scholarly journals Properties of cyclic nucleotide-gated channels mediating olfactory transduction. Activation, selectivity, and blockage.

1992 ◽  
Vol 100 (1) ◽  
pp. 45-67 ◽  
Author(s):  
S Frings ◽  
J W Lynch ◽  
B Lindemann
Keyword(s):  
Cyclic Nucleotide ◽  
Olfactory Receptor ◽  
Cation Binding ◽  
Ph Effects ◽  
Induced Current ◽  
Cyclic Nucleotide Gated Channels ◽  
Olfactory Receptor Cells ◽  
Receptor Cells ◽  
Cation Binding Site ◽  
Voltage Dependent

Cyclic nucleotide-gated channels (cng channels) in the sensory membrane of olfactory receptor cells, activated after the odorant-induced increase of cytosolic cAMP concentration, conduct the receptor current that elicits electrical excitation of the receptor neurons. We investigated properties of cng channels from frog and rat using inside-out and outside-out membrane patches excised from isolated olfactory receptor cells. Channels were activated by cAMP and cGMP with activation constants of 2.5-4.0 microM for cAMP and 1.0-1.8 for cGMP. Hill coefficients of dose-response curves were 1.4-1.8, indicating cooperativity of ligand binding. Selectivity for monovalent alkali cations and the Na/Li mole-fraction behavior identified the channel as a nonselective cation channel, having a cation-binding site of high field strength in the pore. Cytosolic pH effects suggest the presence of an additional titratable group which, when protonated, inhibits the cAMP-induced current with an apparent pK of 5.0-5.2. The pH effects were not voltage dependent. Several blockers of Ca2+ channels also blocked olfactory cng channels. Amiloride, D 600, and diltiazem inhibited the cAMP-induced current from the cytosolic side. Inhibition constants were voltage dependent with values of, respectively, 0.1, 0.3, and 1 mM at -60 mV, and 0.03, 0.02, and 0.2 mM at +60 mV. Our results suggest functional similarity between frog and rat cng channels, as well as marked differences to cng channels from photoreceptors and other tissues.

scholarly journals Cyclic nucleotide-gated channels of rat olfactory receptor cells: divalent cations control the sensitivity to cAMP.

1994 ◽  
Vol 103 (1) ◽  
pp. 87-106 ◽  
Author(s):  
J W Lynch ◽  
B Lindemann
Keyword(s):  
Cyclic Nucleotide ◽  
Olfactory Receptor ◽  
Divalent Cations ◽  
Membrane Surface ◽  
Regulatory Protein ◽  
Induced Response ◽  
Intact Cells ◽  
Cyclic Nucleotide Gated Channels ◽  
Olfactory Receptor Cells ◽  
Receptor Cells

cAMP-gated channels were studied in inside-out membrane patches excised from the apical cellular pole of isolated olfactory receptor cells of the rat. In the absence of divalent cations the dose-response curve of activation of patch current by cAMP had a KM of 4.0 microM at -50 mV and of 2.5 microM at +50 mV. However, addition of 0.2 or 0.5 mM Ca2+ shifted the KM of cAMP reversibly to the higher cAMP concentrations of 33 or 90 microM, respectively, at -50 mV. Among divalent cations, the relative potency for inducing cAMP affinity shifts was: Ca2+ > Sr2+ > Mn2+ > Ba2+ > Mg2+, of which Mg2+ (up to 3 mM) did not shift the KM at all. This potency sequence corresponds closely to that required for the activation of calmodulin. However, the Ca(2+)-sensitivity is lower than expected for a calmodulin-mediated action. Brief (60 s) transient exposure to 3 mM Mg2+, in the absence of other divalent cations, had a protective effect in that following washout of Mg2+, subsequent exposure to 0.2 mM Ca2+ no longer caused affinity shifts. This protection effect did not occur in intact cells and was probably a consequence of patch excision, possibly representing ablation of a regulatory protein from the channel cyclic nucleotide binding site. Thus, the binding of divalent cations, probably via a regulatory protein, controls the sensitivity of the cAMP-gated channels to cAMP. The influx of Ca2+ through these channels during the odorant response may rise to a sufficiently high concentration at the intracellular membrane surface to contribute to the desensitization of the odorant-induced response. The results also indicate that divalent cation effects on cyclic nucleotide-gated channels may depend on the sequence of pre-exposure to other divalent cations.

scholarly journals Na+-dependent Ca2+ Extrusion Governs Response Recovery in Frog Olfactory Receptor Cells

1998 ◽  
Vol 112 (5) ◽  
pp. 529-535 ◽  
Author(s):  
Johannes Reisert ◽  
H.R. Matthews
Keyword(s):  
Cyclic Nucleotide ◽  
Olfactory Receptor ◽  
Ionic Composition ◽  
External Solution ◽  
Free Solution ◽  
Olfactory Cilia ◽  
Olfactory Receptor Cells ◽  
Receptor Cells ◽  
Response Recovery ◽  
Extrusion Mechanism

To study the mechanism by which Ca2+, which enters during the odor response, is extruded during response recovery, recordings were made from isolated frog olfactory receptor cells using the suction pipette technique, while superfusing the olfactory cilia with solutions of modified ionic composition. When external Na+ was substituted with another cation, the response to odor was greatly prolonged. This prolongation of the response was similar irrespective of whether Na+ was replaced with Li+, which permeates the cyclic nucleotide-gated conductance, or choline, which does not. The prolonged current was greatly reduced by exposure to 300 μM niflumic acid, a blocker of the calcium-activated chloride channel, indicating that it is carried by this conductance, and abolished if Ca2+ was omitted from the external solution, demonstrating that Ca2+ influx is required for its generation. When the cilia were exposed to Na+-free solution after odor stimulation, the recovery of the response to a second stimulus from the adaptation induced by the first was greatly reduced. We conclude that a Na+-dependent Ca2+ extrusion mechanism is present in frog olfactory cilia and that it serves as the main mechanism that returns cytoplasmic Ca2+ concentration to basal levels after stimulation and mediates the normally rapid recovery of the odor response and the restoration of sensitivity after adaptation.

scholarly journals A Point Mutation in the Pore Region Alters Gating, Ca2+Blockage, and Permeation of Olfactory Cyclic Nucleotide–Gated Channels

2000 ◽  
Vol 116 (3) ◽  
pp. 311-326 ◽  
Author(s):  
Paola Gavazzo ◽  
Cristiana Picco ◽  
Elisabeth Eismann ◽  
U. Benjamin Kaupp ◽  
Anna Menini
Keyword(s):  
Cyclic Nucleotide ◽  
Single Channel ◽  
Channel Gating ◽  
Cation Binding ◽  
Xenopus Laevis Oocytes ◽  
Wild Type ◽  
Pore Region ◽  
Α Subunit ◽  
Cyclic Nucleotide Gated Channels ◽  
Cation Binding Site

Upon stimulation by odorants, Ca2+ and Na+ enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotide–gated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the α subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotide–gated channels play a similar role. We studied the corresponding glutamate (E340) of the α subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca2+ and Mg2+. E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca2+ and Mg2+ powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca2+ relative to Na+ was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca2+ and Mg2+, and affects channel gating by cAMP more than by cGMP.

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