, and DRASIC), and their expression in DRG, suggest that they might underlie these H(+)-gated currents and function as sensory transducers. However, it is uncertain which of these DEG/ENaC subunits generate the currents, and whether they function as homomultimers or heteromultimers. We found that the biophysical properties of transient H(+)-gated currents from medium to large mouse DRG neurons differed from BNC1, ASIC, or DRASIC expressed individually, but were reproduced by coexpression of the subunits together. To test the contribution of each subunit, we studied DRG from three strains of mice, each bearing a targeted disruption of BNC1, ASIC, or DRASIC. Deletion of any one subunit did not abolish H(+)-gated currents, but altered currents in a manner consistent with heteromultimerization of the two remaining subunits. These data indicate that combinations of two or more DEG/ENaC subunits coassemble as heteromultimers to generate transient H(+)-gated currents in mouse DRG neurons.

Animals↗
PubMed2002

Discovery of five conserved beta -defensin gene clusters using a computational search strategy.

The innate immune system includes antimicrobial peptides that protect multicellular organisms from a diverse spectrum of microorganisms. beta-Defensins comprise one important family of mammalian antimicrobial peptides. The annotation of the human genome fails to reveal the expected diversity, and a recent query of the draft sequence with the blast search engine found only one new beta-defensin gene (DEFB3). To define better the beta-defensin gene family, we adopted a genomics approach that uses hmmer, a computational search tool based on hidden Markov models, in combination with blast. This strategy identified 28 new human and 43 new mouse beta-defensin genes in five syntenic chromosomal regions. Within each syntenic cluster, the gene sequences and organization were similar, suggesting each cluster pair arose from a common ancestor and was retained because of conserved functions. Preliminary analysis indicates that at least 26 of the predicted genes are transcribed. These results demonstrate the value of a genomewide search strategy to identify genes with conserved structural motifs. Discovery of these genes represents a new starting point for exploring the role of beta-defensins in innate immunity.

Amino Acid Motifs↗
PubMed2002

CFTR with a partially deleted R domain corrects the cystic fibrosis chloride transport defect in human airway epithelia in vitro and in mouse nasal mucosa in vivo.

In developing gene therapy for cystic fibrosis (CF) airways disease, a transgene encoding a partially deleted CF transmembrane conductance regulator (CFTR) Cl- channel could be of value for vectors such as adeno-associated virus that have a limited packaging capacity. Earlier studies in heterologous cells indicated that the CFTR R (regulatory) domain is predominantly random coil and that parts of the R domain can be deleted without abolishing channel function. Therefore, we designed a series of CFTR variants with shortened R domains (between residues 708 and 835) and expressed them in well-differentiated cultures of CF airway epithelia. All of the variants showed normal targeting to the apical membrane, and for the constructs we tested, biosynthesis was like wild type. Moreover, all constructs generated transepithelial Cl- current in CF epithelia. Comparison of the Cl- transport suggested that the length of the R domain, the presence of phosphorylation sites, and other factors contribute to channel activity. A variant deleting residues 708-759 complemented CF airway epithelia to the same extent as wild-type CFTR and showed no current in the absence of cAMP stimulation. In addition, expression in nasal mucosa of CF mice corrected the Cl- transport defect. These data provide insight into the structure and function of the R domain and identify regions that can be deleted with retention of function. Thus they suggest a strategy for shortening the transgene used in CF gene therapy.

Animals↗
PubMed2002

Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel).

