Intrinsic versus extrinsic voltage sensitivity of blocker interaction with an ion channel pore

Abstract

Many physiological and synthetic agents act by occluding the ion conduction pore of ion channels. A hallmark of charged blockers is that their apparent affinity for the pore usually varies with membrane voltage. Two models have been proposed to explain this voltage sensitivity. One model assumes that the charged blocker itself directly senses the transmembrane electric field, i.e., that blocker binding is intrinsically voltage dependent. In the alternative model, the blocker does not directly interact with the electric field; instead, blocker binding acquires voltage dependence solely through the concurrent movement of permeant ions across the field. This latter model may better explain voltage dependence of channel block by large organic compounds that are too bulky to fit into the narrow (usually ion-selective) part of the pore where the electric field is steep. To date, no systematic investigation has been performed to distinguish between these voltage-dependent mechanisms of channel block. The most fundamental characteristic of the extrinsic mechanism, i.e., that block can be rendered voltage independent, remains to be established and formally analyzed for the case of organic blockers. Here, we observe that the voltage dependence of block of a cyclic nucleotide-gated channel by a series of intracellular quaternary ammonium blockers, which are too bulky to traverse the narrow ion selectivity filter, gradually vanishes with extreme depolarization, a predicted feature of the extrinsic voltage dependence model. In contrast, the voltage dependence of block by an amine blocker, which has a smaller "diameter" and can therefore penetrate into the selectivity filter, follows a Boltzmann function, a predicted feature of the intrinsic voltage dependence model. Additionally, a blocker generates (at least) two blocked states, which, if related serially, may preclude meaningful application of a commonly used approach for investigating channel gating, namely, inferring the properties of the activation gate from the kinetics of channel block.

Figure 1.

cGMP activation of CNGA1 channels. (A) Macroscopic current traces recorded in symmetric 130 mM Na⁺ from an inside-out patch containing CNGA1 channels in the presence of the indicated concentrations of intracellular cGMP. Currents were elicited by stepping from the 0-mV holding potential to voltages between −200 and 200 mV in 50-mV increments. Currents in the absence of cGMP were used as templates for subsequent offline background current corrections. Dotted lines indicate 0 current levels. (B) Fraction of maximal current (I/I_max; mean ± SEM; n = 3–7) plotted against cGMP concentration for −100 mV (squares) and 100 mV (circles). Solid curves are Hill equation fits yielding EC₅₀ = 79 ± 1 µM and h = 1.37 ± 0.03 at −100 mV, and EC₅₀ = 64 ± 2 µM and h = 1.39 ± 0.05 at 100 mV.

Figure 2.

Voltage-dependent CNGA1 block by extracellular PhTx. (A) Structures of PhTx and spermine (SPM). (B) Macroscopic current traces recorded from inside-out patches containing CNGA1 channels in the presence of the indicated concentrations of extracellular PhTx (in the pipette solution). Currents were activated with 2 mM of intracellular cGMP and elicited with the voltage protocol shown. Dotted lines indicate 0 current levels. (C) Mean I-V curves (mean ± SEM; n = 3–11) determined at the end of the test pulses in the absence or presence of three concentrations of extracellular PhTx. (D) Fraction of current not blocked (mean ± SEM; n = 4–6) by extracellular PhTx is plotted against membrane voltage. Curves are fits of a Boltzmann function (Eq. 1) to the three datasets simultaneously with parameters: ^appK_d (0 mV) = 2.71 ± 0.20 × 10⁻⁵ M and Z = 1.76 ± 0.02.

Figure 3.

Kinetics of hyperpolarization-induced CNGA1 block by extracellular PhTx. (A) Current transient elicited by stepping membrane voltage from 100 to −150 mV in the continued presence of 0.1 µM of extracellular PhTx. The superimposed curve is a single-exponential fit. (B) Reciprocal of the time constant (mean ± SEM; n = 4–6) for channel block (1/τ_on), obtained from fits as shown in A, is plotted against the extracellular concentration of PhTx for eight voltages. The (unresolved) lines through the data are linear fits whose slope represents the apparent second-order rate constant (k_on) for blocker binding. (C) Natural logarithm of k_on from B is plotted against membrane voltage. The plot is fitted with the equation ln k_on = ln k_on (0 mV) − z_onVF/RT, yielding k_on (0 mV) = 1.04 ± 0.06 × 10⁹ M⁻¹s⁻¹ (open circle) and z_on = 0.01 ± 0.01.

