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.

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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 .

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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.

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Species referenced: Xenopus laevis
Genes referenced: camp clta

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	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 â80 to +80 mV in 20-mV steps. Currents were activated by cyclic nucleotide concentrations eliciting maximal currents (see doseâresponse relations in DâE): (A) cGMP concentration was 1 mM, (B) cAMP concentration was 3 mM for E340G and E340D, 5 mM for E340N and E340Q, and 500 Î¼M for wild-type channels. (C) Currentâvoltage relations from recordings shown in A and B for cGMP (â¡) and cAMP (âµ). (DâE) Doseâresponse relations. Currents activated by cGMP or by cAMP at â80 mV were measured in the same patch, normalized to the maximal current activated by cGMP at â80 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 Î¼M, ncG = 2.7, K1/2,cA = 97 Î¼M, ncA = 2.2; E340D: Imax,cA/Imax,cG = 0.97, K1/2,cG = 4.5 Î¼M, ncG = 2.7, K1/2,cA = 137 Î¼M, ncA = 2.3; E340Q: Imax,cA/Imax,cG = 0.87, K1/2,cG = 4.8 Î¼M, ncG = 2.4, K1/2,cA = 189 Î¼M, ncA = 2.0; E340N: Imax,cA/Imax,cG = 0.81, K1/2,cG = 6.6 Î¼M, ncG = 2.3, K1/2,cA = 251 Î¼M, ncA = 1.9; E340G: Imax,cA/Imax,cG = 0.37, K1/2,cG = 18.5 Î¼M, ncG = 1.6, K1/2,cA = 335 Î¼M, ncA = 2.5.
	Figure 2. Single-channel doseâresponse relation for the E340G mutant. Current recordings from a membrane patch containing one E340G channel activated at â80 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) Doseâresponse 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 Î¼M, ncG = 2.4, Pmax,cA = 0.46, K1/2,cA = 360 Î¼M, 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 Î¼M cGMP at â80 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 Î¼M (B) or 100 Î¼M (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 Î¼M cGMP: Pclosed = 0.765, Po,low = 0.22, ilow = 1.1 pA, Po,high = 0.015, ihigh = 2.5 pA; for 100 Î¼M 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 â80 to + 80 mV and normalized to unity at +80 mV. Average values of normalized macroscopic currents (â¡) and of single-channel amplitudes (â´) 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 doseâresponse relations for wild-type, E340D, E340N, and E340G channels. Continuous curves show fits of to the doseâresponse relations using the following parameters. Wild-type: LcG = 334, Kd,cG = 29 Î¼M, ncG = 2.3, LcA = 70, Kd,cA = 333 Î¼M, ncA = 2.9; E340D: LcG = 66, Kd,cG = 31 Î¼M, ncG = 2.6, LcA = 12, Kd,cA = 558 Î¼M, ncA = 3; E340G: LcG = 4.6, Kd,cG = 24 Î¼M, ncG = 2.4, LcA = 0.9, Kd,cA = 472 Î¼M, ncA = 2.3; E340N: LcG = 10, Kd,cG = 24 Î¼M, ncG = 3.5, LcA = 3, Kd,cA = 400 Î¼M, 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 Î¼M cGMP in the patch pipette. Voltage ramps were from â80 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 â80 mV, normalized to the current measured in the absence of divalent cations at the same voltage and plotted as a function of external Ca2+ (CâD) or Mg2+ (EâF) concentrations. Continuous lines were the best fit of to the data with the following values. (C) At +80 mV, wild type (â¦): Ki,Ca = 380 Î¼M, n = 1; E340D (â¡): Ki,Ca = 16 Î¼M, n = 1; E340G (â´): Ki,Ca = 21 mM, n = 0.8; E340N (â): Ki,Ca = 28 mM, n = 0.9. (D) At â80 mV, wild type (â¦): Ki,Ca = 42 Î¼M, n = 0.8; E340D (â¡): Ki,Ca = 70 Î¼M, n = 0.9; E340G (â´): Ki,Ca = 3 mM, n = 0.6; E340N (â): Ki,Ca = 6.3 mM, n = 0.6. (E) At +80 mV, wild type (â¦): Ki,Mg = 650 Î¼M, n = 0.7; E340D (â¡): Ki,Mg = 112 Î¼M, n = 0.7; E340G (â´): Ki,Mg = 13 mM, n = 0.8; E340N (â): Ki,Mg = 51 mM, n = 0.7. (F) At â80 mV, wild type (â¦): Ki,Mg = 90 Î¼M, n = 1.1; E340D (â¡): Ki,Mg = 48 Î¼M, n = 0.8; E340G (â´): Ki,Mg = 1.4 mM, n = 0.8; E340N (â): 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 â80 mV activated by cGMP in outside-out patches in the presence of the indicated concentrations of external Ca2+. The cGMP concentration was 5 Î¼M 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 â80 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 (â¦): Ki,Ca = 250 Î¼M, n = 1; E340G (â´): Ki,Ca = 32 mM, n = 0.7. At â80 mV, (D) wild type (â¦): Ki,Ca = 31 Î¼M, n = 1.1; E340G (â´): 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 â80 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. Currentâvoltage relations are plotted in C and D for wild-type and E340G, respectively. Symbols indicate the bi-ionic conditions: symmetrical NaCl (â), internal NaCl and external CaCl2 (â´), and internal NaCl and external MgCl2 (â¡).

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