Terlau H et al. (1999), The block of Shaker K+ channels by kappa-conoto...

XB-ART-12733

J Gen Physiol 1999 Jul 01;1141:125-40.

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The block of Shaker K+ channels by kappa-conotoxin PVIIA is state dependent.

Terlau H , Boccaccio A , Olivera BM , Conti F .

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kappa-conotoxin PVIIA is the first conotoxin known to interact with voltage-gated potassium channels by inhibiting Shaker-mediated currents. We studied the mechanism of inhibition and concluded that PVIIA blocks the ion pore with a 1:1 stoichiometry and that binding to open or closed channels is very different. Open-channel properties are revealed by relaxations of partial block during step depolarizations, whereas double-pulse protocols characterize the slower reequilibration of closed-channel binding. In 2.5 mM-[K+]o, the IC50 rises from a tonic value of approximately 50 to approximately 200 nM during openings at 0 mV, and it increases e-fold for about every 40-mV increase in voltage. The change involves mainly the voltage dependence and a 20-fold increase at 0 mV of the rate of PVIIA dissociation, but also a fivefold increase of the association rate. PVIIA binding to Shaker Delta6-46 channels lacking N-type inactivation or to wild phenotypes appears similar, but inactivation partially protects the latter from open-channel unblock. Raising [K+]o to 115 mM has little effect on open-channel binding, but increases almost 10-fold the tonic IC50 of PVIIA due to a decrease by the same factor of the toxin rate of association to closed channels. In analogy with charybdotoxin block, we attribute the acceleration of PVIIA dissociation from open channels to the voltage-dependent occupancy by K+ ions of a site at the outer end of the conducting pore. We also argue that the occupancy of this site by external cations antagonizes on binding to closed channels, whereas the apparent competition disappears in open channels if the competing cation can move along the pore. It is concluded that PVIIA can also be a valuable tool for probing the state of ion permeation inside the pore.