Figure 4.

Kinetics of depolarization-induced recovery from CNGA1 block by extracellular PhTx. (A) Current transient elicited by stepping the membrane voltage from −200 to −50 mV in the presence of 0.1 µM of extracellular PhTx. The superimposed curve is a single-exponential fit. (B) Natural logarithm of the reciprocal of the time constant (mean ± SEM; n = 4–6) for channel unblock (1/τ_off, an estimate of the apparent off-rate constant k_off) at four voltages, obtained from fits as shown in A, is plotted against the concentration of extracellular PhTx. Lines through the data represent averages over the three concentrations tested at each voltage. (C) Natural logarithm of k_off from B is plotted against membrane voltage. The plot is fitted with the equation ln k_off = ln k_off (0 mV) + z_offVF/RT, yielding k_off (0 mV) = 9.65 ± 0.35 × 10³ s⁻¹ and z_off = 1.31 ± 0.02.

Figure 5.

Voltage-dependent block by intracellular PhTx. (A) Macroscopic current traces recorded from an inside-out patch containing CNGA1 channels in the absence or presence of 0.3 µM of intracellular PhTx. Currents were activated with 2 mM of intracellular cGMP and elicited with the voltage protocol shown. Dotted line indicates 0 current level. (B) Fraction of current not blocked (mean ± SEM; n = 5–10) by intracellular PhTx is plotted against membrane voltage. Curves are fits of a single Boltzmann function to the four datasets simultaneously with parameters: ^appK_d (0 mV) = 8.58 ± 0.38 × 10⁻⁷ M and Z = 2.67 ± 0.04. (C) Kinetics of depolarization-induced CNGA1 block by intracellular PhTx. Natural logarithm of second-order rate constant k_on, determined as in Fig. 3, at three concentrations of intracellular PhTx (0.3, 1, and 10 µM; n = 8) is plotted against membrane voltage. The plot is fitted with the equation ln k_on = ln k_on (0 mV) + z_onVF/RT, yielding k_on (0 mV) = 4.23 ± 0.34 × 10⁷ M⁻¹s⁻¹ and z_on = 0.23 ± 0.01. (D) Kinetics of hyperpolarization-induced recovery from CNGA1 block by intracellular PhTx. Natural logarithm of k_off, determined as in Fig. 4, at three concentrations of intracellular PhTx (0.1, 0.3, and 1 µM; n = 5) is plotted against membrane voltage. The plot is fitted with the equation ln k_off = ln k_off (0 mV) − z_offVF/RT, yielding k_off (0 mV) = 33 ± 1 s⁻¹ and z_off = 1.60 ± 0.01.

Figure 6.

