Gavazzo P et al. (2000), A point mutation in the pore region alters gati...

XB-ART-10433

J Gen Physiol 2000 Sep 01;1163:311-26.

Show Gene links Show Anatomy links

A point mutation in the pore region alters gating, Ca(2+) blockage, and permeation of olfactory cyclic nucleotide-gated channels.

Gavazzo P , Picco C , Eismann E , Kaupp UB , Menini A .

???displayArticle.abstract???
Upon stimulation by odorants, Ca(2+) 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 alpha 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 alpha subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca(2+) and Mg(2+). 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 Ca(2+) and Mg(2+) 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 Ca(2+) 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 Ca(2+) and Mg(2+), and affects channel gating by cAMP more than by cGMP.

???displayArticle.pubmedLink??? 10962010
???displayArticle.pmcLink??? PMC2233693

Species referenced: Xenopus laevis
Genes referenced: camp clta

???attribute.lit??? ???displayArticles.show???

	Scheme S1.
	Scheme S2.
	Figure 1. Maximal currents activated by cGMP or cAMP for wild-type and mutant olfactory CNG channels. Currents in each row were from the same excised inside-out patch. Voltage steps of 150-ms duration from a holding potential of 0 mV were given from −80 to +80 mV in 20-mV steps. Currents were activated by cyclic nucleotide concentrations eliciting maximal currents (see dose–response relations in D–E): (A) cGMP concentration was 1 mM, (B) cAMP concentration was 3 mM for E340G and E340D, 5 mM for E340N and E340Q, and 500 μM for wild-type channels. (C) Current–voltage relations from recordings shown in A and B for cGMP (□) and cAMP (▵). (D–E) Dose–response relations. Currents activated by cGMP or by cAMP at −80 mV were measured in the same patch, normalized to the maximal current activated by cGMP at −80 mV, and plotted versus cGMP (D) or cAMP (E) concentrations. Continuous lines represent the best fit of the Hill equation () to the data with the following values. Wild-type: Imax,cA/Imax,cG = 0.99, K1/2,cG = 3.2 μM, ncG = 2.7, K1/2,cA = 97 μM, ncA = 2.2; E340D: Imax,cA/Imax,cG = 0.97, K1/2,cG = 4.5 μM, ncG = 2.7, K1/2,cA = 137 μM, ncA = 2.3; E340Q: Imax,cA/Imax,cG = 0.87, K1/2,cG = 4.8 μM, ncG = 2.4, K1/2,cA = 189 μM, ncA = 2.0; E340N: Imax,cA/Imax,cG = 0.81, K1/2,cG = 6.6 μM, ncG = 2.3, K1/2,cA = 251 μM, ncA = 1.9; E340G: Imax,cA/Imax,cG = 0.37, K1/2,cG = 18.5 μM, ncG = 1.6, K1/2,cA = 335 μM, ncA = 2.5.
	Figure 2. Single-channel dose–response relation for the E340G mutant. Current recordings from a membrane patch containing one E340G channel activated at −80 mV by various concentrations of cGMP (A) or cAMP (B). The continuous lines indicate the closed channel level and the dotted lines indicate the open level. (C) Dose–response relations were plotted as the open probability versus cyclic nucleotide concentration from the patch shown in A and B. Continuous curves were the best fit of the Hill equation () to the data with the following values: Pmax,cG = 0.82, K1/2,cG = 11.6 μM, ncG = 2.4, Pmax,cA = 0.46, K1/2,cA = 360 μM, ncA = 2.4.
	Figure 3. Single-channel voltage and concentration dependence of E340N mutant. (A) Current recordings from a membrane patch containing one E340N channel activated by 3 or 100 μM cGMP at −80 or +80 mV. The continuous lines indicate the closed channel level and the dotted lines indicate two conductance open levels. Amplitude histograms at +80 mV for 3 μM (B) or 100 μM (C) cGMP were fitted as the sum of three Gaussians and shown enlarged in the insets. Histograms were fitted with the following parameters. For 3 μM cGMP: Pclosed = 0.765, Po,low = 0.22, ilow = 1.1 pA, Po,high = 0.015, ihigh = 2.5 pA; for 100 μM cGMP: Pclosed = 0.01, Po,low = 0.925, ilow = 1 pA, Po,high = 0.065, ihigh = 2.5 pA.
