Fodor AA et al. (1997), Tetracaine reports a conformational change in t...

XB-ART-15860

J Gen Physiol 1997 Nov 01;1105:591-600. doi: 10.1085/jgp.110.5.591.

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Tetracaine reports a conformational change in the pore of cyclic nucleotide-gated channels.

Fodor AA , Black KD , Zagotta WN .

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Local anesthetics are a diverse group of clinically useful compounds that act as pore blockers of both voltage- and cyclic nucleotide-gated (CNG) ion channels. We used the local anesthetic tetracaine to probe the nature of the conformational change that occurs in the pore of CNG channels during the opening allosteric transition. When applied to the intracellular side of wild-type rod CNG channels expressed in Xenopus oocytes from the alpha subunit, the local anesthetic tetracaine exhibits state-dependent block, binding with much higher affinity to closed states than to open states. Here we show that neutralization of a glutamic acid in the conserved P region (E363G) eliminated this state dependence of tetracaine block. Tetracaine blocked E363G channels with the same effectiveness at high concentrations of cGMP, when the channel spent more time open, and at low concentrations of cGMP, when the channel spent more time closed. In addition, Ni2+, which promotes the opening allosteric transition, decreased the effectiveness of tetracaine block of wild-type but not E363G channels. Similar results were obtained in a chimeric CNG channel that exhibits a more favorable opening allosteric transition. These results suggest that E363 is accessible to internal tetracaine in the closed but not the open configuration of the pore and that the conformational change that accompanies channel opening includes a change in the conformation or accessibility of E363.

