29 research outputs found
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The tarantula toxin GxTx detains K+ channel gating charges in their resting conformation.
Allosteric ligands modulate protein activity by altering the energy landscape of conformational space in ligand-protein complexes. Here we investigate how ligand binding to a K+ channel's voltage sensor allosterically modulates opening of its K+-conductive pore. The tarantula venom peptide guangxitoxin-1E (GxTx) binds to the voltage sensors of the rat voltage-gated K+ (Kv) channel Kv2.1 and acts as a partial inverse agonist. When bound to GxTx, Kv2.1 activates more slowly, deactivates more rapidly, and requires more positive voltage to reach the same K+-conductance as the unbound channel. Further, activation kinetics are more sigmoidal, indicating that multiple conformational changes coupled to opening are modulated. Single-channel current amplitudes reveal that each channel opens to full conductance when GxTx is bound. Inhibition of Kv2.1 channels by GxTx results from decreased open probability due to increased occurrence of long-lived closed states; the time constant of the final pore opening step itself is not impacted by GxTx. When intracellular potential is less than 0 mV, GxTx traps the gating charges on Kv2.1's voltage sensors in their most intracellular position. Gating charges translocate at positive voltages, however, indicating that GxTx stabilizes the most intracellular conformation of the voltage sensors (their resting conformation). Kinetic modeling suggests a modulatory mechanism: GxTx reduces the probability of voltage sensors activating, giving the pore opening step less frequent opportunities to occur. This mechanism results in K+-conductance activation kinetics that are voltage-dependent, even if pore opening (the rate-limiting step) has no inherent voltage dependence. We conclude that GxTx stabilizes voltage sensors in a resting conformation, and inhibits K+ currents by limiting opportunities for the channel pore to open, but has little, if any, direct effect on the microscopic kinetics of pore opening. The impact of GxTx on channel gating suggests that Kv2.1's pore opening step does not involve movement of its voltage sensors
The tarantula toxin GxTx detains K<sup>+</sup> channel gating charges in their resting conformation.
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What keeps Kv channels small? The molecular physiology of modesty.
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Azide-Alkyne Click Conjugation on Quantum Dots by Selective Copper Coordination.
Functionalization of nanocrystals is essential for their practical application, but synthesis on nanocrystal surfaces is limited by incompatibilities with certain key reagents. The copper-catalyzed azide-alkyne cycloaddition is among the most useful methods for ligating molecules to surfaces, but has been largely useless for semiconductor quantum dots (QDs) because Cu+ ions quickly and irreversibly quench QD fluorescence. To discover nonquenching synthetic conditions for Cu-catalyzed click reactions on QD surfaces, we developed a combinatorial fluorescence assay to screen >2000 reaction conditions to maximize cycloaddition efficiency while minimizing QD quenching. We identify conditions for complete coupling without significant quenching, which are compatible with common QD polymer surfaces and various azide/alkyne pairs. Based on insight from the combinatorial screen and mechanistic studies of Cu coordination and quenching, we find that superstoichiometric concentrations of Cu can promote full coupling if accompanied by ligands that selectively compete with the Cu from the QD surface but allow it to remain catalytically active. Applied to the conjugation of a K+ channel-specific peptidyl toxin to CdSe/ZnS QDs, we synthesize unquenched QD conjugates and image their specific and voltage-dependent affinity for K+ channels in live cells
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Azide–Alkyne Click Conjugation on Quantum Dots by Selective Copper Coordination
Functionalization
of nanocrystals is essential for their practical
application, but synthesis on nanocrystal surfaces is limited by incompatibilities
with certain key reagents. The copper-catalyzed azide–alkyne
cycloaddition is among the most useful methods for ligating molecules
to surfaces, but has been largely useless for semiconductor quantum
dots (QDs) because Cu<sup>+</sup> ions quickly and irreversibly quench
QD fluorescence. To discover nonquenching synthetic conditions for
Cu-catalyzed click reactions on QD surfaces, we developed a combinatorial
fluorescence assay to screen >2000 reaction conditions to maximize
cycloaddition efficiency while minimizing QD quenching. We identify
conditions for complete coupling without significant quenching, which
are compatible with common QD polymer surfaces and various azide/alkyne
pairs. Based on insight from the combinatorial screen and mechanistic
studies of Cu coordination and quenching, we find that superstoichiometric
concentrations of Cu can promote full coupling if accompanied by ligands
that selectively compete with the Cu from the QD surface but allow
it to remain catalytically active. Applied to the conjugation of a
K<sup>+</sup> channel-specific peptidyl toxin to CdSe/ZnS QDs, we
synthesize unquenched QD conjugates and image their specific and voltage-dependent
affinity for K<sup>+</sup> channels in live cells
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Tarantula toxins use common surfaces for interacting with Kv and ASIC ion channels.
Tarantula toxins that bind to voltage-sensing domains of voltage-activated ion channels are thought to partition into the membrane and bind to the channel within the bilayer. While no structures of a voltage-sensor toxin bound to a channel have been solved, a structural homolog, psalmotoxin (PcTx1), was recently crystalized in complex with the extracellular domain of an acid sensing ion channel (ASIC). In the present study we use spectroscopic, biophysical and computational approaches to compare membrane interaction properties and channel binding surfaces of PcTx1 with the voltage-sensor toxin guangxitoxin (GxTx-1E). Our results show that both types of tarantula toxins interact with membranes, but that voltage-sensor toxins partition deeper into the bilayer. In addition, our results suggest that tarantula toxins have evolved a similar concave surface for clamping onto α-helices that is effective in aqueous or lipidic physical environments