	Figure 4. Comparison of normalized I-V relations from macroscopic and single-channel currents for wild-type and mutant channels. Macroscopic currents were measured at saturating cGMP in a voltage range from −80 to + 80 mV and normalized to unity at +80 mV. Average values of normalized macroscopic currents (□) and of single-channel amplitudes (▴) were plotted versus applied voltage. Each point is the average from at least four patches. Bars indicate standard deviation. Error bars smaller than the size of the symbols were not plotted.
	Figure 5. Single-channel dose–response relations for wild-type, E340D, E340N, and E340G channels. Continuous curves show fits of to the dose–response relations using the following parameters. Wild-type: LcG = 334, Kd,cG = 29 μM, ncG = 2.3, LcA = 70, Kd,cA = 333 μM, ncA = 2.9; E340D: LcG = 66, Kd,cG = 31 μM, ncG = 2.6, LcA = 12, Kd,cA = 558 μM, ncA = 3; E340G: LcG = 4.6, Kd,cG = 24 μM, ncG = 2.4, LcA = 0.9, Kd,cA = 472 μM, ncA = 2.3; E340N: LcG = 10, Kd,cG = 24 μM, ncG = 3.5, LcA = 3, Kd,cA = 400 μM, ncA = 3.5.
	Figure 6. External Ca2+ and Mg2+ blockage in wild-type and mutant channels. cGMP-gated currents were measured in outside-out patches in the presence of the indicated concentrations of Ca2+ (A) or Mg2+ (B) in the extracellular solution. Currents were activated by 100 μM cGMP in the patch pipette. Voltage ramps were from −80 to +80 mV. Currents from the experiments shown in A and B in the presence of various Ca2+ or Mg2+ concentrations were measured at +80 or −80 mV, normalized to the current measured in the absence of divalent cations at the same voltage and plotted as a function of external Ca2+ (C–D) or Mg2+ (E–F) concentrations. Continuous lines were the best fit of to the data with the following values. (C) At +80 mV, wild type (♦): Ki,Ca = 380 μM, n = 1; E340D (□): Ki,Ca = 16 μM, n = 1; E340G (▴): Ki,Ca = 21 mM, n = 0.8; E340N (○): Ki,Ca = 28 mM, n = 0.9. (D) At −80 mV, wild type (♦): Ki,Ca = 42 μM, n = 0.8; E340D (□): Ki,Ca = 70 μM, n = 0.9; E340G (▴): Ki,Ca = 3 mM, n = 0.6; E340N (○): Ki,Ca = 6.3 mM, n = 0.6. (E) At +80 mV, wild type (♦): Ki,Mg = 650 μM, n = 0.7; E340D (□): Ki,Mg = 112 μM, n = 0.7; E340G (▴): Ki,Mg = 13 mM, n = 0.8; E340N (○): Ki,Mg = 51 mM, n = 0.7. (F) At −80 mV, wild type (♦): Ki,Mg = 90 μM, n = 1.1; E340D (□): Ki,Mg = 48 μM, n = 0.8; E340G (▴): Ki,Mg = 1.4 mM, n = 0.8; E340N (○): Ki,Mg = 26 mM, n = 0.6.
	Figure 7. External Ca2+ blockage on single-channel current for wild-type and E340G mutant channels. (A and B) Single-channel currents at −80 mV activated by cGMP in outside-out patches in the presence of the indicated concentrations of external Ca2+. The cGMP concentration was 5 μM for wild-type (A) and 1mM for E340G (B) mutant channels. (C and D) Currents from each patch were determined from amplitude histograms in the presence of various Ca2+ concentrations at +80 or −80 mV, normalized to the current measured at the same voltage in the absence of divalent cations, and plotted as a function of external Ca2+ concentrations (C and D). Continuous lines were the best fit of to the data with the following values. At +80 mV, (C) wild type (♦): Ki,Ca = 250 μM, n = 1; E340G (▴): Ki,Ca = 32 mM, n = 0.7. At −80 mV, (D) wild type (♦): Ki,Ca = 31 μM, n = 1.1; E340G (▴): Ki,Ca = 8.8 mM, n = 0.8.
	Figure 8. Permeation of external Ca2+ and Mg2+ in wild-type and E340G mutant channels. Current recordings from individual outside-out patches in bi-ionic conditions. The patch pipette contained 1 mM cGMP and 110 mM NaCl, while the external solution contained either 110 mM NaCl, 73.3 mM CaCl2, or 73.3 mM MgCl2, as indicated in the figure. Voltage was changed in 20-mV steps from −80 to +80 mV from a holding potential of 0 mV. The dashed lines indicate the zero-current level. Raw data are illustrated without leak subtraction. Capacitance artifacts were not subtracted. Current–voltage relations are plotted in C and D for wild-type and E340G, respectively. Symbols indicate the bi-ionic conditions: symmetrical NaCl (○), internal NaCl and external CaCl2 (▴), and internal NaCl and external MgCl2 (□).

References [+] :

Altenhofen, Control of ligand specificity in cyclic nucleotide-gated channels from rod photoreceptors and olfactory epithelium. 1991, Pubmed, Xenbase

Altenhofen, Control of ligand specificity in cyclic nucleotide-gated channels from rod photoreceptors and olfactory epithelium. 1991, Pubmed , Xenbase
Becchetti, Cyclic nucleotide-gated channels. Pore topology studied through the accessibility of reporter cysteines. 1999, Pubmed , Xenbase
Bradley, Heteromeric olfactory cyclic nucleotide-gated channels: a subunit that confers increased sensitivity to cAMP. 1994, Pubmed
Brown, Movement of gating machinery during the activation of rod cyclic nucleotide-gated channels. 1998, Pubmed , Xenbase
Brown, Specific labeling and permanent activation of the retinal rod cGMP-activated channel by the photoaffinity analog 8-p-azidophenacylthio-cGMP. 1993, Pubmed
Bucossi, Single-channel properties of ionic channels gated by cyclic nucleotides. 1997, Pubmed , Xenbase
Bönigk, The native rat olfactory cyclic nucleotide-gated channel is composed of three distinct subunits. 1999, Pubmed
Colamartino, Blockage and permeation of divalent cations through the cyclic GMP-activated channel from tiger salamander retinal rods. 1991, Pubmed
Dhallan, Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons. 1990, Pubmed
Dzeja, Ca2+ permeation in cyclic nucleotide-gated channels. 1999, Pubmed
Eismann, A single negative charge within the pore region of a cGMP-gated channel controls rectification, Ca2+ blockage, and ionic selectivity. 1994, Pubmed , Xenbase
Fabiato, Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. 1988, Pubmed
Finn, Cyclic nucleotide-gated ion channels: an extended family with diverse functions. 1996, Pubmed
Fodor, Tetracaine reports a conformational change in the pore of cyclic nucleotide-gated channels. 1997, Pubmed , Xenbase
Frings, Profoundly different calcium permeation and blockage determine the specific function of distinct cyclic nucleotide-gated channels. 1995, Pubmed , Xenbase
Gavazzo, Mechanisms of modulation by internal protons of cyclic nucleotide-gated channels cloned from sensory receptor cells. 1997, Pubmed , Xenbase
Gordon, Direct interaction between amino- and carboxyl-terminal domains of cyclic nucleotide-gated channels. 1997, Pubmed
Gordon, Localization of regions affecting an allosteric transition in cyclic nucleotide-activated channels. 1995, Pubmed , Xenbase
Goulding, Molecular cloning and single-channel properties of the cyclic nucleotide-gated channel from catfish olfactory neurons. 1992, Pubmed
Goulding, Molecular mechanism of cyclic-nucleotide-gated channel activation. 1994, Pubmed , Xenbase
Hackos, Divalent cation selectivity is a function of gating in native and recombinant cyclic nucleotide-gated ion channels from retinal photoreceptors. 1999, Pubmed , Xenbase
Herlitze, A general and rapid mutagenesis method using polymerase chain reaction. 1990, Pubmed
Kaupp, Family of cyclic nucleotide gated ion channels. 1995, Pubmed
Kaupp, Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel. 1989, Pubmed , Xenbase
Kleene, Both external and internal calcium reduce the sensitivity of the olfactory cyclic-nucleotide-gated channel to CAMP. 1999, Pubmed
