Coupling Neuropeptide Levels to Structural Plasticity in Drosophila Clock Neurons

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Salt bridges and gating in the COOH-terminal region of HCN2 and CNGA1 channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels and cyclic nucleotide-gated (CNG) channels are activated by the direct binding of cyclic nucleotides. The intracellular COOH-terminal regions exhibit high sequence similarity in all HCN and CNG channels. This region contains the cyclic nucleotide-binding domain (CNBD) and the C-linker region, which connects the CNBD to the pore. Recently, the structure of the HCN2 COOH-terminal region was solved and shown to contain intersubunit interactions between C-linker regions. To explore the role of these intersubunit interactions in intact channels, we studied two salt bridges in the C-linker region: an intersubunit interaction between C-linkers of neighboring subunits, and an intrasubunit interaction between the C-linker and its CNBD. We show that breaking these salt bridges in both HCN2 and CNGA1 channels through mutation causes an increase in the favorability of channel opening. The wild-type behavior of both HCN2 and CNGA1 channels is rescued by switching the position of the positive and negative residues, thus restoring the salt bridges. These results suggest that the salt bridges seen in the HCN2 COOH-terminal crystal structure are also present in the intact HCN2 channel. Furthermore, the similar effects of the mutations on HCN2 and CNGA1 channels suggest that these salt bridge interactions are also present in the intact CNGA1 channel. As disrupting the interactions leads to channels with more favorable opening transitions, the salt bridges appear to stabilize a closed conformation in both the HCN2 and CNGA1 channels. These results suggest that the HCN2 COOH-terminal crystal structure contains the C-linker regions in the resting configuration even though the CNBD is ligand bound, and channel opening involves a rearrangement of the C-linkers and, thus, disruption of the salt bridges. Discovering that one portion of the COOH terminus, the CNBD, can be in the activated configuration while the other portion, the C-linker, is not activated has lead us to suggest a novel modular gating scheme for HCN and CNG channels.     

Conformational rearrangements in the S6 domain and C-linker during gating in CNGA1 channels

This work completes previous findings and, using cysteine scanning mutagenesis (CSM) and biochemical methods, provides detailed analysis of conformational changes of the S6 domain and C-linker during gating of CNGA1 channels. Specific residues between Phe375 and Val424 were mutated to a cysteine in the CNGA1 and CNGA1_cys-free background and the effect of intracellular Cd²⁺ or cross-linkers of different length in the open and closed state was studied. In the closed state, Cd²⁺ ions inhibited mutant channels A406C and Q409C and the longer cross-linker reagent M-4-M inhibited mutant channels A406C_cys-free and Q409C_cys-free. Cd²⁺ ions inhibited mutant channels D413C and Y418C in the open state, both constructed in a CNGA1 and CNGA1_cys-free background. Our results suggest that, in the closed state, residues from Phe375 to approximately Ala406 form a helical bundle with a three-dimensional (3D) structure similar to those of the KcsA; furthermore, in the open state, residues from Ser399 to Gln409 in homologous subunits move far apart, as expected from the gating in K⁺ channels; in contrast, residues from Asp413 to Tyr418 in homologous subunits become closer in the open state. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evaluation of the validity of a maximum likelihood adaptive staircase procedure for measurement of olfactory detection threshold in mice

Threshold is defined as the stimulus intensity necessary for a subject to reach a specified percent correct on a detection test. MLPEST (maximum likelihood parameter estimation by sequential testing) is a method that is able to determine threshold accurately and more rapidly than many other methods. Originally developed for human auditory and visual tasks, it has been adapted for human olfactory and gustatory tests. In order to utilize this technique for olfactory testing in mice, we have adapted MLPEST methodology for use with computerized olfactometry as a tool to estimate odor detection thresholds. Here we present Monte Carlo simulations and operant conditioning data that demonstrate the potential utility of this technique in mice, we explore the ramifications of altering MLPEST test parameters on performance, and we discuss the advantages and disadvantages of using MLPEST compared to other methods for the estimation of thresholds in rodents. Using MLPEST, we find that olfactory detection thresholds in mice deficient for the cyclic nucleotide-gated channel subunit A2 are similar to those of wild-type animals for odorants the knockout animals are able to detect.     

shRNA knockdown of guanylate cyclase 2e or cyclic nucleotide gated channel alpha 1 increases photoreceptor survival in a cGMP phosphodiesterase mouse model of retinitis pigmentosa

In vertebrate rods, dark and light conditions produce changes in guanosine 3′,5′‐cyclic monophosphate (cGMP) and calcium (Ca²⁺) levels, which are regulated by the opposing function of several proteins. During the recovery of a bright flash, guanylate cyclase (GUCY) helps raise cGMP to levels that open cGMP‐gated calcium sodium channels (CNG) to increase Na⁺ and Ca²⁺ influx in the outer segment. In contrast, light activates cGMP phosphodiesterase 6 (PDE6) causing rapid hydrolysis of cGMP, CNG closure, and reduced Na⁺ and Ca²⁺ levels. In Pde6b mouse models of retinitis pigmentosa (RP), photoreceptor death is preceded by abnormally high cGMP and Ca²⁺ levels, likely because of continued synthesis of cGMP by guanylate cyclases and unregulated influx of Ca²⁺ to toxic levels through CNG channels. To reverse the effects of Pde6b loss of function, we employed an shRNA knockdown approach to reduce the expression of Gucy2e or Cnga1 in Pde6b^H620Q photoreceptors prior to degeneration. Gucy2e‐ or Cnga1‐shRNA lentiviral‐mediated knockdown GUCY2E and CNGA1 expression increase visual function and photoreceptor survival in Pde6b^H620Q mice. We demonstrated that effective knockdown of GUCY2E and CNGA1 expression to counteract loss of PDE6 function may develop into a valuable approach for treating some patients with RP.     

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade.     

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade.     

Mutations in olfactory signal transduction genes are not a major cause of human congenital general anosmia

Anosmia affects the western world population, mostly the elderly, reaching to 5% in subjects over the age of 45 years and strongly lowering their quality of life. A smaller minority (about 0.01%) is born without a sense of smell, afflicted with congenital general anosmia (CGA). No causative genes for human CGA have been identified yet, except for some syndromic cases such as Kallman syndrome. In mice, however, deletion of any of the 3 main olfactory transduction components (guanidine triphosphate binding protein, adenylyl cyclase, and the cyclic adenosine monophosphate-gated channel) causes profound reduction of physiological responses to odorants. In an attempt to identify human CGA-related mutations, we performed whole-genome linkage analysis in affected families, but no significant linkage signals were observed, probably due to the small size of families analyzed. We further carried out direct mutation screening in the 3 main olfactory transduction genes in 64 unrelated anosmic individuals. No potentially causative mutations were identified, indicating that transduction gene variations underlie human CGA rarely and that mutations in other genes have to be identified. The screened genes were found to be under purifying selection, suggesting that they play a crucial functional role not only in olfaction but also potentially in additional pathways.