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	Scheme S1.
	Scheme S2.
	Figure 1. The effect of PVIIA on Shaker-H4 currents in NFR. (A) Superimposed records of voltage-clamp currents from the same oocyte before (top) and after (bottom) the addition of 500 nM PVIIA to the bath solution. All peak currents under toxin are reduced by about the same factor of 10.5, corresponding to a dissociation constant of the toxin from the blocking site of closed channels, . (B) Toxin to control current ratios, U, for the indicated voltage steps. After the time to peak U(t) is well fitted by a single exponential (solid lines) with a plateau level increasing with voltage. Fitted asymptotic values and time constants (ms): 0.20, 27 (0 mV); 0.27, 16 (20 mV); 0.35, 9.7 (40 mV); and 0.46, 6.3 (60 mV). Interpreted as toxin-free probabilities, the asymptotic values indicate apparent toxin dissociation constants, K(O)app, of 125, 185, 270, and 430 nM.
	Figure 2. The effect of PVIIA on Shaker–Δ6-46 channels. (A) Voltage-clamp currents recorded from the same oocyte before (top) and after (bottom) the bath-addition of 200 nM PVIIA. Apart from the longer pulse length and PVIIA concentration, the other experimental conditions are as in Fig. 1. The toxin causes a strong reduction of the early currents, but has a much smaller effect on the steady state conductance activated by step depolarizations. (B) Current ratios, or unblock probabilities, U(t), for the indicated voltage steps. At the half-activation time of the control responses, U is ∼0.19 for all voltages and corresponds to a toxin dissociation constant from closed channels, .
	Figure 5. Relaxation of PVIIA binding to closed Shaker-Δ channels after a depolarizing pulse. (Top) Superimposed records of responses to double-pulse stimulations before (Control) and after the addition of 200 nM PVIIA to NFR; each stimulation consisted of a conditioning 40-ms pulse to 40 mV, followed by an identical test pulse with a variable pulse interval, Ti. Successive stimulations were separated by 3-s pauses at −100 mV. The vertical bars represent 3 μA. (Bottom left) The amplitude of the second response at the half-activation time of control is normalized to the first response and plotted as a function of Ti; control data (○) are virtually unity for all Ti. (Bottom right) Plot of the toxin-to-control ratio of the early second response as a function of Ti; the steady state value of 0.22 for s is the toxin-to-control ratio of the first response; the solid line represents a single-exponential decay from 0.86 to 0.22 with a time constant of 190 ms.
	Figure 3. Dose response of PVIIA effects on Sh-Δ channels. (A) Voltage-clamp currents for a voltage step to +20 mV from a holding potential of −100 mV recorded from the same oocyte exposed at increasing values of [T] as indicated. The trace marked “ from the fit of U(O) data in C.
	Figure 9. PVIIA block of the Shaker K+ channel. The cartoon depicts a Shaker channel blocked by PVIIA, occluding a pore that contains three different binding sites for K+ in its narrowest region. When PVIIA is bound to the channel, it experiences a strong or much weaker repulsion, depending on whether the outer-most site is occupied by a K ion (C:K+:Tx) or not (C:Tx). The transition C:Tx → C:K+:Tx involves the concerted movement of two K+, each traversing a small fraction (∼1/3?) of the membrane voltage.
	Figure 6. Recovery of tonic PVIIA effects in Shaker-H4 channels. Same experimental protocol as in Fig. 5. (Top) Superimposed double-pulse responses before (Control) and after addition of 100 nM PVIIA to NFR. Successive stimulations were separated by 2-s pauses at −100 mV. (Bottom left) The second peak amplitude is normalized to the first and plotted as a function of Ti; control data (○) show the usual recovery from inactivation, which is well fitted by a single exponential (solid line) with a time constant, is the mean ratio of the first peak responses; the solid line represents a single-exponential decay from 0.71 to 0.44 with a time constant of 270 ms.
	Figure 7. Relaxation of PVIIA-block of open Sh-Δ channels in symmetric high-K+ solutions. (A) Currents in response to a standard current–voltage protocol recorded from an outside-out patch without (left) and with (middle) 1 μM PVIIA in the bath K+-Ringer. Plots of the late currents (right) show that most of the inward currents, but not so much the outward currents at large depolarizations, are blocked by 1 μM PVIIA. (B) Direct comparison of three of the control records with the respective records under toxin scaled by a constant factor of 3.3. The records at +20 mV match almost exactly, whereas the others match only during the early rising phase. (C) Current ratios for different voltage steps show a block increase (−30 and +10 mV) or decrease (+50 and +70 mV) that is well fitted by a single exponential (smooth lines). The extrapolated ratio at the half-activation time of the control records is ∼0.3 for all voltages and corresponds to . Fitted time constants and asymptotic values: 6.8 ms, 0.09 (−30 mV); 5.6 ms, 0.25 (10 mV); 4.0 ms, 0.50 (50 mV); and 2.8 ms, 0.62 (70 mV).
	Figure 8. Recovery of tonic PVIIA binding to closed Sh-Δ channels in . (A) Superimposed records of responses to double pulses of 40 ms at −20 mV with variable pulse interval, Ti; the stimulation intervals at −100 mV were 3 s; the inset shows a sample of double responses in real time, whereas the main graph shows the same data with the second response translated to the time of onset of the first. The first pulse induces an increase of toxin block seen as a peak in the response; the peak reappears clearly in the second response only for Ti larger than hundreds of milliseconds. (B) The early amplitude of the second response is normalized to the first response and plotted as a function of Ti to follow the recovery of tonic block (the early amplitude was defined as the mean value of the current during the fixed interval in which the toxin free responses rise from 25 to 75% of their maximum); the solid line is the best fit with a single exponential with a time constant of 430 ms.
	Figure 4. Voltage dependence of PVIIA-binding to open channels. (A) Semi-logarithmic plot of estimates of the first-order dissociation rate constant, k(O)off, derived from U(O) and τ(O) data from the experiment of Fig. 2 (○) and from similar data in high [K]o (•, see Fig. 7). The straight lines are best fits with the equations: .
	Scheme S3.
	Scheme S4.

References [+] :

Anderson, Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength. 1988, Pubmed