Kleene, Block by external calcium and magnesium of the cyclic-nucleotide-activated current in olfactory cilia. 1995, Pubmed
Liman, A second subunit of the olfactory cyclic nucleotide-gated channel confers high sensitivity to cAMP. 1994, Pubmed , Xenbase
Liu, Calcium-calmodulin modulation of the olfactory cyclic nucleotide-gated cation channel. 1994, Pubmed
Melton, Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. 1984, Pubmed
Menini, Cyclic nucleotide-gated channels in visual and olfactory transduction. 1995, Pubmed
Menini, Calcium signalling and regulation in olfactory neurons. 1999, Pubmed
Nakamura, A cyclic nucleotide-gated conductance in olfactory receptor cilia. , Pubmed
Paoletti, C-Linker of cyclic nucleotide-gated channels controls coupling of ligand binding to channel gating. 1999, Pubmed
Park, Divalent cation selectivity in a cyclic nucleotide-gated ion channel. 1995, Pubmed , Xenbase
Perry, Response properties of cones from the retina of the tiger salamander. 1991, Pubmed
Picones, Permeability and interaction of Ca2+ with cGMP-gated ion channels differ in retinal rod and cone photoreceptors. 1995, Pubmed
Root, Identification of an external divalent cation-binding site in the pore of a cGMP-activated channel. 1993, Pubmed , Xenbase
Root, Two identical noninteracting sites in an ion channel revealed by proton transfer. 1994, Pubmed , Xenbase
Sanger, DNA sequencing with chain-terminating inhibitors. 1977, Pubmed
Sautter, An isoform of the rod photoreceptor cyclic nucleotide-gated channel beta subunit expressed in olfactory neurons. 1998, Pubmed
Scott, Three residues predicted by molecular modeling to interact with the purine moiety alter ligand binding and channel gating in cyclic nucleotide-gated channels. 1998, Pubmed
Seifert, Molecular determinants of a Ca2+-binding site in the pore of cyclic nucleotide-gated channels: S5/S6 segments control affinity of intrapore glutamates. 1999, Pubmed , Xenbase
Sun, Exposure of residues in the cyclic nucleotide-gated channel pore: P region structure and function in gating. 1996, Pubmed
Sunderman, Mechanism of allosteric modulation of rod cyclic nucleotide-gated channels. 1999, Pubmed , Xenbase
Sunderman, Sequence of events underlying the allosteric transition of rod cyclic nucleotide-gated channels. 1999, Pubmed , Xenbase
Tibbs, Allosteric activation and tuning of ligand efficacy in cyclic-nucleotide-gated channels. 1997, Pubmed , Xenbase
Varnum, Molecular mechanism for ligand discrimination of cyclic nucleotide-gated channels. 1995, Pubmed
Varnum, Interdomain interactions underlying activation of cyclic nucleotide-gated channels. 1997, Pubmed , Xenbase
Zagotta, Structure and function of cyclic nucleotide-gated channels. 1996, Pubmed
Zimmerman, Cyclic nucleotide gated channels. 1995, Pubmed
Zimmerman, Hindered diffusion in excised membrane patches from retinal rod outer segments. 1988, Pubmed
Zimmerman, Cation interactions within the cyclic GMP-activated channel of retinal rods from the tiger salamander. 1992, Pubmed
Zong, Three amino acids in the C-linker are major determinants of gating in cyclic nucleotide-gated channels. 1998, Pubmed