Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

Abstract

Significance Electrically excitable cells, such as neurons, exhibit tremendous variation in their patterns of electrical signals. These variations arise from the collection of ion channels present in any specific cell, but understanding which ion channels are at the root of particular electrical signals remains a significant challenge. Here, we describe novel probes, derived from a tarantula venom peptide, that are able to report the activity of voltage-gated ion channels in living cells. This technology uses state-selective binding to optically monitor the activation of ion channels during cellular electrical signaling. Activity-reporting probes based on these prototypes could potentially identify when endogenous ion channels contribute to electrical signaling, thus facilitating the identification of ion channel targets for therapeutic drug intervention.

Cell Cycle-dependent Changes in Localization and Phosphorylation of the Plasma Membrane Kv2.1 K+ Channel Impact Endoplasmic Reticulum Membrane Contact Sites in COS-1 Cells

“…We next assessed whether the M phase Kv2.1 clusters are found on the cell surface. If Kv2.1 clusters are plasma membrane localized, they are targeted by guangxitoxin-1E, a tarantula toxin that selectively binds to Kv2 channels (51,59). We applied guangxitoxin-1E conjugated to a DyLight 550 fluorophore (GxTX-550) to live COS-1 cells expressing GFP-Kv2.1.…”

“…Conductance, G, was determined from the current level at the end of a 100-ms step to the indicated voltage, normalized by the driving force from a calculated reversal potential of Ϫ97 mV. Conductance change with voltage was fit from Ϫ80 to ϩ40 mV with the fourth power of a Boltzmann distribution described previously (51). V mid is the voltage when the fit reaches half-maximal amplitude, z is the number of electronic charges determining slope, A is maximal amplitude.…”

Azide–Alkyne Click Conjugation on Quantum Dots by Selective Copper Coordination

“…A CHO-K1 cell line expressing rat Kv2.1 voltage-gated potassium channels 13 was cultured with 1 μg/mL blasticidin (ThermoFisher) and 25 μg/mL zeocin (Invitrogen) to retain transfected vectors. Before experiments, 1 μg/mL minocycline was added to the cell media 2 days prior to imaging to induce Kv2.1 expression.…”

“…13 The Cu-catalyzed azide-alkyne 1,3-dipolar click coupling (CuAAC) is among the most widely used reactions for both bioconjugation 14–16 and for modification of surfaces. 17–18 CuAAC reactions are bioorthogonal, work well in dilute aqueous conditions, and require only a Cu ion catalyst and mild reducing agent to form covalent bonds between terminal alkynes and azides.…”

Optogenetic Approaches to Drug Discovery in Neuroscience and Beyond

“…Fluorescently labeled toxins with state-dependent binding probe activation of native K v 2.1 ion channels [73]. FRET-based reporters can probe activation of specific kinases, GPCRs, and other targets [13, 14].…”