R. Lane Brown

Ciliary Targeting of Olfactory CNG Channels Requires the CNGB1b Subunit and the Kinesin-2 Motor Protein, KIF17

Nonmotile cilia on olfactory sensory neurons (OSNs) compartmentalize signaling molecules, including odorant receptors and cyclic nucleotide-gated (CNG) channels, allowing for efficient, spatially confined responses to sensory stimuli . Little is known about the mechanisms of the ciliary targeting of olfactory CNG channels, composed of three subunits: CNGA2, CNGA4, and CNGB1b . Recent reports suggest that subunit composition of the retinal CNG channel influences localization, leading to disease . However, the mechanistic role of subunits in properly targeting native olfactory CNG channels remains unclear. Here, we show that heteromeric assembly with CNGB1b, containing a critical carboxy-terminal motif (RVxP), is required for ciliary trafficking of olfactory CNG channels. Movement of proteins within the cilia is governed by intraflagellar transport (IFT), a process that facilitates bidirectional movement of cargo along microtubules. Work in C. elegans has established that heterotrimeric and homodimeric kinesin-2 family members play a critical role in anterograde transport . In mammalian systems, the heterotrimeric KIF3a/KIF3b/KAP-3 complex plays a clear role in IFT; however, no role has been established for KIF17, the mammalian homolog of OSM-3 . Here, we demonstrate that KIF17 is required for olfactory CNG channel targeting, providing novel insights into mechanisms of mammalian ciliary transport.

TRPM1 is required for the depolarizing light response in retinal ON-bipolar cells

The ON pathway of the visual system, which detects increases in light intensity, is established at the first retinal synapse between photoreceptors and ON-bipolar cells. Photoreceptors hyperpolarize in response to light and reduce the rate of glutamate release, which in turn causes the depolarization of ON-bipolar cells. This ON-bipolar cell response is mediated by the metabotropic glutamate receptor, mGluR6, which controls the activity of a depolarizing current. Despite intensive research over the past two decades, the molecular identity of the channel that generates this depolarizing current has remained elusive. Here, we present evidence indicating that TRPM1 is necessary for the depolarizing light response of ON-bipolar cells, and further that TRPM1 is a component of the channel that generates this light response. Gene expression profiling revealed that TRPM1 is highly enriched in ON-bipolar cells. In situ hybridization experiments confirmed that TRPM1 mRNA is found in cells of the retinal inner nuclear layer, and immunofluorescent confocal microscopy showed that TRPM1 is localized in the dendrites of ON-bipolar cells in both mouse and macaque retina. The electroretinogram (ERG) of TRPM1-deficient (TRPM1−/−) mice had a normal a-wave, but no b-wave, indicating a loss of bipolar cell response. Finally, whole-cell patch-clamp recording from ON-bipolar cells in mouse retinal slices demonstrated that genetic deletion of TRPM1 abolished chemically simulated light responses from rod bipolar cells and dramatically altered the responses of cone ON-bipolar cells. Identification of TRPM1 as a mGluR6-coupled cation channel reveals a key step in vision, expands the role of the TRP channel family in sensory perception, and presents insights into the evolution of vertebrate vision.

Intrinsic light responses of retinal ganglion cells projecting to the circadian system

In mammals, light entrainment of the circadian clock, located in the suprachiasmatic nuclei (SCN), requires retinal input. Traditional rod and cone photoreceptors, however, are not required. Instead, the SCN‐projecting retinal ganglion cells (RGCs) function as autonomous photoreceptors and exhibit light responses independent of rod‐ and cone‐driven input. Using whole‐cell patch‐clamp recording techniques, we have investigated the morphological and electrophysiological properties of this unique class of RGCs. Although SCN‐projecting RGCs resemble Type III cells in form, they display strikingly different physiological properties from these neurons. First, in response to the injection of a sustained depolarizing current, SCN‐projecting cells fired in a transient fashion, in contrast to most RGCs which fired robust trains of action potentials. Second, in response to light, SCN‐projecting RGCs exhibited an intensity‐dependent transient depolarization in the absence of rod and cone input. This depolarization reached a peak within 5 s and generated increased spiking activity before decaying to a plateau. Voltage‐clamp recordings were used to characterize the light‐activated conductance which generated this depolarization. In response to varying light intensities, SCN‐projecting RGCs exhibited a graded transient inward current which peaked within 5 s and decayed to a plateau. The voltage dependence of the light‐activated current was obtained by subtracting currents elicited by a voltage ramp before and during illumination. The light‐activated current displayed both inward and outward rectification and was largely unaffected by substitution of extracellular Na+ with choline. In both respects, the intrinsic light‐activated current observed in SCN‐projecting RGCs resembles currents carried by ion channels of the transient receptor potential (trp) family, which are known to mediate the light response of invertebrate photoreceptors.

