A novel binding site between the voltage-dependent calcium channel CaV1.2 subunit and CaVß2 subunit discovered using a new analysis method for protein-protein interactions.

We developed a new method to analyze protein-protein interactions using a dual-inducible prokaryotic expression system. To evaluate protein-protein binding, a chimeric fusion toxin gene was constructed using a DNase-treated short DNA fragment (epitope library) and CcdB, which encodes a DNA topoisomerase II toxin. Protein-protein interactions would affect toxin activity, resulting in colony formation. Using this novel system, we found a new binding site in the voltage-dependent calcium channel α1 subunit (CaV1.2) for the voltage-dependent calcium channel ß2 subunit. Prokaryotic expression screening of the ß2 subunit using an epitope library of CaV1.2 resulted in two overlapping clones of the C-terminal sequence of CaV1.2. In vitro overlay and immunoprecipitation analyses revealed preferential binding of the C-terminal sequences of CaV1.2 and ß2.

Rapid method for plasmid DNA recombination (Murakami-system).

DNA recombination is a useful technology for cloning and subsequent functional analysis, while standard techniques for plasmid DNA recombination have remained unchanged. In the present study, we introduced rapid method for plasmid DNA recombination, which we named "Murakami-system", to complete the experiments in under 33 h. For this purpose, we selected the following: PCR amplification with 25 cycles and E. coli strain with rapid growth (incubation time of 6-8 h). In addition, we selected rapid plasmid DNA purification (mini-prep; ∼10 min) and rapid restriction enzyme incubation (20 min). This recombination system enabled rapid plasmid DNA recombination within 24-33 h, which could be useful in various fields. We also established a 1-day method for competent cell preparation. Our rapid recombination system allowed several sessions of plasmid DNA recombination to be performed every week, which improves the functional analysis of various genes.•"Rapid method for plasmid DNA recombination (Murakami-system).•E. coli strain with rapid growth (incubation time of 6-8 h).•Combination of rapid protocols (PCR, electrophoresis, DNA purification, ligation, and mini-prep) enabled plasmid DNA recombination within 24-33 h.

Dopamine D1 Receptor Immunoreactivity on Fine Processes of GFAP-Positive Astrocytes in the Substantia Nigra Pars Reticulata of Adult Mouse.

Substantia nigra pars reticulata (SNr), the major output nucleus of the basal ganglia, receives dopamine from dendrites extending from dopaminergic neurons of the adjacent nucleus pars compacta (SNc), which is known for its selective degeneration in Parkinson's disease. As a recipient for dendritically released dopamine, the dopamine D1 receptor (D1R) is a primary candidate due to its very dense immunoreactivity in the SNr. However, the precise location of D1R remains unclear at the cellular level in the SNr except for that reported on axons/axon terminals of presumably striatal GABAergic neurons. To address this, we used D1R promotor-controlled, mVenus-expressing transgenic mice. When cells were acutely dissociated from SNr of mouse brain, prominent mVenus fluorescence was detected in fine processes of glia-like cells, but no such fluorescence was detected from neurons in the same preparation, except for the synaptic bouton-like structure on the neurons. Double immunolabeling of SNr cells dissociated from adult wild-type mice brain further revealed marked D1R immunoreactivity in the processes of glial fibrillary acidic protein (GFAP)-positive astrocytes. Such D1R imunoreactivity was significantly stronger in the SNr astrocytes than that in those of the visual cortex in the same preparation. Interestingly, GFAP-positive astrocytes dissociated from the striatum demonstrated D1R immunoreactivity, either remarkable or minimal, similarly to that shown in neurons in this nucleus. In contrast, in the SNr and visual cortex, only weak D1R immunoreactivity was detected in the neurons tested. These results suggest that the SNr astrocyte may be a candidate recipient for dendritically released dopamine. Further study is required to fully elucidate the physiological roles of divergent dopamine receptor immunoreactivity profiles in GFAP-positive astrocytes.

Uptake of a fluorescent L-glucose derivative 2-NBDLG into three-dimensionally accumulating insulinoma cells in a phloretin-sensitive manner.

Of two stereoisomers of glucose, only D- and not L-glucose is abundantly found in nature, being utilized as an essential fuel by most organisms. The uptake of D-glucose into mammalian cells occurs through glucose transporters such as GLUTs, and this process has been effectively monitored by a fluorescent D-glucose derivative 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) at the single cell level. However, since fluorescence is an arbitrary measure, we have developed a fluorescent analog of L-glucose 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-L-glucose (2-NBDLG), as a negative control substrate for more accurately identifying the stereoselectivity of the uptake. Interestingly, a small portion of mouse insulinoma cells MIN6 abundantly took up 2-NBDLG at a late culture stage (≳ 10 days in vitro, DIV) when multi-cellular spheroids exhibiting heterogeneous nuclei were formed, whereas no such uptake was detected at an early culture stage (≲ 6 DIV). The 2-NBDLG uptake was persistently observed in the presence of a GLUT inhibitor cytochalasin B. Neither D- nor L-glucose in 50 mM abolished the uptake. No significant inhibition was detected by inactivating sodium/glucose cotransporters (SGLTs) with Na(+)-free condition. To our surprise, the 2-NBDLG uptake was totally inhibited by phloretin, a broad spectrum inhibitor against transporters/channels including GLUTs and aquaporins. From these, a question might be raised if non-GLUT/non-SGLT pathways participate in the 2-NBDLG uptake into spheroid-forming MIN6 insulinoma. It might also be worthwhile investigating whether 2-NBDLG can be used as a functional probe for detecting cancer, since the nuclear heterogeneity is among critical features of malignancy.

