An analysis of philanthotoxin block for recombinant rat GluR6(Q) glutamate receptor channels

Abstract

1. The action of philanthotoxin 343 (PhTX) on rat homomeric GluR6(Q) recombinant glutamate receptor channels was analysed using concentration-jump techniques and outside-out patches from HEK 293 cells. Both onset and recovery from block by external PhTX were dependent on the presence of agonist, indicating that channels must open for PhTX to bind and that channel closure can trap PhTX. 2. Block by external PhTX developed with double-exponential kinetics. The rate of onset of the fast component of block showed an exponential increase per 27 mV hyperpolarization over the range -40 to -100 mV. The rate of onset of the slow component of block showed a non-linear concentration dependence indicating a rate-limiting step in the blocking mechanism. 3. The extent of block by 1 microM external PhTX was maximal at -40 mV and did not increase with further hyperpolarization; the rate of recovery from block by external PhTX increased 6-fold on hyperpolarization from -40 to -100 mV suggesting that PhTX permeates at negative membrane potentials. 4. Apparent Kd values for block by external PhTX estimated from dose-inhibition experiments decreased 300-fold on hyperpolarization from +40 mV (Kd, 19.6 microM) to -40 mV (Kd, 69 nM); there was little further increase in affinity with hyperpolarization to -80 mV (Kd, 56 nM), consistent with permeation of PhTX at negative membrane potentials. 5. Block by internal PhTX showed complex kinetics and voltage dependence. Analysis with voltage ramps from -120 to +120 mV indicated a Kd at 0 mV of 20 microM, decreasing e-fold per 16 mV depolarization. However, at +90 mV the extent of block by 1 and 10 microM internal PhTX (73 % and 95 %, respectively) reached a maximum and did not increase with further depolarization. 6. Voltage-jump analysis of block by 100 microM internal PhTX revealed partial trapping. With 100 ms jumps from -100 to -40 mV, onset and recovery from block were complete within 5 ms. With jumps of longer duration the extent of block increased, with a time constant of 8.1 s, reaching 84 % at 30 s. On repolarization to -100 mV, recovery from block showed fast and slow components. 7. The amplitude of the slow component of block by internal PhTX showed a biphasic voltage dependence, first increasing then decreasing with progressive depolarization. Maximum block was obtained at 0 mV. 8. Our results suggest that PhTX acts as an open channel blocker; however, provided that the toxin remains bound to the channel, an allosteric mechanism destabilizes the open state, inducing channel closing and trapping PhTX. Strong depolarization for internal PhTX, or strong hyperpolarization for external PhTX, forces the toxin to permeate before it triggers entry into closed blocked states.

Figure 1. Block by external PhTX requires activation by agonist

A, rapid onset of block by 1 μM external PhTX (PhTX_o, open bar) applied to an outside-out patch after channels have opened in response to 50 μM domoate (filled bar). B, in the same patch PhTX failed to produce block of the response to domoate when applied to closed channels prior to the application of agonist, but rapidly attenuated the inward current generated by domoate as soon as channels opened in response to a concentration-jump application of agonist. Both responses were recorded at -60 mV. Dotted lines indicate the holding current prior to application of agonist. Curves fitted to the onset of block by PhTX represent double-exponential functions of time constants: τ_fast = 370, τ_slow = 790 ms (36 %, A) and τ_fast = 360, τ_slow = 890 ms (13 %, B). C, mean values from 4 experiments for block by 1 μM PhTX plotted relative to the amplitude of control responses to 50 μM domoate. The large amplitude of peak responses to domoate after prior application of PhTX in the absence of agonist (97 ± 7 %) indicates minimal closed channel block; steady-state (ss) current during block by 1 μM PhTX is similar when toxin is applied prior to or after application of agonist.

