Spatial Distribution of Calcium-Gated Chloride Channels in Olfactory Cilia

Background: In vertebrate olfactory receptor neurons, sensory cilia transduce odor stimuli into changes in neuronal membrane potential. The voltage changes are primarily caused by the sequential openings of two types of channel: a cyclic-nucleotide-gated (CNG) cationic channel and a calcium-gated chloride channel. In frog, the cilia are 25 to 200 mm in length, so the spatial distributions of the channels may be an important determinant of odor sensitivity. Principal Findings: To determine the spatial distribution of the chloride channels, we recorded from single cilia as calcium was allowed to diffuse down the length of the cilium and activate the channels. A computational model of this experiment allowed an estimate of the spatial distribution of the chloride channels. On average, the channels were concentrated in a narrow band centered at a distance of 29 % of the ciliary length, measured from the base of the cilium. This matches the location of the CNG channels determined previously. This non-uniform distribution of transduction proteins is consistent with similar findings in other cilia. Conclusions: On average, the two types of olfactory transduction channel are concentrated in the same region of the cilium

Limits of Calcium Clearance by Plasma Membrane Calcium ATPase in Olfactory Cilia

BACKGROUND: In any fine sensory organelle, a small influx of Ca(2+) can quickly elevate cytoplasmic Ca(2+). Mechanisms must exist to clear the ciliary Ca(2+) before it reaches toxic levels. One such organelle has been well studied: the vertebrate olfactory cilium. Recent studies have suggested that clearance from the olfactory cilium is mediated in part by plasma membrane Ca(2+)-ATPase (PMCA). PRINCIPAL FINDINGS: In the present study, electrophysiological assays were devised to monitor cytoplasmic free Ca(2+) in single frog olfactory cilia. Ca(2+) was allowed to enter isolated cilia, either through the detached end or through membrane channels. Intraciliary Ca(2+) was monitored via the activity of ciliary Ca(2+)-gated Cl(-) channels, which are sensitive to free Ca(2+) from about 2 to 10 microM. No significant effect of MgATP on intraciliary free Ca(2+) could be found. Carboxyeosin, which has been used to inhibit PMCA, was found to substantially increase a ciliary transduction current activated by cyclic AMP. This increase was ATP-independent. CONCLUSIONS: Alternative explanations are suggested for two previous experiments taken to support a role for PMCA in ciliary Ca(2+) clearance. It is concluded that PMCA in the cilium plays a very limited role in clearing the micromolar levels of intraciliary Ca(2+) produced during the odor response

A Selective PMCA Inhibitor Does Not Prolong the Electroolfactogram in Mouse

Within the cilia of vertebrate olfactory receptor neurons, Ca(2+) accumulates during odor transduction. Termination of the odor response requires removal of this Ca(2+), and prior evidence suggests that both Na(+)/Ca(2+) exchange and plasma membrane Ca(2+)-ATPase (PMCA) contribute to this removal.In intact mouse olfactory epithelium, we measured the time course of termination of the odor-induced field potential. Replacement of mucosal Na(+) with Li(+), which reduces the ability of Na(+)/Ca(2+) exchange to expel Ca(2+), prolonged the termination as expected. However, treating the epithelium with the specific PMCA inhibitor caloxin 1b1 caused no significant increase in the time course of response termination.Under these experimental conditions, PMCA does not contribute detectably to the termination of the odor response

The Na(+)/Ca(2+) exchanger NCKX4 governs termination and adaptation of the mammalian olfactory response

Sensory perception requires accurate encoding of stimulus information by sensory receptor cells. We identified NCKX4, a potassium-dependent Na(+)/Ca(2+) exchanger, as being necessary for rapid response termination and proper adaptation of vertebrate olfactory sensory neurons (OSNs). Nckx4(-/-) (also known as Slc24a4) mouse OSNs displayed substantially prolonged responses and stronger adaptation. Single-cell electrophysiological analyses revealed that the majority of Na(+)-dependent Ca(2+) exchange in OSNs relevant to sensory transduction is a result of NCKX4 and that Nckx4(-/-) mouse OSNs are deficient in encoding action potentials on repeated stimulation. Olfactory-specific Nckx4(-/-) mice had lower body weights and a reduced ability to locate an odorous source. These results establish the role of NCKX4 in shaping olfactory responses and suggest that rapid response termination and proper adaptation of peripheral sensory receptor cells tune the sensory system for optimal perception

