Milescu M et al. (2013), Opening the shaker K+ channel with hanatoxin.

XB-ART-48204

J Gen Physiol 2013 Feb 01;1412:203-16. doi: 10.1085/jgp.201210914.

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Opening the shaker K+ channel with hanatoxin.

Milescu M , Lee HC , Bae CH , Kim JI , Swartz KJ .

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Voltage-activated ion channels open and close in response to changes in membrane voltage, a property that is fundamental to the roles of these channels in electrical signaling. Protein toxins from venomous organisms commonly target the S1-S4 voltage-sensing domains in these channels and modify their gating properties. Studies on the interaction of hanatoxin with the Kv2.1 channel show that this tarantula toxin interacts with the S1-S4 domain and inhibits opening by stabilizing a closed state. Here we investigated the interaction of hanatoxin with the Shaker Kv channel, a voltage-activated channel that has been extensively studied with biophysical approaches. In contrast to what is observed in the Kv2.1 channel, we find that hanatoxin shifts the conductance-voltage relation to negative voltages, making it easier to open the channel with membrane depolarization. Although these actions of the toxin are subtle in the wild-type channel, strengthening the toxin-channel interaction with mutations in the S3b helix of the S1-S4 domain enhances toxin affinity and causes large shifts in the conductance-voltage relationship. Using a range of previously characterized mutants of the Shaker Kv channel, we find that hanatoxin stabilizes an activated conformation of the voltage sensors, in addition to promoting opening through an effect on the final opening transition. Chimeras in which S3b-S4 paddle motifs are transferred between Kv2.1 and Shaker Kv channels, as well as experiments with the related tarantula toxin GxTx-1E, lead us to conclude that the actions of tarantula toxins are not simply a product of where they bind to the channel, but that fine structural details of the toxin-channel interface determine whether a toxin is an inhibitor or opener.

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Species referenced: Xenopus laevis
Genes referenced: kcnb1

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	Figure 2. Generating enhanced hanatoxin receptors in the Shaker Kv channel. (A) Sequence alignment for S3 helices in Kv2.1 (blue) and Shaker (black) Kv channels. Conserved residues are shown in bold lettering. In the Shaker Î3 construct, three residues (asterisks) were mutated to the corresponding residues in Kv2.1 (L327I, A328F, and V331E). (B and C) Voltage-activated Kv channel currents in the absence (black) and presence (red) of 2 ÂµM hanatoxin. Recordings in B and C are from the same cell. The gray line indicates the level of zero current. (D) Normalized G-V relations in the absence (black circles) and presence of 200 nM (red triangles) or 2 ÂµM (red circles) hanatoxin. Conductance was measured using tail currents. Smooth curves are Boltzmann fits to the data normalized to the maximal conductance in control external solution, with the following V1/2 and z values: â15 mV and 3 for control, â36 mV and 2.9 for 200 nM, and â39 mV and 2.8 for 2 ÂµM hanatoxin. Error bars indicate SEM. (n = 5). (E) Concentration dependence of shifting the V1/2 for activation of the wild-type Shaker Kv channel (black circles) and the Shaker Î3 Kv channel (red circles). The external solution contained 50 mM Rb+. Error bars indicate SEM (n = 3).
	Figure 3. Recovery of Shaker Kv channels after removal of hanatoxin from the external solution. (A) Voltage-activated Rb+ currents recorded for the Shaker Î3 Kv channel in control solution before applying toxin (gray trace), after applying 2 ÂµM hanatoxin to the external recording solution and allowing currents to reach equilibrium (red trace), or after removal of the toxin from the recording chamber (black traces). (B) Time course for recovery after removing hanatoxin from the external recording solution for the wild-type Shaker Kv channel (open black symbols) or for the Shaker Î3 Kv channel (closed black symbols). Tail current amplitude was measured and normalized to the value obtained when the channel was in equilibrium with hanatoxin (red symbol).
	Figure 4. Influence of hanatoxin on the kinetics of activation and deactivation for the Shaker Î3 Kv channel. (A) Kinetics of channel opening in the absence (black traces) or presence (red traces) of 2 ÂµM hanatoxin. (B) Mean time constant (Ï) from single-exponential fits to channel activation in the absence (black) or presence of 100 nM (red squares), 200 nM (red triangles), and 2 ÂµM (red circles) hanatoxin. (C) Kinetics of channel closing in the absence (black traces) or presence (red traces) of 2 ÂµM hanatoxin. (D) Mean time constant (Ï) from single-exponential fits to channel deactivation in the absence (black circles) or presence of 2 ÂµM hanatoxin (red circles). The external solution contained 50 mM Rb+, and error bars indicate SEM (n = 3).
	Figure 5. Influence of hanatoxin on gating charge movement in the Shaker Î3/W434F channel. (A) Families of gating currents recorded from one cell bathed in control external solution (black) and another preequilibrated with 200 nM hanatoxin (red). Leak, background, and capacitive currents were subtracted using a P/â4 protocol. (B) Normalized Q-V relation in the absence (black circles) and presence of 200 nM hanatoxin (red circles). In both cases, on and off gating currents were integrated, and their average was normalized to the maximal charge measured for depolarizations to +50 mV. Smooth curves are fits of Boltzmann functions to the data with the following V1/2 and z values: â40 mV and 3.7 for control, and â53 mV and 1.3 for 200 nM hanatoxin. Error bars indicate SEM (n = 4). (C) Mean time constants (Ï) for charge movement obtained by fitting single-exponential functions to the decay of on gating currents in the absence (black circles) and presence (red circles) of 200 nM hanatoxin. Error bars indicate SEM (n = 6).
	Figure 6. Hanatoxin facilitates opening in the Shaker Î3/ILT channel. (A) Opening of the Shaker Î3/ILT channel with a depolarizing step from 0 mV to +100 mV in the absence (black) and presence of 2 ÂµM hanatoxin (red). The gray line indicates the level of zero current. (B) Normalized I-V relation in the absence (black circles) and presence of 2 ÂµM hanatoxin (red circles). The external solution contained 50 mM Rb+. Error bars indicate SEM (n = 5).
	Figure 7. Influence of hanatoxin on paddle chimeras between Kv2.1 and Shaker Kv channels. (A) Sequence alignments for the S3-S4 regions of Kv2.1 (blue), Shaker Î3 (black), and two chimeras wherein the S3b-S4 paddle motifs were swapped between the two Kv channels. Conserved residues are shown using bold lettering. (B) Voltage-activated Kv channel currents for two chimeras in the absence (black) and presence (red) of hanatoxin. The top sets of traces are for depolarization to 0 mV, whereas the lower are for depolarization to +60 mV. The gray line indicates the level of zero current. (C) Normalized G-V relations in the absence (black circles) and presence of (red circles) 2 ÂµM hanatoxin. The external solution contained 50 mM K+. Error bars indicate SEM (n = 5).
	Figure 8. Engineering sensitivity to the tarantula toxin GxTx-1E into the Shaker Kv channel. (A) Sequence alignment for S3 helices in Kv2.1 (blue) and Shaker (black) Kv channels. Conserved residues are shown in bold lettering. In the Shaker Î5 construct, five residues (asterisks) were mutated to the corresponding residues in Kv2.1 (L327I, A328F, V330T, V331E, and A332S). (B and C) Normalized G-V relations in the absence (black circles) and presence (red circles) of hanatoxin or GxTx-1E, both applied to the external solution at a concentration of 2 ÂµM. Conductance was measured using tail currents and the external solution contained 50 mM Rb+. Error bars indicate SEM (n = 6).

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase