Mechanosensitive (MS) ion channels are present in a variety of cells. However, very little is known about the ion channels that account for mechanical sensitivity in sensory neurons. We identified the two most frequently encountered but distinct types of MS channels in 1390 of 2962 membrane patches tested in cultured dorsal root ganglion neurons. The two MS channels exhibited different thresholds, thus named as low-threshold (LT) and high-threshold (HT) MS channels, and sensitivity to pressure. The two channels retained different single-channel conductances and current-voltage relationships: LT and HT channels elicited large- and small-channel conductance with outwardly rectifying and linear I-V relationships, respectively. Both LT and HT MS channels were permeable to monovalent cations and Ca2+ and were blocked by gadolinium, a blocker of MS channels. Colchicine and cytochalasin D markedly reduced the activities of the two MS channels, indicating that cytoskeletal elements support the mechanosensitivity. Both types of MS channels were found primarily in small sensory neurons with diameters of <30 microm. Furthermore, HT MS channels were sensitized by a well known inducer of mechanical hyperalgesia, prostaglandin E2, via the protein kinase A pathway. We identified two distinct types of MS channels in sensory neurons that probably give rise to the observed MS whole-cell currents and transduce mechanical stimuli to neural signals involved in somatosensation, including pain.

The DagA product of Streptomyces coelicolor is an agarase with a primary translation product (35 kDa) of 309 amino acids, including a 30-amino acid signal peptide. Although dagA expression in Streptomyces lividans under the control of its own set of promoters was previously reported, its enzymatic properties have never been elucidated. To develop an improved expression system for dagA, three types of strong promoters for the Streptomyces host were linked to dagA, and their efficiencies in DagA production were compared in S. lividans TK24. All of the transformants with dagA grew at improved rates and produced larger amounts of DagA in the modified R2YE medium containing 0.5% agar as the sole carbon source. Of the three transformants, the S. lividans TK24/pUWL201-DagA (ermE promoter) produced the highest agarase activity (A (540)=4.24), and even the S. lividans TK24/pHSEV1-DagA (tipA promoter) and S. lividans TK24/pWHM3-DagA (sprT promoter) produced higher agarase activity (A (540)=0.24 and 0.12, respectively) than the control (A (540)=0.01) in the modified R2YE medium. The mature form of DagA protein (32 kDa) was successfully purified by one-step affinity column chromatography by using agarose beads with excellent yield. The purified DagA was found to exhibit maximal agarase activity at 40 °C and pH 7.0. The K(m), V(max), and K(cat) values for agarose were 2.18 mg/ml (approximately 1.82 × 10(-5) M), 39.06 U/mg of protein, and 9.5 × 10(3)/s, respectively. Thin layer chromatography (TLC) analysis, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry, and Fourier transform nuclear magnetic resonance (FT-NMR) spectrometry of the hydrolyzed products of agarose by DagA revealed that DagA is an endo-type β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose.

