Voltage-gated Na(+) channel β-subunits are multifunctional molecules that modulate Na(+) channel activity and regulate cell adhesion, migration and neurite outgrowth. β-subunits including β4 are known to be highly concentrated in the nodes of Ranvier and axon initial segments in myelinated axons. Here we show diffuse β4 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 β4 dimer expression in the mouse brain, with high levels of β4 dimers in the striatal projection fascicles, suggesting a specific role of β4 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 β4 in the striatum.
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 i 2؉ 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.
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.Electronic supplementary materialThe online version of this article (doi:10.1007/s13577-015-0125-3) contains supplementary material, which is available to authorized users.
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.
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