Auto-oxidation products of epigallocatechin gallate activate TRPA1 and TRPV1 in sensory neurons.

The sensation of astringency is elicited by catechins and their polymers in wine and tea. It has been considered that catechins in green tea are unstable and auto-oxidized to induce more astringent taste. Here, we examined how mammalian transient receptor potential V1 (TRPV1) and TRPA1, which are nociceptive sensors, are activated by green tea catechins during the auto-oxidation process. Neither TRPV1 nor TRPA1 could be activated by any of the freshly prepared catechin. When one of the major catechin, epigallocatechin gallate (EGCG), was preincubated for 3h in Hank's balanced salt solution, it significantly activated both TRP channels expressed in HEK293 cells. Even after incubation, other catechins showed much less effects. Results suggest that only oxidative products of EGCG activate both TRPV1 and TRPA1. Dorsal root ganglion (DRG) sensory neurons were also activated by the incubated EGCG through TRPV1 and TRPA1 channels. Liquid chromatography-mass spectrometry revealed that theasinensins A and D are formed during incubation of EGCG. We found that purified theasinensin A activates both TRPV1 and TRPA1, and that it stimulates DRG neurons through TRPV1 and TRPA1 channels. Results suggested a possibility that TRPV1 and TRPA1 channels are involved in the sense of astringent taste of green tea.

Singular localization of sodium channel ß4 subunit in unmyelinated fibres and its role in the striatum.

Voltage-gated Na(+) channel ß-subunits are multifunctional molecules that modulate Na(+) channel activity and regulate cell adhesion, migration and neurite outgrowth. ß-subunits including ß4 are known to be highly concentrated in the nodes of Ranvier and axon initial segments in myelinated axons. Here we show diffuse ß4 localization in striatal projection fibres using transgenic mice that express fluorescent protein in those fibres. These axons are unmyelinated, forming large, inhibitory fibre bundles. Furthermore, we report ß4 dimer expression in the mouse brain, with high levels of ß4 dimers in the striatal projection fascicles, suggesting a specific role of ß4 in those fibres. Scn4b-deficient mice show a resurgent Na(+) current reduction, decreased repetitive firing frequency in medium spiny neurons and increased failure rates of inhibitory postsynaptic currents evoked with repetitive stimulation, indicating an in vivo channel regulatory role of ß4 in the striatum.

Syntheses of 2-NBDG analogues for monitoring stereoselective uptake of D-glucose.

2-NBDG is a widely used fluorescent tracer for monitoring d-glucose uptake into single living cells. However, 2-NBDG alone is not sufficient for monitoring the net stereoselective uptake of d-glucose, unless its possible non-stereoselective uptake is properly evaluated. l-Glucose derivatives, which emit fluorescence distinct from that of 2-NBDG, should provide valuable information on the stereoselective uptake, when used with 2-NBDG in combination. In the present study, we synthesized Texas Red (sulforhodamine 101 acid)-coupled and [2-(benz-2-oxa-1,3-diazol-4-yl)amino]-coupled 2-deoxy-D-glucose, referred to as [2-TRG] and [2-BDG], respectively. These derivatives showed emission wavelength longer and shorter than that of 2-NBDG, respectively. 2-TRLG, an antipode of 2-TRG, proved to be an effective tracer for evaluating the extent of non-stereoselective uptake of 2-NBDG when used simultaneously with 2-NBDG. On the other hand, 2-BDG exhibited very weak fluorescence, but the application of a novel cross coupling in the presence of a benzoxadiazole group may be useful for the future development of effective glucose tracers.

The Met268Pro mutation of mouse TRPA1 changes the effect of caffeine from activation to suppression.