Voltage dependence of intracellular PhTx block under low Na⁺ conditions. (A) Macroscopic current traces recorded in an inside-out patch containing CNGA1 channels in the absence or presence of 5 µM of intracellular PhTx in symmetrical 30 mM Na⁺. Currents were activated with 2 mM of intracellular cGMP and elicited with the voltage protocol shown. Dotted line indicates 0 current level. (B) Fraction of current not blocked (mean ± SEM; n = 4–8) by the indicated concentrations of intracellular PhTx is plotted against membrane voltage. Curves are fits of Eq. 3 to the three datasets simultaneously with Z₁ fixed at 0. The best-fit parameters were: K₁ = 1.04 ± 0.03 × 10⁻⁵ M, K₂ = 2.03 ± 0.14 × 10⁻³, and Z₂ = 2.08 ± 0.04. (C) Fraction of current not blocked (mean ± SEM; n = 8) by 1 µM of intracellular PhTx in the presence of 0.02 mM (filled circles) or 2 mM (open circles; taken from B) cGMP is plotted against membrane voltage. Curves are fits of Eq. 3 to both datasets simultaneously, with Z₁ set to 0 and K₁ common to both cGMP curves. The best-fit parameters were: K₁ = 7.65 ± 0.22 × 10⁻⁶ M for both cGMP concentrations; K₂ = 1.29 ± 0.08 × 10⁻² and Z₂ = 1.92 ± 0.05 for 0.02 mM cGMP; and K₂ = 1.72 ± 0.18 × 10⁻³ and Z₂ = 2.29 ± 0.06 for 2 mM cGMP. (D) Kinetics of depolarization-induced CNGA1 block by intracellular PhTx in 30 mM Na⁺. Natural logarithm of k_on, determined as in Fig. 3, at four concentrations (0.03, 0.1, 0.3, and 1 µM; n = 6) of intracellular PhTx is plotted against membrane voltage. The plot is fitted with the equation ln k_on = ln k_on (0 mV) + z_onVF/RT, yielding k_on (0 mV) = 8.42 ± 0.68 × 10⁸ M⁻¹s⁻¹ and z_on = 0.14 ± 0.01. (E) Kinetics of hyperpolarization-induced recovery from CNGA1 block by intracellular PhTx in 30 mM Na⁺. Natural logarithm of k_off, determined as in Fig. 4, at four concentrations (0.1, 0.3, 0.6, and 1 µM; n = 6) of intracellular PhTx is plotted against membrane voltage. The plot is fitted with the equation ln k_off = ln k_off (0 mV) − z_offVF/RT, yielding k_off (0 mV) = 27 ± 2 s⁻¹ and z_off = 1.10 ± 0.02.

Figure 7.

Voltage dependence of intracellular and extracellular PhTx block at saturating and subsaturating cGMP concentrations. (A) Normalized current transients elicited by stepping membrane voltage from −80 to 120 mV in the absence (top) or presence (bottom) of 0.3 µM of intracellular PhTx and of 2 mM (black) or 20 µM cGMP (blue; average of 20 consecutive traces from the same patch). The current in 20 µM cGMP is sevenfold smaller than that in 2 mM. (B) Normalized current transients, elicited by stepping membrane voltage from 120 to −80 mV, in the presence of 0.3 µM of intracellular PhTx and of 2 mM (black; average of five consecutive traces from the same patch) or 20 µM cGMP (blue; average of 10 consecutive traces from the same patch). (C) Normalized current transients elicited by stepping membrane voltage from 100 to −150 mV in the presence of 0.1 µM of extracellular PhTx and of 2 mM (black) or 20 µM cGMP (blue; average of 20 consecutive traces from the same patch). (D) Normalized current transients elicited by stepping membrane voltage from −200 to −50 mV in the presence of 0.1 µM of intracellular PhTx and of 2 mM (black) or 20 µM cGMP (blue; average of 20 consecutive traces from the same patch).

Figure 8.

Voltage-dependent block by intracellular bis-QA_C10. (A) Chemical structure of bis-QA_C10. (B) Macroscopic current traces recorded from an inside-out patch containing CNGA1 channels in the absence or presence of 5 mM of intracellular bis-QA_C10. Currents were elicited by first stepping the voltage from the 0-mV holding potential to −150 mV, and then testing voltages between −150 and 150 mV in 10-mV increments before returning to the holding potential. For clarity, only traces every 20 mV are shown. Dotted line indicates 0 current level. (C) I-V curves (mean ± SEM; n = 5) determined at the end of the test pulses in the absence or presence of 5 mM bis-QA_C10. (D) Fraction of current not blocked (mean ± SEM; n = 5) by 5 mM bis-QA_C10 is plotted against membrane voltage. The solid curve is a fit of Eq. 10 to the data from −150 to 90 mV (arrow) with K_B1 = 3.17 ± 0.05 × 10⁻² M, K_B2 = 2.23 ± 0.14 × 10⁻², K_B2-Na = 2.67 ± 0.09 M⁻¹, and Z = 1.11 ± 0.02. Dotted curve is a fit to the data from −150 to 90 mV (arrow) of a Boltzmann function (similar to Eq. 1, except for a nonunity asymptote at hyperpolarized potentials) with parameters ^appK_d = 1.23 ± 0.08 × 10⁻² M and Z = 0.83 ± 0.03.

