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Mechanism of tetracaine block of cyclic nucleotide-gated channels.
Fodor AA
,
Gordon SE
,
Zagotta WN
.
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Local anesthetics are a diverse group of ion channel blockers that can be used to probe conformational changes in the pore. We examined the effects of the local anesthetic tetracaine on rod and olfactory cyclic nucleotide-gated channels expressed from subunit 1 in Xenopus oocytes. We found that 40 microM tetracaine effectively blocked the bovine rod channel but not the rat olfactory channel at saturating concentrations of cGMP. By testing chimeric channels containing regions of sequence from both rod and olfactory channels, we found that determinants of apparent affinity for tetracaine at saturating cGMP did not map to any one region of the channel sequence. Rather, the differences in apparent affinity could be explained by differences between the chimeras in the free energy of the opening allosteric transition. If a channel construct (such as the rod channel) spent appreciable time in the closed state at saturating cGMP, then it had a high apparent affinity for tetracaine. If, on the other hand, a channel construct (such as the olfactory channel) spent little time in the closed state at saturating cGMP, then it had a low apparent affinity for tetracaine. Furthermore, tetracaine became more effective at low concentrations of cGMP and at saturating concentrations of cAMP, conditions which permit the channels to spend more time in the closed configuration. These results were well fit by a model in which tetracaine binds more tightly to the closed channel than to the open channel. Dose-response curves for tetracaine in the presence of saturating cGMP are well fit with a Michaelis-Menten binding scheme indicating that a single tetracaine molecule is sufficient to produce block. In addition, tetracaine block is voltage dependent with an effective z delta of +0.56. These data are consistent with a pore-block hypothesis. The finding that tetracaine is a state-dependent pore blocker suggests that the inner mouth of the pore of cyclic nucleotide-gated channels undergoes a conformational change during channel opening.
Figure 2. The KD cGMP for cGMP and the KD Tet for tetracaine are inversely correlated for a number of rod-olfactory channel chimeras. KD cGMP's for cGMP are indicated by shaded boxes. KD Tet's for tetracaine are indicated by open boxes. Values for KD Tetwere obtained from fits to the Michaelis-Menten equation and values for KD cGMP were obtained from fits to the Hill Equation (see Results). KD cGMPis defined as the concentration of cGMP which produces half the current seen at saturating cGMP. KD Tet is defined as the concentration of tetracaine that blocks half the current at saturating cGMP. In this box plot, the left and right edges of the boxes shows the 25th and 75thpercentiles of the data. The whiskers coming out of some of the boxes show the 5th and 95th percentiles. The line in the middle of the boxes shows the median.
Figure 4. Tetracaine becomes a more effective blocker at low concentrations of cGMP. (A) Current traces recorded from the olfactory channel. All traces were recorded from the same patch. The top set of traces shows currents recorded at saturating cGMP in the presence and absence of 4 μM tetracaine. The bottom set of traces shows currents recorded at 2 μM cGMP in the presence and absence of 4 μM tetracaine. (B and C) cGMP dose-response curves for the rod (B) and olfactory (C) channels in the presence (squares) and absence (circles) of 4 μM tetracaine. Currents recorded at +60 mV. The curves are generated from the model in Fig. 3 A with the following parameters: for the rod channel, K = 4,500 Mâ1, L = 17, KDc = 280 nM, KDo = 185 μM, ISatâcGMP = 13,630 pA. For the olfactory channel: K = 4,500 Mâ1, L = 30000, KDc = 440 nM, KDo = 185 μM, ISat cGMP= 1,781 pA. All currents were measured at +60 mV.