The transient receptor potential A1 channel (TRPA1) is activated by various compounds, including isothiocyanates, menthol, and cinnamaldehyde. The sensitivities of the rodent and human isoforms of TRPA1 to menthol and the cysteine-attacking compound CMP1 differ, and the molecular determinants for these differences have been identified in the 5th transmembrane region (TM5) for menthol and TM6 for CMP1. We recently reported that caffeine activates mouse TRPA1 (mTRPA1) but suppresses human TRPA1 (hTRPA1). Here we aimed to identify the molecular determinant that is responsible for species-specific differences in the response to caffeine by analyzing the functional properties of various chimeras expressed in Xenopus oocytes. We initially found that the region between amino acids 231 and 287, in the distal N-terminal cytoplasmic region of mTRPA1, is critical. In a mutagenesis study of this region, we subsequently observed that introduction of a Met268Pro point mutation into mTRPA1 changed the effect of caffeine from activation to suppression. Because the region including Met-268 is different from other reported ligand-binding sites and from the EF-hand motif, these results suggest that the caffeine response is mediated by a unique mechanism, and confirm the importance of the distal N-terminal region for regulation of TRPA1 channel activity.

Effects of spinophilin on the function of RGS8 regulating signals from M2 and M3-mAChRs.

We found that a scaffold protein, spinophilin (SPL), can interact with M2 and M3-muscarinic acetylcholine receptors (mAChRs). As SPL can also bind to RGS8 by using the different region of SPL, we investigated the effects of SPL on the function of RGS8 regulating signals from M2 and M3 receptors. M2 receptor-mediated Gi-signaling was studied by monitoring G-protein-coupled inwardly rectifying K+ channels, and M3 receptor-mediated Gq-signaling was monitored by the increase of Ca2+-activated Cl(-) current. The expression of SPL could enhance the regulatory function of RGS8 on the M3-mAChR, but the acceleration function of RGS8 on the M2-mediated signaling could not be enhanced by SPL. Results showed that the recruitment of RGS8 to the receptor differentially affects the function of RGS8 among receptors.

Caffeine activates mouse TRPA1 channels but suppresses human TRPA1 channels.

Caffeine has various well-characterized pharmacological effects, but in mammals there are no known plasma membrane receptors or ion channels activated by caffeine. We observed that caffeine activates mouse transient receptor potential A1 (TRPA1) in heterologous expression systems by Ca(2+)(i) imaging and electrophysiological analyses. These responses to caffeine were confirmed in acutely dissociated dorsal root ganglion sensory neurons from WT mice, which are known to express TRPA1, but were not seen in neurons from TRPA1 KO mice. Expression of TRPA1 was detected immunohistochemically in nerve fibers and bundles in the mouse tongue. Moreover, WT mice, but not KO mice, showed a remarkable aversion to caffeine-containing water. These results demonstrate that mouse TRPA1 channels expressed in sensory neurons cause an aversion to drinking caffeine-containing water, suggesting they mediate the perception of caffeine. Finally, we observed that caffeine does not activate human TRPA1; instead, it suppresses its activity.

Alternative splicing of RGS8 gene changes the binding property to the M1 muscarinic receptor to confer receptor type-specific Gq regulation.

RGS proteins constitute a large family that modulates heterotrimeric G-protein signaling. We previously showed that RGS8 suppressed Gq signaling in a receptor type-specific manner. To elucidate molecular mechanisms underlying receptor-specific attenuation by RGS8, we examined whether RGS8 can interact with certain G-protein-coupled receptors. By pull-down assay, we showed that RGS8 directly binds to the third intracellular (i3) loop of M1 and M3 muscarinic acetylcholine receptors (mAChRs). The binding of RGS8S, a splice variant with a different N-terminus, was weaker. RGS8 could bind specifically to the C-terminal part of M1i3 (containing amino acids of 304-353 of i3 of human M1-mAChR), but RGS8S could not. Moreover, deletion of the N-terminal 9 amino acids and substitution of both Arg-8 and Arg-9 of RGS8 with Ala resulted in reduced binding to M1i3. BRET experiments revealed that RGS8 actually interacts with M1-mAChR, but RGS8S does not interact in the living cells. The RGS8 mutant, which had less binding ability to M1i3, showed a reduced inhibitory function of Gq signaling through M1-mAChR. These results demonstrated that RGS8 can directly interact with M1-mAChR via its N-terminus and the i3 loop of the receptor, and this binding must play an essential role in receptor-specific suppression by RGS8.

Molecular cloning and characterization of a new RGS protein of Medaka.

We identified eight genes of putative RGS proteins in skin of Medaka fish using PCR amplification with degenerate primers for the RGS domain of known RGS proteins. Then, we cloned a full-length cDNA for a new RGS protein. This RGS protein was similar to human RGS3 within the RGS domain, but other parts were unique among known RGS proteins. RT-PCR analysis demonstrated that this Medaka RGS3-like protein (MeRGS3L) is mainly expressed in skin and heart. When coexpressed in Xenopus oocytes, MeRGS3L accelerated the turning-on and -off of Gi/o-mediated modulation of GIRK channels without apparent desensitization in the presence of ligand. MeRGS3L also decreased the response of Gq signaling upon activation of m1 muscarinic receptor. This new RGS protein may play important roles in regulation of melanophore responses in Medaka skin.