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	Figure 2. Tetracaine shifted the cGMP dose– response curve to the right in wild-type but not E363G channels. (A and B) cGMP dose–response curves in the absence and presence of 10 μM tetracaine for a patch containing wild-type (A) or E363G (B) channels. Currents in the absence of tetracaine are normalized to currents obtained in the presence of saturating (2 mM) cGMP. For comparison, currents in the presence of 10 μM tetracaine are normalized to currents obtained in the presence of saturating (2 mM) cGMP + 10 μM tetracaine. All currents were measured at +60 mV. Fits are to Eq. 1. For the wild-type rod channel in the absence of tetracaine: K1/2cGMP = 60.5 μM, n = 2.0. For the wild-type rod channel in the presence of 10 μM tetracaine, K1/2cGMP = 228 μM, n = 2.0. For the rod E363G channel both in the presence and absence of tetracaine, K1/2cGMP = 251 μM, n = 1.7.
	Figure 3. Ni2+ substantially increases the currents produced by saturating (4 mM) cGMP in E363G but not in wild-type channels. Currents are in response to families of voltage steps from a holding potential of 0 to between −80 and +80 mV in increments of +20 mV. Leak currents in the absence of cGMP have been subtracted.
	Figure 4. The E363G mutation eliminated Ni2+ relief of tetracaine block. (A) Block of wild-type and E363G channels in the absence and presence of Ni2+. Currents are in response to a voltage step to +60 mV from a holding potential of 0 mV for four separate patches. In each panel, the top trace shows current in the absence of tetracaine and the bottom trace shows current in the presence of 40 μM tetracaine. The shaded line represents the zero current level. (B) Dose–response curves for tetracaine from three separate patches containing wild-type rod channels in the presence of 0 (•), 1 (▪), and 10 (▴) μM Ni2+. All currents were measured at +60 mV. All currents were recorded in the presence of saturating (2 mM) cGMP and normalized by currents recorded in the absence of tetracaine. Fits are to Eq. 2. For 0 μM Ni2+: K1/2Tet = 3.4 μM, n = 1.0; for 1 μM Ni2+: K1/2Tet = 13.3 μM, n = 1.1; and for 10 μM Ni2+: K1/2Tet = 37.7 μM, n = 1.0. (C) Dose–response curves for tetracaine for three separate patches recorded from E363G channels in the presence of 0 (•), 1 (▪), and 10 (▴) μM Ni2+. The fit is to Eq. 2 with K1/2Tet = 15.19 μM, n = 1.6. (D) Box plots showing range of tetracaine apparent affinities for wild-type and E363G channels in 0, 1, and 10 μM Ni2+. The line in the middle of the boxes shows the median. The top and bottom edges of the boxes show the 25th and 75th percentiles of the data. The whiskers coming out of some of the boxes show the 5th and 95th percentiles. Circles represent outliers.
	Figure 5. Tetracaine shifted the cGMP dose–response curve to the right in CHM15 but not CHM15-E363G channels. (A and B) cGMP dose–response curves in the absence and presence of 40 μM tetracaine for a patch containing CHM15 (A) or CHM15-E363G (B) channels. Currents in the absence of tetracaine are normalized by currents obtained in the presence of saturating (2 mM) cGMP. For comparison, currents in the presence of 40 μM tetracaine are normalized by currents obtained in the presence of saturating (2 mM) cGMP + 40 μM tetracaine. All currents were measured at +60 mV. Fits are to Eq. 1. For the CHM15 channel in the absence of tetracaine, K1/2cGMP = 15.6 μM, n = 2.1. For the CHM15 channel in the presence of 40 μM tetracaine, K1/2cGMP = 108.7 μM, n = 2.1. And for the CHM15-E363G channel both in the presence and absence of tetracaine, K1/2cGMP = 117.0 μM, n = 2.0.
	Figure 6. Tetracaine becomes more effective at low concentrations of cGMP for CHM15 but not for CHM15-E363G channels. (A and B) Comparison of the effect of 40 μM tetracaine on CHM15 (A) and CHM15-E363G (B) channels. Currents are in response to a voltage step to +60 mV from a holding potential of 0 mV. The currents recorded in the absence of tetracaine (top traces) are superimposed with the currents recorded in the presence of 40 μM tetracaine (bottom traces). Shaded lines show the zero current level. Leak current in the absence of cGMP has been subtracted from all traces. (C) The effect of 40 μM tetracaine at different concentrations of cGMP for CHM15 (□, n = 5) or CHM15-E363G (•, n = 4) channels. Data were pooled from a number of different patches. On the y-axis is the current, recorded with 40 μM tetracaine normalized by the current recorded in the absence of tetracaine at the concentration of cGMP indicated on the x-axis. All currents were measured at +60 mV. Fits to the data are from Scheme 1, with K = 4,500 M−1, L = 300, KDc = 200 nM, KDo = 200 μM for CHM15 channels and KDc = KDo = 25 μM (i.e., no state dependence of tetracaine block) for CHM15-E363G channels.
	Figure 7. The effects of Ni2+ on CHM15 and CHM15-E363G. Currents are in response to families of voltage steps from a holding potential of 0 to between −80 and +80 mV in increments of +20 mV. Leak currents in the absence of cGMP have been subtracted.
	Figure 8. The E363G mutation in CHM15 eliminated Ni2+ relief of tetracaine block. (A) Block of CHM15 and CHM15-E363G channels in the absence and presence of Ni2+. Currents are in response to a voltage step to +60 mV from a holding potential of 0 mV for four separate patches. In each panel, the top trace shows current in the absence of tetracaine and the bottom trace shows current in the presence of 40 μM tetracaine. The shaded line represents the zero current level. (B) Dose–response curves for tetracaine from two separate patches containing CHM15 channels in the presence of 0 (•) or 10 (▴) μM Ni2+. All currents were measured at +60 mV. All currents were recorded in the presence of saturating (2 mM) cGMP and normalized by currents recorded in the absence of tetracaine. Fits are to Eq. 2. For 0 μM Ni2+: K1/2Tet = 26.5 μM, n = 1.0; for 10 μM Ni2+: K1/2Tet = 59.4 μM, n = 1.0. (C) Dose–response curves for tetracaine for two separate patches recorded from CHM15-E363G channels in the presence of 0 (•) or 10 (▴) μM Ni2+. The fit is to Eq. 2 with K1/2Tet = 31.0 μM, n = 1.3. (D) Box plot showing range of tetracaine-apparent affinities for CHM15 and CHM15-E363G channels in 0 and 10 μM Ni2+. The line in the middle of the boxes shows the median. The top and bottom edges of the boxes show the 25th and 75th percentiles of the data. The whiskers coming out of some of the boxes show the 5th and 95th percentiles.
	Figure 9. State-dependent tetracaine block in wild-type channels. See text.

References [+] :

Bucossi, Time-dependent current decline in cyclic GMP-gated bovine channels caused by point mutations in the pore region expressed in Xenopus oocytes. 1996, Pubmed, Xenbase