Journal Article

Conotoxin κM-RIIIJ, a tool targeting asymmetric heteromeric K v 1 channels

Markushina, Anna, Song, Jie, Finol-Urdaneta, Rocio K., Terlau, Heinrich, Cordeiro, Sönke, French, Robert J., Kopec, Wojciech, Olivera, Baldomero M., Giacobassi, Mario J., Leavitt, Lee S., Teichert, Russell W., Köpfer, David, Raghuraman, Shrinivasan, de Groot, Bert L.

2019

Overview

Most ion channels are multimeric (typically comprising 3–5 subunits). The subunits are encoded by homologous members of a gene family, generating an enormous set of possible heteromeric combinations. In this study, we provide evidence that the preferred target of conopeptide κM-RIIIJ is a heteromeric K v 1 channel consisting of three K v 1.2 subunits and one K v 1.1 or K v 1.6 subunit. We define the molecular interaction of κM-RIIIJ with these asymmetric K v 1 channels and show that in dorsal root ganglia (DRG) neurons, different κM-RIIIJ concentrations can distinguish discrete subpopulations of neurons. Our results highlight the potential of natural products and venom components, such as conopeptides, to generally elucidate native physiological roles of specific heteromeric ion channel isoforms at the cellular, circuit, and systems level. The vast complexity of native heteromeric K + channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K + channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this “zone of ignorance” in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets “asymmetric” K v channels composed of three K v 1.2 subunits and one K v 1.1 or K v 1.6 subunit with 100-fold higher apparent affinity compared with homomeric K v 1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional K v 1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative K v 1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by ( i ) covalent linkage of members of the mammalian Shaker-related K v 1 family to K v 1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K + channel-mediated currents in heterologous expression systems; ( ii ) molecular dynamics simulations of asymmetric K v 1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and ( iii ) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric K v channel complexes that operate in native tissues.

• Subject: Conotoxins - chemistry / Conotoxins - metabolism / Ganglia, Spinal - chemistry / Ganglia, Spinal - metabolism / HEK293 Cells / Humans / Ion Transport / Membrane Potentials / Molecular Dynamics Simulation / Neurons - chemistry / Neurons - metabolism / Protein Binding / Shaker Superfamily of Potassium Channels - antagonists & inhibitors / Shaker Superfamily of Potassium Channels - chemistry / Shaker Superfamily of Potassium Channels - metabolism