Inflammation-induced changes in BK(Ca) currents in cutaneous dorsal root ganglion neurons from the adult rat.

BACKGROUND: Inflammation-induced sensitization of primary afferents is associated with a decrease in K(+) current. However, the type of K(+) current and basis for the decrease varies as a function of target of innervation. Because glabrous skin of the rat hindpaw is used often to assess changes in nociception in models of persistent pain, the purpose of the present study was to determine the type and extent to which K(+) currents contribute to the inflammation-induced sensitization of cutaneous afferents. Acutely dissociated retrogradely labeled cutaneous dorsal root ganglion neurons from naïve and inflamed (3 days post complete Freund's adjuvant injection) rats were studied with whole cell and perforated patch techniques. RESULTS: Inflammation-induced sensitization of small diameter cutaneous neurons was associated with an increase in action potential duration and rate of decay of the afterhyperpolarization. However, no changes in voltage-gated K(+) currents were detected. In contrast, Ca(2+) modulated iberiotoxin sensitive and paxilline sensitive K(+) (BK(Ca)) currents were significantly smaller in small diameter IB4+ neurons. This decrease in current was not associated with a detectable change in total protein levels of the BK(Ca) channel α or ß subunits. Single cell PCR analysis revealed a significant change in the pattern of expression of α subunit splice variants and ß subunits that were consistent, at least in part, with inflammation-induced changes in the biophysical properties of BK(Ca) currents in cutaneous neurons. CONCLUSIONS: Results of this study provide additional support for the conclusion that it may be possible, if not necessary to selectively treat pain arising from specific body regions. Because a decrease in BK(Ca) current appears to contribute to the inflammation-induced sensitization of cutaneous afferents, BK(Ca) channel openers may be effective for the treatment of inflammatory pain.

Calcium-dependent dynamics of cadherin interactions at cell-cell junctions.

Cadherins play a key role in the dynamics of cell-cell contact formation and remodeling of junctions and tissues. Cadherin-cadherin interactions are gated by extracellular Ca(2+), which serves to rigidify the cadherin extracellular domains and promote trans junctional interactions. Here we describe the direct visualization and quantification of spatiotemporal dynamics of N-cadherin interactions across intercellular junctions in living cells using a genetically encodable FRET reporter system. Direct measurements of transjunctional cadherin interactions revealed a sudden, but partial, loss of homophilic interactions (τ = 1.17 ± 0.06 s(-1)) upon chelation of extracellular Ca(2+). A cadherin mutant with reduced adhesive activity (W2A) exhibited a faster, more substantial loss of homophilic interactions (τ = 0.86 ± 0.02 s(-1)), suggesting two types of native cadherin interactions--one that is rapidly modulated by changes in extracellular Ca(2+) and another with relatively stable adhesive activity that is Ca(2+) independent. The Ca(2+)-sensitive dynamics of cadherin interactions were transmitted to the cell interior where ß-catenin translocated to N-cadherin at the junction in both cells. These data indicate that cadherins can rapidly convey dynamic information about the extracellular environment to both cells that comprise a junction.

BKCa currents are enriched in a subpopulation of adult rat cutaneous nociceptive dorsal root ganglion neurons.

The biophysical properties and distribution of voltage-dependent, Ca(2+) -modulated K(+) (BK(Ca)) currents among subpopulations of acutely dissociated DiI-labeled cutaneous sensory neurons from the adult rat were characterized with whole-cell patch-clamp techniques. BK(Ca) currents were isolated from total K(+) current with iberiotoxin, charybdotoxin or paxilline. There was considerable variability in biophysical properties of BK(Ca) currents. There was also variability in the distribution of BK(Ca) current among subpopulations of cutaneous dorsal root ganglia (DRG) neurons. While present in each of the subpopulations defined by cell body size, IB4 binding or capsaicin sensitivity, BK(Ca) current was present in the vast majority (> 90%) of small-diameter IB4+ neurons, but was present in only a minority of neurons in subpopulations defined by other criteria (i.e. small-diameter IB4-). Current-clamp analysis indicated that in IB4+ neurons, BK(Ca) currents contribute to the repolarization of the action potential and adaptation in response to sustained membrane depolarization, while playing little role in the determination of action potential threshold. Reverse transcriptase-polymerase chain reaction analysis of mRNA collected from whole DRG revealed the presence of multiple splice variants of the BK(Ca) channel alpha-subunit, rslo and all four of the accessory beta-subunits, suggesting that heterogeneity in the biophysical and pharmacological properties of BK(Ca) current in cutaneous neurons reflects, at least in part, the differential distribution of splice variants and/or beta-subunits. Because even a small decrease in BK(Ca) current appears to have a dramatic influence on excitability, modulation of this current may contribute to sensitization of nociceptive afferents observed following tissue injury.

Plasticity of neuron-glial interactions mediated by astrocytic EphARs.

Ephrin (Eph) signaling via Eph receptors affects neuronal structure and function. We report here that exogenous ephrinAs (EphAs) induce outgrowth of filopodial processes from astrocytes within minutes in rat hippocampal slice cultures. Identical effects were induced by release of endogenous ephrinAs by cleavage of their glycosylphosphatidylinositol anchor. Reverse transcription-PCR and immunocytochemistry revealed the expression of multiple EphA receptors (EphARs) in astrocytes. Exogenous and endogenous ephrins did not induce process outgrowth from astrocytes transfected with a kinase-dead EphAR construct, indicating that the critical EphARs were located on glia. Concomitant with these morphological changes, ephrinA reduced the frequency of (S)-3,5-dihydroxyphenylglycine-evoked NMDA receptor-mediated inward currents in CA1 pyramidal cells, elicited by release of glutamate from glial cells. The sensitivity of CA1 cell synaptic or extrasynaptic NMDA receptors was unaffected by ephrinA, indicating that this effect was mediated by inhibition of glutamate release from glial cells. Finally, ephrinA application decreased the frequency and increased the duration of spontaneous oscillations of the intracellular [Ca2+] in astrocytes. We conclude that ephrinA-EphA signaling is a pluripotent regulator of neuron-astrocyte interactions mediating rapid structural and functional plasticity.

