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.egregation of visual signals into ON and OFF pathways originates in BCs, the second-order neurons in the retina (1, 2). ON and OFF BCs express metabotropic glutamate receptors, mGluR6, and ionotropic glutamate receptors (iGluRs), respectively, on their dendrites (3-5). Reduction of glutamate released from photoreceptors by light stimulation depolarizes ON BCs and hyperpolarizes OFF BCs (6-8) mediated through respective glutamate receptors. The mGluR6 couples to a heterotrimeric G protein complex, Go (9, 10). Signals require Goα, which ultimately closes a downstream nonselective cation channel in ON BCs (6, 9, 11-13). However, this transduction cation channel in ON BCs has not been identified, despite intensive investigation.In our screen to identify functionally important molecules in the retina, we found that TRPM1 is predominantly expressed in retinal BCs. Most members of the TRP superfamily, which are found in a variety of sense organs, are non-voltage-gated cation channels (14-16). The founding member of the TRP family was discovered as a key component of the light response in Drosophila photoreceptors (17). TRPM1, also known as melastatin, was the first member of the melanoma-related transient receptor potential (TRPM) subfamily to be discovered (18,19). TRPM1 is alternatively spliced, resulting in the production of a long form (TRPM1-L) and a short N-terminal form devoid of transmembrane segments (TRPM1-S) (18,20). Although mouse TRPM1-S was previously identified as melastatin, mouse TRPM1-L has not been identified (18). The distinct physiological and biological functions of TRPM1 still remain elusive, although some recent evidences including us suggested that TRPM1 might contribute to retinal BC function (21-23). Here, we show that TRPM1-L is the transduction cation channel of retinal ON BCs in the downstream of mGluR6 cascade.

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 beta-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 beta construct beta-AID dominant negative, which disrupts the RIM1-beta 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 beta 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.

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.

Assembly of voltage-dependent Ca