Capsaicin (CAP)-activated ion channel plays a key role in generating nociceptive neural signals in sensory neurons. Here we present evidence that intracellular ATP upregulates the activity of capsaicin receptor channel. In inside-out membrane patches isolated from sensory neurons, application of CAP activated a nonselective cation channel (i cap ). Further addition of ATP to the bath caused a significant increase in i cap , with a K 1/2 of 3.3 mM. Capsaicin (CAP), the pungent ingredient of hot peppers, excites small sensory neurons and thereby causes pain or neurogenic inflammation (Bevan and Szolcsanyi, 1990;Szallasi and Blumberg, 1999). In cultured dorsal root ganglion (DRG) neurons, CAP has been shown to activate a nonselective cation channel and produce influx of cations (Bevan and Szolcsanyi, 1990;Oh et al., 1996). The activation of this cation channel is probably responsible for the excitation of a group of sensory neurons. A cDNA, vanilloid receptor 1 (VR1), that encodes a channel sensitive to CAP has recently been cloned (Caterina et al., 1997). A striking property of VR1 is that it is also activated by heat (Caterina et al., 1997;Tominaga et al., 1998). Another interesting property of VR1 is its activation by acid, indicating that protons can activate the channel (Tominaga et al., 1998). It is now known that disruption of VR1 gene reduces inflammation-induced heat hyperalgesia (Caterina et al., 2000;Davis et al., 2000). As a result, the view of the CAP receptor as a potential nociceptive heat and chemical sensor has gained a wide acceptance (Kress and Zeilhofer, 1999;Szallasi and Blumberg, 1999;Caterina et al., 2000;Davis et al., 2000).Recently, endogenous lipids such as anandamide and various metabolic products of lipoxygenases such as 12-hydroperoxyeicosatetraenoic acid and leukotriene B 4 have been shown to activate the CAP channel (Zygmunt et al., 1999;Hwang et al., 2000). The three-dimensional structure of 12-hydroperoxyeicosatetraenoic acid, one of the lipoxygenase products, was found to superimpose reasonably well with that of capsaicin, suggesting that these lipids may potentially act as endogenous capsaicin-like substances (Hwang et al., 2000).During the course of our studies on the CAP channel, we observed that the channel activity in the cell-attached state with CAP in the pipette is always significantly higher than that after formation of the inside-out state. This difference in channel activity could not be explained by a change in membrane potential or ionic composition. Therefore, we speculated that there exists a cytosolic substance that helps to maintain the channel activity at a higher level and that its washout reduces channel activity. Activity of many ion channels is often modulated by phosphorylation (Levitan, 1994). For example, activity of large conductance Ca 2ϩ -activated K ϩ channel in the brain increases after the addition of ATP via phosphorylation mediated by cAMP-dependent protein kinase A (PKA) (Baraban et al., 1985;Chung et al., 1991) or protein kinase C (PKC) (De Peyer ...

A vanilloid receptor (VR1, now known as TRPV1) is an ion channel activated by vanilloids, including capsaicin (CAP) and resiniferatoxin (RTX), which are pungent ingredients of plants. Putative endogenous activators (anandamide and metabolites of arachidonic acid) are weak activators of VR1 compared to capsaicin and RTX, and the concentrations of the physiological condition of those activators are not sufficient to induce significant activation of VR1. One way to overcome the weak activation of endogenous activators would be the sensitization of VR1, with the phosphorylation of the channel being one possibility. The phosphorylation of VR1 by several kinases has been reported, mostly by indirect evidence. Here, using an in vivo phosphorylation method, the VR1 channel was shown to be sensitized by phosphorylation of the channel itself by multiple pathways involving PKA, PKC and acid. Also, in sensitizing VR1, BK appeared to show activation of PKC for the sensitization of VR1 by phosphorylation of the channel.

Alteromonas sp. GNUM-1 is known to degrade agar, the main cell wall component of red macroalgae, for their growth. A putative agarase gene (agaG1) was identified from the mini-library of GNUM-1, when extracellular agarase activity was detected in a bacterial transformant. The nucleotide sequence revealed that AgaG1 had significant homology to GH16 agarases. agaG1 encodes a primary translation product (34.7 kDa) of 301 amino acids, including a 19-amino-acid signal peptide. For intracellular expression, a gene fragment encoding only the mature form (282 amino acids) was cloned into pGEX-5X-1 in Escherichia coli, where AgaG1 was expressed as a fusion protein with GST attached to its N-terminal (GST-AgaG1). GST-AgaG1 purified on a glutathione sepharose column had an apparent molecular weight of 59 kDa on SDS-PAGE, and this weight matched with the estimated molecular weight (58.7 kDa). The agarase activity of the purified protein was confirmed by the zymogram assay. GST-AgaG1 could hydrolyze the artificial chromogenic substrate, p-nitrophenyl-β-D-galactopyranoside but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for GST-AgaG1 activity were identified as 7.0 and 40 °C, respectively. GST-AgaG1 was stable up to 40 °C (100 %), and it retained more than 70 % of its initial activity at 45 °C after heat treatment for 30 min. The K m and V max for agarose were 3.74 mg/ml and 23.8 U/mg, respectively. GST-AgaG1 did not require metal ions for its activity. Thin layer chromatography analysis, mass spectrometry, and (13)C-nuclear magnetic resonance spectrometry of the GST-AgaG1 hydrolysis products revealed that GST-AgaG1 is an endo-type β-agarase that hydrolyzes agarose and neoagarotetraose into neoagarobiose.