Figure 1. At saturating cGMP, tetracaine effectively blocks the rod but not the olfactory channel. (A) Current families are recorded in response to voltage steps from 0 mV to between â80 and +80 mV in 20-mV steps. The top set of traces are from the bovine rod channel and the bottom set of traces are from the rat olfactory channel. All currents were recorded in the presence of 2 mM cGMP. The left traces are in the absence of tetracaine and the right traces are in identical solutions with 40 μM tetracaine. (B) Dose- response curves for tetracaine block. Circles represent data from the rod channel and squares from the olfactory channel. Smooth curves are fits to the data with the Michaelis-Menten equation (see results) with KD Tet = 2.63 μM for the rod channel and KD Tet = 102.4 μM for the olfactory channel. All currents were measured at +60 mV.
Figure 8. The voltage dependence of tetracaine block. (A) Current families recorded from the rod channels in the presence of saturating cGMP (2 mM) in response to voltage steps from 0 mV to between â80 and +80 mV in 20 mV steps. In these recordings, the duration of each voltage step was extended to 800 ms in order to allow currents from hyperpolarized voltages to achieve steady state. All data were recorded from the same patch. (B) The current in the presence of 40 μM tetracaine at each voltage was normalized by the current recorded in the absence of tetracaine at that voltage. The curve is a fit from the model in Fig. 3 A except that KDc was replaced with an expression that varied with voltage (see results). Parameters from the fit are KDc-0 = 365 nM, zδ = 0.56 and L = 20.
Figure 5. The effect of 4 μM tetracaine at different concentrations of cGMP for a number of different patches containing rod (circles, N = 7) and olfactory (squares, N = 4) channels. On the abscissa is the current recorded with 4 μM tetracaine normalized by the current recorded without tetracaine at the concentration of cGMP indicated on the ordinate. Curves are fits from the model shown in Fig. 3 A with the following parameters: K = 4,500 Mâ1, LROD = 17, LOLF = 30000, KDc = 280 nM and KDo = 180 μM. All currents were measured at +60 mV.
Figure 6. For olfactory channels, 40 μM tetracaine is more effective at blocking currents produced by saturating cAMP than for currents produced by saturating cGMP. All traces were recorded from the same patch. The top set of traces shows currents recorded in the absence of tetracaine. The bottom set of traces show currents recorded in the presence of 40 μM tetracaine. The left traces show currents evoked by a saturating concentration of cGMP while the right traces show current evoked by a saturating concentration of cAMP.
Figure 7. The effect of tetracaine block on single channels. All data shown are from a single patch containing two rod channels recorded at +80 mV. Amplitude histograms were constructed from 30 s of continuous recording. c represents the current present when both channels are closed. Dashed lines represent the currents present when one or both of the channels are open. The position of the lines representing the open states were derived from fits to the histograms and are the same in the presence and absence of tetracaine. Fits to binned data are to an equation for the sum of two Gaussians constrained so that the distribution of open channels follows a binomial distribution with N = 2 (see methods). (A) In the absence of cGMP, both channels are closed. Fits to the histogram are with Po= 0.0 and Ï = 0.38 pA. (B) At saturating (2 mM) cGMP, both channels spend most of their time open. Fits to the histogram are with i = 2.74 pA, Ï = 0.27 pA, É = 0.25 pA, Po = 0.83. (C) Addition of 10 μM tetracaine causes the channels to spend less time in the open state. Fits to the histogram are with the same values as in the absence of tetracaine except that Po = 0.17. Tetracaine does not cause the single-channel amplitude, i, to change.
Figure 9. (A) Structure of tetracaine. (B) Model of tetracaine block. The greater affinity of tetracaine for closed channels is envisioned as arising from two distinct mechanisms. Interactions between tetracaine and the inner mouth of the channel pore are seen as leading to tighter binding of tetracaine to closed channels and therefore to stabilization of the closed state. An increase in the width of the pore in the open conformation disrupts these interactions leading to the lower affinity of the open state for the blocker. In addition, the open channel is seen as containing an ion in the pore, not present in the closed channel, which stabilizes the open state. The positively charged ion electrostatically repels tetracaine's positively charged amine group causing tetracaine to bind to the open state with low affinity.
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