Ca2+ Extrusion by NCX Is Compromised in Olfactory Sensory Neurons of OMP−/− Mice

The role of olfactory marker protein (OMP), a hallmark of mature olfactory sensory neurons (OSNs), has been poorly understood since its discovery. The electrophysiological and behavioral phenotypes of OMP knockout mice indicated that OMP influences olfactory signal transduction. However, the mechanism by which this occurs remained unknown.We used intact olfactory epithelium obtained from WT and OMP(-/-) mice to monitor the Ca(2+) dynamics induced by the activation of cyclic nucleotide-gated channels, voltage-operated Ca(2+) channels, or Ca(2+) stores in single dendritic knobs of OSNs. Our data suggested that OMP could act to modulate the Ca(2+)-homeostasis in these neurons by influencing the activity of the plasma membrane Na(+)/Ca(2+)-exchanger (NCX). Immunohistochemistry verifies colocalization of NCX1 and OMP in the cilia and knobs of OSNs. To test the role of NCX activity, we compared the kinetics of Ca(2+) elevation by stimulating the reverse mode of NCX in both WT and OMP(-/-) mice. The resulting Ca(2+) responses indicate that OMP facilitates NCX activity and allows rapid Ca(2+) extrusion from OSN knobs. To address the mechanism by which OMP influences NCX activity in OSNs we studied protein-peptide interactions in real-time using surface plasmon resonance technology. We demonstrate the direct interaction of the XIP regulatory-peptide of NCX with calmodulin (CaM).Since CaM also binds to the Bex protein, an interacting protein partner of OMP, these observations strongly suggest that OMP can influence CaM efficacy and thus alters NCX activity by a series of protein-protein interactions

Computational Model of the Insect Pheromone Transduction Cascade

A biophysical model of receptor potential generation in the male moth olfactory receptor neuron is presented. It takes into account all pre-effector processes—the translocation of pheromone molecules from air to sensillum lymph, their deactivation and interaction with the receptors, and the G-protein and effector enzyme activation—and focuses on the main post-effector processes. These processes involve the production and degradation of second messengers (IP3 and DAG), the opening and closing of a series of ionic channels (IP3-gated Ca2+ channel, DAG-gated cationic channel, Ca2+-gated Cl− channel, and Ca2+- and voltage-gated K+ channel), and Ca2+ extrusion mechanisms. The whole network is regulated by modulators (protein kinase C and Ca2+-calmodulin) that exert feedback inhibition on the effector and channels. The evolution in time of these linked chemical species and currents and the resulting membrane potentials in response to single pulse stimulation of various intensities were simulated. The unknown parameter values were fitted by comparison to the amplitude and temporal characteristics (rising and falling times) of the experimentally measured receptor potential at various pheromone doses. The model obtained captures the main features of the dose–response curves: the wide dynamic range of six decades with the same amplitudes as the experimental data, the short rising time, and the long falling time. It also reproduces the second messenger kinetics. It suggests that the two main types of depolarizing ionic channels play different roles at low and high pheromone concentrations; the DAG-gated cationic channel plays the major role for depolarization at low concentrations, and the Ca2+-gated Cl− channel plays the major role for depolarization at middle and high concentrations. Several testable predictions are proposed, and future developments are discussed

Regulation of Bestrophins by Ca2+: A Theoretical and Experimental Study

Bestrophins are a recently discovered family of Cl− channels, for which no structural information is available. Some family members are activated by increased intracellular Ca2+ concentration. Bestrophins feature a well conserved Asp-rich tract in their COOH terminus (Asp-rich domain), which is homologous to Ca2+-binding motifs in human thrombospondins and in human big-conductance Ca2+- and voltage-gated K+ channels (BKCa). Consequently, the Asp-rich domain is also a candidate for Ca2+ binding in bestrophins. Based on these considerations, we constructed homology models of human bestrophin-1 (Best1) Asp-rich domain using human thrombospondin-1 X-ray structure as a template. Molecular dynamics simulations were used to identify Asp and Glu residues binding Ca2+ and to predict the effects of their mutations to alanine. We then proceeded to test selected mutations in the Asp-rich domain of the highly homologous mouse bestrophin-2. The mutants expressed in HEK-293 cells were investigated by electrophysiological experiments using the whole-cell voltage-clamp technique. Based on our molecular modeling results, we predicted that Asp-rich domain has two defined binding sites and that D301A and D304A mutations may impact the binding of the metal ions. The experiments confirmed that these mutations do actually affect the function of the protein causing a large decrease in the Ca2+-activated Cl− current, fully consistent with our predictions. In addition, other studied mutations (E306A, D312A) did not decrease Ca2+-activated Cl− current in agreement with modeling results

