Boccaccio A et al. (2004), Binding of kappa-conotoxin PVIIA to Shaker K+ c...

XB-ART-3347

J Gen Physiol 2004 Jul 01;1241:71-81. doi: 10.1085/jgp.200409048.

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Binding of kappa-conotoxin PVIIA to Shaker K+ channels reveals different K+ and Rb+ occupancies within the ion channel pore.

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

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The x-ray structure of the KcsA channel at different [K(+)] and [Rb(+)] provided insight into how K(+) channels might achieve high selectivity and high K(+) transit rates and showed marked differences between the occupancies of the two ions within the ion channel pore. In this study, the binding of kappa-conotoxin PVIIA (kappa-PVIIA) to Shaker K(+) channel in the presence of K(+) and Rb(+) was investigated. It is demonstrated that the complex results obtained were largely rationalized by differences in selectivity filter occupancy of this 6TM channels as predicted from the structural work on KcsA. kappa-PVIIA inhibition of the Shaker K(+) channel differs in the closed and open state. When K(+) is the only permeant ion, increasing extracellular [K(+)] decreases kappa-PVIIA affinity for closed channels by decreasing the "on" binding rate, but has no effect on the block of open channels, which is influenced only by the intracellular [K(+)]. In contrast, extracellular [Rb(+)] affects both closed- and open-channel binding. As extracellular [Rb(+)] increases, (a) binding to the closed channel is slightly destabilized and acquires faster kinetics, and (b) open channel block is also destabilized and the lowest block seems to occur when the pore is likely filled only by Rb(+). These results suggest that the nature of the permeant ions determines both the occupancy and the location of the pore site from which they interact with kappa-PVIIA binding. Thus, our results suggest that the permeant ion(s) within a channel pore can determine its functional and pharmacological properties.

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	SCHEME I.
	Figure 1. . Îº-PVIIA blocks Shaker currents differently in symmetrical K+ and Rb+. (Left) Current responses to voltage steps from a holding potential of â100 mV to â20 and +60 mV, under control conditions and with 200 nM Îº-PVIIA in symmetrical K+ (top) and 500 nM Îº-PVIIA in symmetrical Rb+ (bottom). (Middle) Current in toxin is scaled to the control to evidence the effect of the toxin. Note that besides a reduction in the current amplitude, also a modification in the kinetics of the current is observed. (Right) Ratio between the toxin and control current traces. The dotted lines correspond to the tonic unblock for closed state, respectively 0.58 in K+ and 0.37 in Rb+, corresponding to a KC of 265 nM and 293 nM, respectively. The single exponential fits are superimposed to the traces: in symmetrical K+ (top) at +60 mV, Ï = 9.56 ms and U = 0.76; at â20 mV, Ï = 27.2 ms and U = 0.26. In symmetrical Rb+ (bottom) at +60 mV, Ï = 4.6 ms and U = 0.87; at â20 mV, Ï = 5.65 ms and U = 0.60.
	Figure 2. . Îº-PVIIA block of Shaker currents in symmetric and asymmetric ionic Rb+ or K+ solutions. Current records in response to IV protocols (VH = â100 mV, VP from â50 mV to +70 mV, every 20 mV) recorded with outside-out patch clamp experiments in symmetrical 115 mM KCl (top left), symmetrical 115 mM RbCl (top right), Kext/Rbint (bottom left), and Rbext/Kint (bottom right) before and after the addition to the bath of Îº-PVIIA (concentration indicated in the figure). The reversal potential is +5 mV in symmetrical 115 mM KCl, +3 mV in symmetrical 115 mM RbCl, +4 mV in Kext/Rbint, and â5 mV in Rbext/Kint.
	Figure 3. . Relaxation of Îº-PVIIA block of Shaker currents depends on the permeant cation, Rb+ or K+. (A) Ratios between toxin and control current traces from the IV experiments in Fig. 2 from IV protocols (VH = â100 mV, VP from â50 mV to +70 mV, every 20 mV) in different ionic conditions (symmetrical K+ with 200 nM Îº-PVIIA, symmetrical Rb+ with 500 nM, Kext/Rbint with 300 nM, and Rbext/Kint with 400 nM). (B) Semilogarithmic plot of the mean values Â± SD of the dissociation constants (KO) versus test potential for symmetrical K+ (empty squares) or symmetrical Rb+ (filled squares). The continuous lines correspond to the exponential fit with KO(V) = KO (0)*exp(âV/vs), with KO = 146 nM at 0 mV and vs = 43 mV for symmetrical K+ and 1023 nM and vs = 76 mV for symmetrical Rb+. (C) KO versus Vp for Kext/Rbint and Rbext/Kint together with the fits from B.
	Figure 4. . Relaxation of Îº-PVIIA binding to closed Shaker-Î channels after a depolarizing pulse. Outside-out patch clamp experiments performed in symmetrical 115 mM KCl (A) or 115 mM RbCl (B; same experiment as in Fig. 2). Superimposed records of responses to double pulse stimulation before and after the addition to the bath respectively of 200 nM and 500 nM Îº-PVIIA. Each stimulation consisted of a conditioning pulse to VP, followed with a variable pulse interval, Ti, by an identical test pulse: (A) duration conditioning pulse = 300 ms, VP = â20 mV, VH = â100 mV, Ti shown 80 and 680 ms, and 5.2 s; (B) duration conditioning pulse = 100 ms, VP = +40 mV, VH = â100 mV, Ti shown 10, 34, 66, and 130 ms. (A) Symmetrical 115 mM KCl. The conditioning pulse induces an increase of toxin block that appears as a hump in the first response; in the second response for small Ti values no peak is observed. (A, inset) Amplitude of the second response at early time normalized to the control response and plotted as a function of Ti. The continuous line is the best exponential fit with Ï = 720 ms, and the dotted horizontal line corresponds to the asymptotic value of U = ITx/ICt = 0.50, giving KC = 200 nM. From U and Ï from Eqs. 2 and 3, we estimate kCoff = 0.69 sâ1 and kCon = 3.5 Î¼Mâ1sâ1. (B) Symmetrical 115 mM RbCl. The toxin equilibrates very fast with the closed state and 60 ms is enough to reach the equilibrium: the responses to the second pulse are indistinguishable after 66 ms. The process of toxin equilibration with the fraction of channels that are in the closed state has the same time scale of the deactivation process. Consequently, a kinetic analysis of the reblock is not possible.
	Figure 5. . Cartoon of Îº-PVIIA binding to Shaker K+ channels in the presence of Rb+ or K+. Îº-PVIIA binding interacts differently with the ion(s)âchannel complex depending on the state of the channel (top panel, closed state; bottom panel, open state) and on the pore occupancy of the permeant ion species (left panel, K+; right panel, Rb+). Ion binding sites within the selectivity filter are numbered from 1 to 4, while S0 is the extracellular binding site for K+. A detailed description is given in the Discussion.

References [+] :

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