The light-activated signaling pathway in SCN-projecting rat retinal ganglion cells

In mammals, the master circadian clock resides in the suprachiasmatic nuclei (SCN) of the hypothalamus. The period and phase of the circadian pacemaker are calibrated by direct photic input from retinal ganglion cells (RGCs). SCN‐projecting RGCs respond to light in the absence of rod‐ and cone‐driven synaptic input, a property for which they are termed intrinsically photosensitive. In SCN‐projecting RGCs, light activates a nonselective cationic current that displays inward and outward rectification. The goal of the present study was to investigate the identity of the light‐activated ion channel and the intracellular signaling pathway leading to its activation. We considered two candidate channels, cyclic nucleotide‐gated (CNG) channels and transient receptor potential (TRP) channels, which mediate vertebrate and invertebrate phototransduction, respectively. We report that the intrinsic light response relies upon a G‐protein‐dependent process. Although our data indicate that cyclic nucleotides modulate the signaling pathway, CNG channels do not appear to conduct the light‐activated current because (i) cyclic nucleotides in the pipette solution do not activate a conductance or completely block the light response, (ii) CNG channel blockers fail to inhibit the light response, (iii) the effects of internal and external divalent cations are inconsistent with their effects on CNG channels, and (iv) immunohistochemistry reveals no CNG channels in SCN‐projecting RGCs. Finally, we show that the pharmacology of the light‐activated channel resembles that of some TRPC channel family members; the response is blocked by lanthanides and ruthenium red and SK&F 96365, and is enhanced by flufenamic acid and 1‐oleoyl‐2‐acetyl‐sn‐glycerol. Furthermore, immunohistochemical experiments reveal that TRPC6 is expressed in many RGCs, including those that express melanopsin.

Photochemistry of retinal chromophore in mouse melanopsin

In mammals, melanopsin is exclusively expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs), which play an important role in circadian photoentrainment and other nonimage-forming functions. These ipRGCs reside in the inner retina, far removed from the pigment epithelium, which synthesizes the 11-cis retinal chromophore used by rod and cone photoreceptors to regenerate opsin for light detection. There has been considerable interest in the identification of the melanopsin chromophore and in understanding the process of photopigment regeneration in photoreceptors that are not in proximity to the classical visual cycle. We have devised an immuno-magnetic purification protocol that allows melanopsin-expressing retinal ganglion cells to be isolated and collected from multiple mouse retinas. Using this technique, we have demonstrated that native melanopsin in vivo exclusively binds 11-cis retinal in the dark and that illumination causes isomerization to the all-trans isoform. Furthermore, spectral analysis of the melanopsin photoproduct shows the formation of a protonated metarhodopsin with a maximum absorbance between 520 and 540 nm. These results indicate that even if melanopsin functions as a bistable photopigment with photo-regenerative activity native melanopsin must also use some other light-independent retinoid regeneration mechanism to return to the dark state, where all of the retinal is observed to be in the 11-cis form.

Synaptic inputs to retinal ganglion cells that set the circadian clock.