Figure 2. Fast and slow components of block by external PhTX

A, responses from the same patch to 4 concentrations of PhTX (open bars) applied during responses to 50 μM domoate (filled bars). Dashed lines represent the holding current at -60 mV prior to application of agonist. The external solution contained 200 and 800 μM external Ca²⁺ and Mg²⁺, respectively, to improve patch stability. The apparent increase in noise preceding the response to PhTX is due to a change in sample clock frequency. Lines drawn through the data points are double-exponential functions fitted to the onset of block by external PhTX. Dotted lines show the exponential for the slow component of onset of block. The time constants were: 0.3 μM PhTX, τ_fast = 1.68 s, τ_slow = 4.12 s (53 %); 1 μM PhTX, τ_fast = 0.58 s, τ_slow = 1.36 s (43 %); 3 μM PhTX, τ_fast = 207 ms, τ_slow = 810 ms (26 %); 30 μM PhTX, τ_fast = 32 ms, τ_slow = 380 ms (9 %). B, rates for the fast component of onset of block (1/τ_fast) plotted against PhTX concentration; the continuous line shows a linear fit to mean values from 4 patches which gives an estimate for the association rate constant for PhTX at -60 mV of 1.3 × 10⁶ M⁻¹ s⁻¹. C, plot of PhTX concentration versus rate of onset of the slow component of block (1/τ_slow, •) and the percentage of the onset of block accounted for by the slow component (○) clearly demonstrates non-linear concentration dependence and suggests saturation at high concentrations of PhTX (lines connecting the data points have no significance).

Figure 3. Recovery from block by PhTX requires activation by agonist

A, slow recovery from block of responses to 50 μM domoate (filled bars) by 10 μM external PhTX (open bar) at -60 mV. Two responses from the same patch are shown superimposed; one was recorded with agonist applied continuously following removal of PhTX, the second was recorded with a 60 s wash with control solution following termination of the application of PhTX before a second application of agonist. The open arrow indicates 80 % recovery from block at 60 s when PhTX was removed in the presence of agonist; the filled arrow recorded after a 60 s wash with control solution indicates the amplitude of the ‘instantaneous’ component of the response to domoate due to activation of a small fraction of unblocked channels, which is followed by a slow increase in current due to recovery from block by PhTX. B summarizes results from 6 patches (means ± s.d.) analysed using the same protocol, and indicates that recovery from block by PhTX is greatly enhanced by the application of agonist.

Scheme 1

Figure 4. Rate of onset of block by external PhTX increases with hyperpolarization

A, block by 1 μM PhTX (open bar) of responses to 50 μM domoate (filled bar) at holding potentials from -100 to +40 mV. The amplitudes of control responses prior to the onset of block are scaled to the response at -100 mV; the upper and lower dotted lines represent the unblocked current at +40 mV and the holding current prior to application of agonist, respectively; the dashed line indicates saturation of block for responses at -100, -60 and -40 mV. Lines through the data points show double-exponential functions fitted to the onset of block by PhTX, except for responses at +40, +20 and -20 mV, which were well fitted by single-exponential functions; to calculate the extent of block at equilibrium at these potentials, fits were extended until they reached steady state. B, time constants for the fast component of onset of block, τ_fast (○), with 1 μM PhTX plotted versus membrane potential (pooled data from 3 to 8 patches; ▵ indicates single exponential fits at +40, +20 and -20 mV); the time constant of onset of block decreased e-fold per 27.1 mV hyperpolarization (the line through the data points was fitted by least squares). Equilibrium block (•, measured relative to control) was well fitted by a modified Boltzmann function (V_½, 21 mV; k,12 mV), accounting for the incompleteness of block at negative membrane potentials (dashed line). The dotted line indicates a Woodhull fit assuming complete block, which gave K_d(0) of 308 nM, zδ = 1.7.

Figure 5. Anomalous voltage dependence for block by external PhTX revealed by dose-response analysis

A, block of responses to 50 μM domoate (filled bars) at -80 mV by 10 nM to 1 μM PhTX (open bars) as indicated; the upper and lower dotted lines represent the unblocked current at -80 mV and the holding current, respectively. B, PhTX dose-inhibition plots at holding potentials between +40 mV and -80 mV (means ± s.d., pooled data from 5 to 13 patches) fitted by a single-binding site model to estimate PhTX affinity as a function of membrane potential. The K_d values were: +40 mV, 19.6 μM; +20 mV, 1.9 μM; -20 mV, 126 nM; -40 mV, 69 nM; -80 mV, 56 nM. C, when plotted on a logarithmic scale, K_d values at negative membrane potentials deviate from the expected exponential increase predicted by extrapolation of K_d values at +40 and +20 mV (dotted line). • indicates the K_d(0) of 308 nM obtained from Woodhull analysis of block by 1 μM PhTX (Fig. 4).