Figure 9.

CNGA1 block by six intracellular QA compounds. Macroscopic currents recorded in the absence (left column) or presence (middle column) of the indicated blocker concentration. Bars, 1 nA and 20 ms. Chemical structures of the blockers are shown on the right.

Figure 10.

Intracellular QA blockers render current–voltage relations of CNGA1 channels inwardly rectifying. Mean I-V curves (mean ± SEM; n = 3–8) in the absence (control) or presence of six QAs, each at two concentrations.

Figure 11.

Voltage-dependent block by six intracellular QAs. Fraction of current not blocked (mean ± SEM; n = 3–8) plotted against voltage for six QAs, each at two concentrations. Curves were obtained by fitting Eq. 10 to the two datasets in each plot simultaneously. The resulting parameters are listed in Table I.

Figure 12.

CNGA1 block by intracellular bis-QA_C10 in 65 and 130 mM of symmetrical Na⁺. (A) The fraction of current not blocked (mean ± SEM; n = 4–7) in the presence of 65 mM (open symbols) and 130 mM (filled symbols) of symmetrical Na⁺ by 5 mM (black symbols) and 25 mM (blue symbols) bis-QA_C10 is plotted against voltage. Curves were obtained by fitting Eq. 10 to the four datasets simultaneously. The resulting parameters are listed in Table I. (B) Data in A scaled to unity at −150 mV. Curves were obtained by fitting Eq. 7 to the four datasets simultaneously. The best-fit parameters were: K_B = 7.97 ± 0.31 × 10⁻⁴ M, K_B-Na = 8.97 ± 0.16 × 10⁻², and Z = 1.00 ± 0.01.

Figure 13.

Simulated curves of a Boltzmann function (gray curve) and of the three-state ion displacement model (black curve), both for the case of a positively charged intracellular blocker. The Boltzmann curve was generated from Eq. 1 with [B] = 5 mM, ^appK_d = 6.5 mM, and Z = 1. The black curve was generated from Eq. 7 with [B] = 5 mM, [Na⁺] = 130 mM, K_B = 1 mM, K_B-Na = 0.05, and Z = 1.

Figure 14.

Models for multistate channel block. Binding of a blocker denoted as “B” to a channel (Ch) produces two blocked states (ChB₁ and ChB₂) in sequential (A) or parallel (B) steps. K_x are equilibrium constants with effective valences Z_x; k_x are rate constants and z_x their effective valences.

Figure 15.

Ion displacement models for channel block. (A) Minimal three-state ion displacement model for voltage-dependent block. The transmembrane voltage drops exclusively across the narrow part of the pore that can only be occupied by permeant ions. The number of permeant ions in the selectivity filter is inconsequential to the model, and the number of ions in the inner pore is set at the minimum of one. The channel can exist in two nonblocked states with (ChNa) or without (Ch) a permeant Na⁺ ion at the inner pore site (internal to the narrow selectivity filter) and one blocked state (ChB). The upper blocking transition is voltage independent as the blocker binds in the empty inner pore with equilibrium constant K_B, whereas the lower transition (equilibrium constant K_B-Na) is voltage dependent as the blocker displaces the Na⁺ ion. (B) Ion displacement model with sequential blocking steps. The two leftmost nonblocked states are equivalent to those of the minimal three-state model in A, with (ChNa) and without (Ch) a Na⁺ ion in the inner pore. The blocker may bind (common equilibrium constant K_B1) to the shallow site of either nonblocked form. It then binds at the deep binding site (ChB₂). The transition that involves Na⁺ displacement is characterized by K_B2-Na and is voltage dependent, whereas the one that does not involve Na⁺ displacement is characterized by K_B2 and is voltage independent.