Melanopsin‐containing retinal ganglion cells (RGCs) project to the suprachiasmatic nuclei (SCN) and mediate photoentrainment of the circadian system. Melanopsin is a novel retinal‐based photopigment that renders these cells intrinsically photosensitive (ip). Although genetic ablation of melanopsin abolishes the intrinsic light response, it has a surprisingly minor effect on circadian photoentrainment. This and other non‐visual responses to light are lost only when the melanopsin deficiency is coupled with mutations that disable classical rod and cone photoreceptors, suggesting that melanopsin‐containing RGCs also receive rod‐ and cone‐driven synaptic inputs. Using whole‐cell patch‐clamp recording, we demonstrate that light triggers synaptic currents in ipRGCs via activation of ionotropic glutamate and γ‐aminobutyric acid (GABA) receptors. Miniature postsynaptic currents (mPSCs) were clearly observed in ipRGCs, although they were less robust and were seen less frequently than those seen in non‐ip cells. Pharmacological treatments revealed that the majority of ipRGCs receive excitatory glutamatergic inputs that were blocked by DNQX and/or kynurenic acid, as well as inhibitory GABAergic inputs that were blocked by bicuculline. Other ipRGCs received either glutamatergic or GABAergic inputs nearly exclusively. Although strychnine (Strych)‐sensitive mPSCs were evident on many non‐ipRGCs, indicating the presence of glycinergic inputs, we saw no evidence of Strych‐sensitive events in ipRGCs. Based on these results, it is clear that SCN‐projecting RGCs can respond to light both via an intrinsic melanopsin‐based signaling cascade and via a synaptic pathway driven by classical rod and/or cone photoreceptors. It remains to be determined how the ipRGCs integrate these temporally distinct inputs to generate the signals that mediate circadian photoentrainment and other non‐visual responses to light.

Interplay between PIP3 and calmodulin regulation of olfactory cyclic nucleotide-gated channels

Phosphatidylinositol-3,4,5-trisphosphate (PIP3) has been proposed to modulate the odorant sensitivity of olfactory sensory neurons by inhibiting activation of cyclic nucleotide-gated (CNG) channels in the cilia. When applied to the intracellular face of excised patches, PIP3 has been shown to inhibit activation of heteromeric olfactory CNG channels, composed of CNGA2, CNGA4, and CNGB1b subunits, and homomeric CNGA2 channels. In contrast, we discovered that channels formed by CNGA3 subunits from cone photoreceptors were unaffected by PIP3. Using chimeric channels and a deletion mutant, we determined that residues 61–90 within the N terminus of CNGA2 are necessary for PIP3 regulation, and a biochemical “pulldown” assay suggests that PIP3 directly binds this region. The N terminus of CNGA2 contains a previously identified calcium–calmodulin (Ca2+/CaM)-binding domain (residues 68–81) that mediates Ca2+/CaM inhibition of homomeric CNGA2 channels but is functionally silent in heteromeric channels. We discovered, however, that this region is required for PIP3 regulation of both homomeric and heteromeric channels. Furthermore, PIP3 occluded the action of Ca2+/CaM on both homomeric and heteromeric channels, in part by blocking Ca2+/CaM binding. Our results establish the importance of the CNGA2 N terminus for PIP3 inhibition of olfactory CNG channels and suggest that PIP3 inhibits channel activation by disrupting an autoexcitatory interaction between the N and C termini of adjacent subunits. By dramatically suppressing channel currents, PIP3 may generate a shift in odorant sensitivity that does not require prior channel activity.

Functional regions of the inhibitory subunit of retinal rod cGMP phosphodiesterase identified by site-specific mutagenesis and fluorescence spectroscopy

In the dark, the activity of the cGMP phosphodiesterase (PDE) of retinal rod outer segments is held in check by its two inhibitory gamma subunits. Following illumination, gamma is rapidly removed from its inhibitory site by transducin, the G-protein of the visual system. In order to probe the functional roles of specific regions in the PDE gamma primary sequence, 10 variants of PDE gamma have been produced by site-specific mutagenesis and expression in bacteria and their properties compared to those of protein containing the wild-type bovine PDE gamma amino acid sequence. Three questions were asked about each mutant: What is its affinity for the alpha beta catalytic subunit of PDE? Does it inhibit catalytic activity? If so, can transducin relieve this inhibition? Binding to PDE alpha beta was determined directly using fluorescein-labeled gamma by measuring the increase in emission anisotropy that occurs when gamma binds to alpha beta. Inhibition of PDE alpha beta was measured by reconstitution of the gamma variants with gamma-free PDE generated by limited digestion with trypsin or endoproteinase Arg-C. Unlike trypsin, the latter enzyme did not remove PDEs ability to bind membranes and be activated by transducin, so that transducin activation of PDE containing specific gamma variants could be assayed directly. The results indicate that mutations in many regions of gamma affect its binding to alpha beta. A mutant missing the last five carboxy-terminal residues (83-87) was totally lacking in inhibitory activity. However, it still bound to PDE alpha beta tightly, although with a 100-fold lower dissociation constant (approximately 5 nM) than that of wild-type gamma (approximately 50 pM).(ABSTRACT TRUNCATED AT 250 WORDS)