Figure 6. Recovery from block by external PhTX is accelerated by hyperpolarization

A, block of responses to 50 μM domoate (filled bar) by 10 μM PhTX (open bar) at -80 mV, with recovery from block measured at -20, -60 and -100 mV (see voltage protocol below current traces). Open arrows indicate the initial amplitude of responses at -80 mV after 30 s recovery from block at the indicated membrane potentials. Lines fitted to the recovery responses represent double-exponential functions of time constants: -100 mV, τ_fast = 2.9 s, τ_slow = 11.2 s (53 %); -60 mV, τ_fast = 4.6 s, τ_slow = 24.7 s (72 %); -20 mV, τ_fast = 14.4 s, τ_slow = 56.6 s (72 %). B, recovery from block measured after 30 s (•) at the indicated membrane potentials (pooled data from 5 patches) shows biphasic voltage dependence. Weighted time constants for recovery from block (○) reveal a striking increase in rate of unblock at hyperpolarized membrane potentials.

Scheme 2

Figure 7. Low-affinity block by internal PhTX

A, currents recorded in response to 50 μM domoate during voltage ramp protocols; the pipette solution contained PhTX at the indicated concentrations (internal PhTX, PhTX_i) and no ATP. To maintain equilibrium block during changes in membrane potential, a slower ramp rate was required with 1 μM internal PhTX (-80 to +120 mV, 0.07 V s⁻¹; upper panel) than with 1000 μM internal PhTX (-120 to +100 mV, 0.44 V s⁻¹; lower panel). Holding potential, -60 mV. Traces are leak subtracted. Dotted lines represent zero current. Note the slow decline in the response to domoate at -60 mV observed with 1000 μM but not 1 μM PhTX. B, G-V curves for block by internal PhTX, normalized to the response recorded at the most negative membrane potential tested, for pooled data from 4 (1 μM), 8 (10 μM), 3 (100 μM) and 5 (1000 #181;M) patches, show a leftward shift with increasing PhTX concentrations (mean V_½ values from Boltzman analysis are +41.5, +11.4, -22.6 and -50.4 mV, with slope factors of 18.4, 15.2, 13.5 and 14.0 mV⁻¹ for 1, 10, 100 and 1000 μM PhTX, respectively). At positive potentials complete block was observed for both 1000 μM and 100 μM internal PhTX, whereas for 1 and 10 μM PhTX maximum block reached an asymptote and failed to increase with further depolarization. Dotted lines show the G-V relationships expected for a non-permeant blocker and represent fits of the Woodhull model of ion channel block to data points more negative than +40 mV for 10 μM PhTX and +30 mV for 1 μM PhTX.

Figure 8. Tail current analysis of partial trapping by internal PhTX

A, voltage steps from -100 to -40 mV for different durations reveal slow block by 100 μM internal PhTX of responses to 50 μM domoate. The voltage protocol is shown below a series of superimposed current traces recorded from the same patch. The dotted line represents zero current. The filled arrow on the left indicates the current amplitude at -40 mV predicted by analysis of ramp I-V relationships (Fig. 7). Lines through the data points are double-exponential functions fitted to the slow relaxations which followed the instantaneous increase in current on return to -100 mV (open arrows). The time constants of unblock, τ_fast and τ_slow, were (respectively): 2.2 s and 7.9 s (45 %) for 3 s steps to -40 mV; 2.3 s and 11.7 s (46 %) for 10 s steps; and 2.4 s and 12.1 s (47 %) for 30 s steps. B, the amplitude of the slow relaxation relative to the control response at -100 mV plotted against test pulse duration for observations from 6 to 7 patches per data point. Fitting an exponential function yielded a time constant of 8.1 s for the development of the slow component of block by internal PhTX. C, responses for voltage steps 10 s in duration from -100 mV to different test potentials as indicated; the upper dotted line shows zero current; because traces were not leak subtracted there was a small outward current at +120 mV which was the same value as that recorded during voltage ramps in the absence of agonist. Note that the amplitude of the slow component of recovery from block (open arrows) decreases with depolarization. D, biphasic voltage dependence for the slow component of block by 100 μM internal PhTX revealed by analysis of tail currents at -100 mV (mean values from 3 to 9 patches per data point). The amplitude of the slow component of the tail current at -100 mV was estimated by fitting exponentials to responses like those shown in C and extrapolating these to the time at which the membrane potential was returned from the test potential to -100 mV.