Neuronal members of the degenerin/epithelial Na(+) channel (DEG/ENaC) family of cation channels include the mammalian brain Na(+) channel 1 (BNC1), acid-sensing ion channel (ASIC) and dorsal-root acid-sensing ion channel (DRASIC). Their response to acidic pH, their sequence similarity to nematode proteins involved in mechanotransduction and their modulation by neuropeptides suggest that they may function as receptors for a number of different stimuli. Using the yeast two-hybrid assay, we found that the PDZ domain-containing protein PICK1 (protein interacting with C kinase) interacts specifically with the C-termini of BNC1 and ASIC, but not DRASIC or the related alphaENaC or betaENaC. In both the yeast two-hybrid system and mammalian cells, deletion of the BNC1 and ASIC C-termini abolished the interaction with PICK1. Likewise, mutating the PDZ domain in PICK1 abolished its interaction with BNC1 and ASIC. In addition, in a heterologous expression system PICK1 altered the distribution of BNC1 channels; this effect was dependent on the PDZ domain of PICK1 and the C-terminus of BNC1. We found crude synaptosomal fractions of brain to be enriched in ASIC, suggesting a possible synaptic localization. Moreover, in transfected hippocampal neurons ASIC co-localized with PICK1 in a punctate pattern at synapses. These data suggest that PICK1 binds ASIC and BNC1 via its PDZ domain. This interaction may be important for the localization and/or function of these channels in both the central and peripheral nervous systems.

Acid Sensing Ion Channels↗
PubMed2002

Mutations that change the position of the putative gamma-phosphate linker in the nucleotide binding domains of CFTR alter channel gating.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is an ATP-binding cassette transporter that contains conserved nucleotide-binding domains (NBDs). In CFTR, the NBDs bind and hydrolyze ATP to open and close the channel. Crystal structures of related NBDs suggest a structural model with an important signaling role for a gamma-phosphate linker peptide that couples bound nucleotide to movement of an alpha-helical subdomain. We mutated two residues in CFTR that the structural model predicts will uncouple effects of nucleotide binding from movement of the alpha-helical subdomain. These residues are Gln-493 and Gln-1291, which may directly connect the ATP gamma-phosphate to the gamma-phosphate linker, and residues Asn-505 and Asn-1303, which may form hydrogen bonds that stabilize the linker. In NBD1, Q493A reduced the frequency of channel opening, suggesting a role for this residue in coupling ATP binding to channel opening. In contrast, N505C increased the frequency of channel opening, consistent with a role for Asn-505 in stabilizing the inactive state of the NBD. In NBD2, Q1291A decreased the effects of pyrophosphate without altering other functions. Mutations of Asn-1303 decreased the rate of channel opening and closing, suggesting an important role for NBD2 in controlling channel burst duration. These findings are consistent with both the bacterial NBD structural model and gating models for CFTR. Our results extend models of nucleotide-induced structural changes from bacterial NBDs to a functional mammalian ATP-binding cassette transporter.

Adenosine Diphosphate↗
PubMed2002

DRASIC contributes to pH-gated currents in large dorsal root ganglion sensory neurons by forming heteromultimeric channels.

For many years it has been observed that extracellular acid activates transient cation currents in large-diameter mechanosensory dorsal root ganglion (DRG) neurons. However, the molecular basis of these currents has not been known. Large DRG neurons express the dorsal root acid sensing ion channel (DRASIC), suggesting that DRASIC might contribute to H+-gated DRG currents. To test this, we examined whole cell currents in large DRG neurons from mice in which the DRASIC gene had been disrupted. We found that DRASIC null neurons retained H+-gated currents, indicating that DRASIC alone was not required for the currents. However, without DRASIC, the properties of the currents changed substantially as compared with wild-type neurons. In DRASIC -/- neurons, the rate of current desensitization in the continued presence of an acid stimulus slowed dramatically. H+-gated currents in DRASIC null neurons showed a decreased sensitivity to pH and an enhanced sensitivity to amiloride. The loss of DRASIC also altered but did not abolish the current potentiation generated by FMRF-related peptides. These data indicate that the DRASIC subunit makes an important contribution to H+-gated currents in large DRG sensory neurons. The results also suggest that related acid-activated DEG/ENaC channel subunits contribute with DRASIC to form heteromultimeric acid-activated channels.

Acid Sensing Ion Channels↗