Gβ5–RGs complexes co-localize with mGluR6 in retinal ON-bipolar cells

The time course of G‐protein‐coupled responses is largely determined by the kinetics of GTP hydrolysis by the G protein α subunit, which is accelerated by interaction with regulator of G‐protein signaling (RGS) proteins. Light responses of ON‐bipolar cells of the vertebrate retina require rapid inactivation of the G protein Gαo, which is activated in the dark by metabotropic glutamate receptor, mGluR6, in their dendritic tips. It is not yet known, however, which RGS protein(s) might be responsible for rapid inactivation kinetics. By immunofluorescence and co‐immunoprecipitation, we have identified complexes of the Gαo‐selective RGS proteins RGS7 and RGS11, with their obligate binding partner, Gβ5, that are localized to the dendritic tips of murine rod and cone ON‐bipolar cells, along with mGluR6. Experiments using pre‐ and post‐synaptic markers, and a dissociated bipolar cell preparation, clearly identified the location of these complexes as the ON‐bipolar cell dendritic tips and not the adjacent photoreceptor terminals or horizontal cell dendrites. In mice lacking mGluR6, the distribution of RGS11, RGS7 and Gβ5 shifts away from the dendritic tips, implying a functional relationship with mGluR6. The precise co‐localization of Gβ5–RGS7 and Gβ5–RGS11 with mGluR6, and the dependence of localization on the presence of mGluR6, suggests that Gβ5–RGS7 and Gβ5–RGS11 function specifically in the mGluR6 signal transduction pathway, where they may stimulate the GTPase activity of Gαo, thus accelerating the ON‐bipolar cell light response, in a manner analogous to the acceleration of photoreceptor light responses by the Gβ5–RGS9‐1 complex.

Movement of gating machinery during the activation of rod cyclic nucleotide-gated channels.

In the visual and olfactory systems, cyclic nucleotide-gated (CNG) ion channels convert stimulus-induced changes in the internal concentrations of cGMP and cAMP into changes in membrane potential. Although it is known that significant activation of these channels requires the binding of three or more molecules of ligand, the detailed molecular mechanism remains obscure. We have probed the structural changes that occur during channel activation by using sulfhydryl-reactive methanethiosulfonate (MTS) reagents and N-ethylmaleimide (NEM). When expressed in Xenopus oocytes, the alpha-subunit of the bovine retinal channel forms homomultimeric channels that are activated by cGMP with a K1/2 of approximately 100 microM. Cyclic AMP, on the other hand, is a very poor activator; a saturating concentration elicits only 1% of the maximum current produced by cGMP. Treatment of excised patches with MTS-ethyltrimethylamine (MTSET) or NEM dramatically potentiated the channels response to both cyclic nucleotides. After MTSET treatment, the dose-response relation for cGMP was shifted by over two orders of magnitude to lower concentrations. The effect on channel activation by cAMP was even more striking. After modification, the channels were fully activated by cAMP with a K1/2 of approximately 60 microM. This potentiation was abolished by conversion of Cys481 to a nonreactive alanine residue. Potentiation occurred more rapidly in the presence of saturating cGMP, indicating that this region of the channel is more accessible when the channel is open. Cys481 is located in a linker region between the transmembrane and cGMP-binding domains of the channel. These results suggest that this region of the channel undergoes significant movement during the activation process and is critical for coupling ligand binding to pore opening. Potentiation, however, is not mediated by the recently reported interaction between the amino- and carboxy-terminal regions of the alpha-subunit. Deletion of the entire amino-terminal domain had little effect on potentiation by MTSET.