Functional Evidence of Multidrug Resistance Transporters (MDR) in Rodent Olfactory Epithelium

Background: P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP1) are membrane transporter proteins which function as efflux pumps at cell membranes and are considered to exert a protective function against the entry of xenobiotics. While evidence for Pgp and MRP transporter activity is reported for olfactory tissue, their possible interaction and participation in the olfactory response has not been investigated. Principal Findings: Functional activity of putative MDR transporters was assessed by means of the fluorometric calcein acetoxymethyl ester (calcein-AM) accumulation assay on acute rat and mouse olfactory tissue slices. Calcein-AM uptake was measured as fluorescence intensity changes in the presence of Pgp or MRP specific inhibitors. Epifluorescence microscopy measured time course analysis in the olfactory epithelium revealed significant inhibitor-dependent calcein uptake in the presence of each of the selected inhibitors. Furthermore, intracellular calcein accumulation in olfactory receptor neurons was also significantly increased in the presence of either one of the Pgp or MRP inhibitors. The presence of Pgp or MRP1 encoding genes in the olfactory mucosa of rat and mouse was confirmed by RT-PCR with appropriate pairs of speciesspecific primers. Both transporters were expressed in both newborn and adult olfactory mucosa of both species. To assess a possible involvement of MDR transporters in the olfactory response, we examined the electrophysiological response to odorants in the presence of the selected MDR inhibitors by recording electroolfactograms (EOG). In both animal species

Regulation of Bestrophins by Ca2+: A Theoretical and Experimental Study

Bestrophins are a recently discovered family of Cl− channels, for which no structural information is available. Some family members are activated by increased intracellular Ca2+ concentration. Bestrophins feature a well conserved Asp-rich tract in their COOH terminus (Asp-rich domain), which is homologous to Ca2+-binding motifs in human thrombospondins and in human big-conductance Ca2+- and voltage-gated K+ channels (BKCa). Consequently, the Asp-rich domain is also a candidate for Ca2+ binding in bestrophins. Based on these considerations, we constructed homology models of human bestrophin-1 (Best1) Asp-rich domain using human thrombospondin-1 X-ray structure as a template. Molecular dynamics simulations were used to identify Asp and Glu residues binding Ca2+ and to predict the effects of their mutations to alanine. We then proceeded to test selected mutations in the Asp-rich domain of the highly homologous mouse bestrophin-2. The mutants expressed in HEK-293 cells were investigated by electrophysiological experiments using the whole-cell voltage-clamp technique. Based on our molecular modeling results, we predicted that Asp-rich domain has two defined binding sites and that D301A and D304A mutations may impact the binding of the metal ions. The experiments confirmed that these mutations do actually affect the function of the protein causing a large decrease in the Ca2+-activated Cl− current, fully consistent with our predictions. In addition, other studied mutations (E306A, D312A) did not decrease Ca2+-activated Cl− current in agreement with modeling results

Automation of a problem list using natural language processing

BACKGROUND: The medical problem list is an important part of the electronic medical record in development in our institution. To serve the functions it is designed for, the problem list has to be as accurate and timely as possible. However, the current problem list is usually incomplete and inaccurate, and is often totally unused. To alleviate this issue, we are building an environment where the problem list can be easily and effectively maintained. METHODS: For this project, 80 medical problems were selected for their frequency of use in our future clinical field of evaluation (cardiovascular). We have developed an Automated Problem List system composed of two main components: a background and a foreground application. The background application uses Natural Language Processing (NLP) to harvest potential problem list entries from the list of 80 targeted problems detected in the multiple free-text electronic documents available in our electronic medical record. These proposed medical problems drive the foreground application designed for management of the problem list. Within this application, the extracted problems are proposed to the physicians for addition to the official problem list. RESULTS: The set of 80 targeted medical problems selected for this project covered about 5% of all possible diagnoses coded in ICD-9-CM in our study population (cardiovascular adult inpatients), but about 64% of all instances of these coded diagnoses. The system contains algorithms to detect first document sections, then sentences within these sections, and finally potential problems within the sentences. The initial evaluation of the section and sentence detection algorithms demonstrated a sensitivity and positive predictive value of 100% when detecting sections, and a sensitivity of 89% and a positive predictive value of 94% when detecting sentences. CONCLUSION: The global aim of our project is to automate the process of creating and maintaining a problem list for hospitalized patients and thereby help to guarantee the timeliness, accuracy and completeness of this information