Yoshitsugu Uriu

TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade

An essential step in intricate visual processing is the segregation of visual signals into ON and OFF pathways by retinal bipolar cells (BCs). Glutamate released from photoreceptors modulates the photoresponse of ON BCs via metabotropic glutamate receptor 6 (mGluR6) and G protein (Go) that regulates a cation channel. However, the cation channel has not yet been unequivocally identified. Here, we report a mouse TRPM1 long form (TRPM1-L) as the cation channel. We found that TRPM1-L localization is developmentally restricted to the dendritic tips of ON BCs in colocalization with mGluR6. TRPM1 null mutant mice completely lose the photoresponse of ON BCs but not that of OFF BCs. In the TRPM1-L-expressing cells, TRPM1-L functions as a constitutively active nonselective cation channel and its activity is negatively regulated by Go in the mGluR6 cascade. These results demonstrate that TRPM1-L is a component of the ON BC transduction channel downstream of mGluR6 in ON BCs.

RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.

The molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca2+ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca2+ channels (VDCCs), that controls neurotransmitter release in mammalian neurons. RIM1 associated with VDCC β-subunits via its C terminus to markedly suppress voltage-dependent inactivation among different neuronal VDCCs. Consistently, in pheochromocytoma neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length and C-terminal RIM1 constructs, but membrane docking of vesicles was enhanced only by the full-length RIM1. The β construct beta-AID dominant negative, which disrupts the RIM1-β association, accelerated the inactivation of native VDCC currents, suppressed vesicle docking and acetylcholine release in PC12 cells, and inhibited glutamate release in cultured cerebellar neurons. Thus, RIM1 association with β in the presynaptic active zone supports release via two distinct mechanisms: sustaining Ca2+ influx through inhibition of channel inactivation, and anchoring neurotransmitter-containing vesicles in the vicinity of VDCCs.

Rim2α Determines Docking and Priming States in Insulin Granule Exocytosis

Insulin secretion is essential for maintenance of glucose homeostasis, but the mechanism of insulin granule exocytosis, the final step of insulin secretion, is largely unknown. Here, we investigated the role of Rim2alpha in insulin granule exocytosis, including the docking, priming, and fusion steps. We found that interaction of Rim2alpha and Rab3A is required for docking, which is considered a brake on fusion events, and that docking is necessary for K(+)-induced exocytosis, but not for glucose-induced exocytosis. Furthermore, we found that dissociation of the Rim2alpha/Munc13-1 complex by glucose stimulation activates Syntaxin1 by Munc13-1, indicating that Rim2alpha primes insulin granules for fusion. Thus, Rim2alpha determines docking and priming states in insulin granule exocytosis depending on its interacting partner, Rab3A or Munc13-1, respectively. Because Rim2alpha(-/-) mice exhibit impaired secretion of various hormones stored as dense-core granules, including glucose-dependent insulinotropic polypeptide, growth hormone, and epinephrine, Rim2alpha plays a critical role in exocytosis of these dense-core granules.

Rab3-interacting Molecule γ Isoforms Lacking the Rab3-binding Domain Induce Long Lasting Currents but Block Neurotransmitter Vesicle Anchoring in Voltage-dependent P/Q-type Ca2+ Channels

Assembly of voltage-dependent Ca2+ channels (VDCCs) with their associated proteins regulates the coupling of VDCCs with upstream and downstream cellular events. Among the four isoforms of the Rab3-interacting molecule (RIM1 to -4), we have previously reported that VDCC β-subunits physically interact with the long α isoform of the presynaptic active zone scaffolding protein RIM1 (RIM1α) via its C terminus containing the C2B domain. This interaction cooperates with RIM1α-Rab3 interaction to support neurotransmitter exocytosis by anchoring vesicles in the vicinity of VDCCs and by maintaining depolarization-triggered Ca2+ influx as a result of marked inhibition of voltage-dependent inactivation of VDCCs. However, physiological functions have not yet been elucidated for RIM3 and RIM4, which exist only as short γ isoforms (γ-RIMs), carrying the C-terminal C2B domain common to RIMs but not the Rab3-binding region and other structural motifs present in the α-RIMs, including RIM1α. Here, we demonstrate that γ-RIMs also exert prominent suppression of VDCC inactivation via direct binding to β-subunits. In the pheochromocytoma PC12 cells, this common functional feature allows native RIMs to enhance acetylcholine secretion, whereas γ-RIMs are uniquely different from α-RIMs in blocking localization of neurotransmitter-containing vesicles near the plasma membrane. γ-RIMs as well as α-RIMs show wide distribution in central neurons, but knockdown of γ-RIMs attenuated glutamate release to a lesser extent than that of α-RIMs in cultured cerebellar neurons. The results suggest that sustained Ca2+ influx through suppression of VDCC inactivation by RIMs is a ubiquitous property of neurons, whereas the extent of vesicle anchoring to VDCCs at the plasma membrane may depend on the competition of α-RIMs with γ-RIMs for VDCC β-subunits.

Expression of N-type calcium channels in human adrenocortical cells and their contribution to corticosteroid synthesis

The inhibition of aldosterone activity is a useful approach for preventing the progression of cardiovascular and renal diseases in hypertensive patients. Although the results of our previous in vivo study suggested that N-type calcium channels may have a role in regulating plasma aldosterone levels, the direct relationship between N-type calcium channels and aldosterone production in adrenocortical cells has not been examined. In this study, the analysis of quantitative reverse transcription-PCR, western blotting, and immunocytological staining indicated the possible presence of N-type calcium channels in human adrenocortical cells (H295R cell line). Patch clamp analysis indicated that omega-conotoxin GVIA (CnTX), an N-type calcium channel inhibitor, suppressed voltage-dependent barium currents. During steroidogenesis, CnTX significantly reduced the transient calcium signaling induced by angiotensin II (Ang II) and partially prevented Ang II-induced aldosterone and cortisol formation with no significant influence on CYP11B2 and CYP11B1 mRNA expression. In addition, in α1B calcium channel subunits, knockdown significantly decreased Ang II-induced aldosterone formation with increments in CYP11B2 mRNA expression. We also investigated the inhibitory activities of some types of dihydropyridine calcium channel blockers (CCBs; cilnidipine: L-/N-type CCB, efonidipine: L-/T-type CCB, and nifedipine: L-type CCB), and these agents showed a dose-dependent inhibition effect on Ang II-induced aldosterone and cortisol production. Furthermore, only cilnidipine failed to suppress CYP11B1 expression in H295R cells. These results suggest that N-type calcium channels have a significant role in transducing the Ang II signal for aldosterone (and cortisol) biosynthesis, which may explain the mechanism by which N-type calcium channels regulate plasma aldosterone levels.

Mutation associated with an autosomal dominant cone-rod dystrophy CORD7 modifies RIM1-mediated modulation of voltage-dependent Ca2+ channels

Genetic analyses have revealed an association between the gene encoding the Rab3A‑interacting molecule (RIM1) and the autosomal dominant cone‑rod dystrophy CORD7. However, the pathogenesis of CORD7 remains unclear. We recently revealed that RIM1 regulates voltage‑dependent Ca2+ channel (VDCC) currents and anchors neurotransmitter‑containing vesicles to VDCCs, thereby controlling neurotransmitter release. We demonstrate here that the mouse RIM1 arginine‑to‑histidine substitution (R655H), which corresponds to the human CORD7 mutation, modifies RIM1 function in regulating VDCC currents elicited by the P/Q‑type Cav2.1 and L‑type Cav1.4 channels. Thus, our data can raise an interesting possibility that CORD7 phenotypes including retinal deficits and enhanced cognition are at least partly due to altered regulation of presynaptic VDCC currents. Addendum to: RIM1 Confers Sustained Activity and Neurotransmitter Vesicle Anchoring to Presynaptic Ca2+ Channels S. Kiyonaka, M. Wakamori, T. Miki, Y. Uriu, M. Nonaka, H. Bito, A.M. Beedle, E. Mori, Y. Hara, M. De Waard, M. Kanagawa, M. Itakura, M. Takahashi, K.P. Campbell, and Y. Mori Nat Neurosci 2007; 10:691-701

Active zone protein RIM1 functionally associates with presynaptic VDCCs

To investigate the physiological roles of glutamate receptors (GluRs) in the mesencephalic trigeminal neurons, the current responses to puff application of glutamate (Glu) were recorded under the whole-cell voltage-clamp condition. When the peak amplitudes were plotted against the holding potentials, an N-shaped I–V relationship was obtained. At <−40 mV, the biphasic responses consisting of inward and outward components were observed. Glu-induced outward component was sensitive to ZD7288 and Cs+, indicating an involvement of h-current (Ih) in generating the outward current. Provided that GluR channels shared the microdomain with h-channels, it is possible that a transient increase in [Na]i in the microdomain by activation of GluR channels would cause a transient reduction of Ih due to the negative shift of its reversal potential, leading to a generation of the outward component of the biphasic response where the N-shaped I–V relationship can be seen. Thus, the biphasic response was seen at <−40 mV.

Functional impact of RIM family sharing C2B domain on gating of voltage-dependent Ca2+ channels

To investigate the physiological roles of glutamate receptors (GluRs) in the mesencephalic trigeminal neurons, the current responses to puff application of glutamate (Glu) were recorded under the whole-cell voltage-clamp condition. When the peak amplitudes were plotted against the holding potentials, an N-shaped I–V relationship was obtained. At <−40 mV, the biphasic responses consisting of inward and outward components were observed. Glu-induced outward component was sensitive to ZD7288 and Cs+, indicating an involvement of h-current (Ih) in generating the outward current. Provided that GluR channels shared the microdomain with h-channels, it is possible that a transient increase in [Na]i in the microdomain by activation of GluR channels would cause a transient reduction of Ih due to the negative shift of its reversal potential, leading to a generation of the outward component of the biphasic response where the N-shaped I–V relationship can be seen. Thus, the biphasic response was seen at <−40 mV.