Evidence for the involvement of dominant-negative Notch molecules in the normal course of Drosophila development.

Notch signaling is used to specify cell types during animal development. A high level specifies one cell type, whereas a low level specifies the alternate type. The effector of Notch signaling is the Notch intracellular domain. Upon its release from the plasma membrane in response to Delta binding the Notch extracellular domain, the Notch intracellular domain combines with the transcription factor Suppressor of Hairless and promotes the expression of target genes. Using a panel of antibodies made against different extracellular and intracellular regions of Notch, we show that cell types and tissues with low levels of Notch signaling are enriched for Notch molecules detected only by the extracellular domain antibodies. This enrichment often follows enrichment for Notch molecules detected only by antibodies made against the Suppressor of Hairless binding region. Notch molecules lacking most of the intracellular domain or containing only the Suppressor of Hairless binding region are produced during development. Such molecules are known to suppress Notch signaling, possibly by taking away Delta or Suppressor of Hairless from the full-length Notch. Thus, it is possible that dominant-negative Notch molecules are produced in the normal course of tissue differentiation in Drosophila as part of an auto-down-regulation mechanism.

Delta activity independent of its activity as a ligand of Notch.

BACKGROUND: Delta, Notch, and Scabrous often function together to make different cell types and refine tissue patterns during Drosophila development. Delta is known as the ligand that triggers Notch receptor activity. Scabrous is known to bind Notch and promote Notch activity in response to Delta. It is not known if Scabrous binds Delta or Delta has activity other than its activity as a ligand of Notch. It is very difficult to clearly determine this binding or activity in vivo as all Notch, Delta, and Scabrous activities are required simultaneously or successively in an inter-dependent manner. RESULTS: Using Drosophila cultured cells we show that the full length Delta promotes accumulation of Daughterless protein, fringe RNA, and pangolin RNA in the absence of Scabrous or Notch. Scabrous binds Delta and suppresses this activity even though it increases the level of the Delta intracellular domain. We also show that Scabrous can promote Notch receptor activity, in the absence of Delta. CONCLUSION: Delta has activity that is independent of its activity as a ligand of Notch. Scabrous suppresses this Delta activity. Scabrous also promotes Notch activity that is dependent on Delta's ligand activity. Thus, Notch, Delta, and Scabrous might function in complex combinatorial or mutually exclusive interactions during development. The data reported here will be of significant help in understanding these interactions in vivo.

The Notch amino terminus regulates protein levels and Delta-induced clustering of Drosophila Notch receptors.

Notch signaling is required for the development of almost all animal tissues. It is a cell surface receptor that generates intracellular signals in response to Delta binding its extracellular domain. Notch response to Delta is affected by mutations in its extracellular domain outside of the Delta binding region. One such region is the Notch amino terminus. Mutations in this region are associated with developmental defects. How a mutation in the Notch amino terminus affects Notch function is unknown. We explored this issue in Drosophila melanogaster. We report that Notch receptors mutated in the amino terminus accumulate to abnormal levels, are deficient in Delta induced receptor clustering, and exhibit reduced rate of internalization and signaling. Notch receptors lacking the whole or the carboxy-terminal half of the intracellular domain are defective in internalization but not in clustering or accumulation. None of the other mutated Notch receptors showed defects in clustering, accumulation, or internalization. These observations suggest that the Notch amino terminus regulates Notch levels and clustering, which could affect the rate of Notch signaling and down-regulation.

The adhesion force of Notch with Delta and the rate of Notch signaling.

Notch signaling is repeatedly used during animal development to specify cell fates. Using atomic force microscopy on live cells, chemical inhibitors, and conventional analyses, we show that the rate of Notch signaling is linked to the adhesion force between cells expressing Notch receptors and Delta ligand. Both the Notch extracellular and intracellular domains are required for the high adhesion force with Delta. This high adhesion force is lost within minutes, primarily due to the action of Presenilin on Notch. Reduced turnover or Delta pulling accelerate this loss. These data suggest that strong adhesion between Notch and Delta might serve as a booster for initiating Notch signaling at a high rate.

Regulation of Notch signaling by a novel mechanism involving suppressor of hairless stability and carboxyl terminus-truncated notch.

Different amounts of Suppressor of Hairless (SuH)-dependent Notch (N) signaling is often used during animal development to produce two different tissues from a population of equipotent cells. During Drosophila melanogaster embryogenesis, cells with high amounts of this signaling differentiate the larval epidermis whereas cells with low amounts, or none, differentiate the central nervous system (CNS). The mechanism by which SuH-dependent N signaling is increased or decreased in these different cells is obscure. The developing epidermis is known to get enriched for the full-length N (NFull) and the developing CNS for the carboxyl terminus-truncated N (NdeltaCterm). Results described here indicate that this differential accumulation of N receptors is part of a mechanism that would promote SuH-dependent N signaling in the developing epidermis but suppress it in the developing CNS. This mechanism involves SuH-dependent stability of NFull, NFull-dependent accumulation of SuH, stage specific stability of SuH, and NdeltaCterm-dependent loss of SuH and NFull.