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Conotoxin ?M-RIIIJ, a tool targeting asymmetric heteromeric Kv1 channels.

ABSTRACT: 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 ?M-conotoxin RIIIJ (?M-RIIIJ) from Conus radiatus precisely targets "asymmetric" Kv channels composed of three Kv1.2 subunits and one Kv1.1 or Kv1.6 subunit with 100-fold higher apparent affinity compared with homomeric Kv1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional Kv1.2 channel complexes: a heteromer, for which ?M-RIIIJ has high affinity, and a putative Kv1.2 homomer, toward which ?M-RIIIJ is less potent. This conclusion was reached by (i) covalent linkage of members of the mammalian Shaker-related Kv1 family to Kv1.2 and systematic assessment of the potency of ?M-RIIIJ block of heteromeric K+ channel-mediated currents in heterologous expression systems; (ii) molecular dynamics simulations of asymmetric Kv1 channels providing insights into the molecular basis of ?M-RIIIJ selectivity and potency toward its targets; and (iii) evaluation of calcium responses of a defined population of DRG neurons to ?M-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric Kv channel complexes that operate in native tissues.

SUBMITTER: Cordeiro S

PROVIDER: S-EPMC6338859 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Conotoxin κM-RIIIJ, a tool targeting asymmetric heteromeric Kv1 channels.

Cordeiro Sönke S Finol-Urdaneta Rocio K RK Köpfer David D Markushina Anna A Song Jie J French Robert J RJ Kopec Wojciech W de Groot Bert L BL Giacobassi Mario J MJ Leavitt Lee S LS Raghuraman Shrinivasan S Teichert Russell W RW Olivera Baldomero M BM Terlau Heinrich H

Proceedings of the National Academy of Sciences of the United States of America 20181228 3

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 clo ...[more]

PMID: 30593566

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Project description:Spiral ganglion neurons (SGNs) relay acoustic code from cochlear hair cells to the brainstem, and their stimulation enables electrical hearing via cochlear implants. Rapid adaptation, a mechanism that preserves temporal precision, and a prominent feature of auditory neurons, is regulated via dendrotoxin-sensitive low-threshold voltage-activated (LVA) K(+) channels. Here, we investigated the molecular physiology of LVA currents in SGNs cultured from mice following the onset of hearing (postnatal days 12-21). Kv1.1- and Kv1.2-specific toxins blocked the LVA currents in a comparable manner, suggesting that both subunits contribute to functional heteromeric channels. Confocal immunofluorescence in fixed cochlear sections localized both Kv1.1 and Kv1.2 subunits to specific neuronal microdomains, including the somatic membrane, juxtaparanodes, and the first heminode, which forms the spike initiation site of the auditory nerve. The spatial distribution of Kv1 immunofluorescence appeared mutually exclusive to that of Kv3.1b subunits, which mediate high-threshold voltage-activated currents. As Kv1.2-containing channels are positively modulated by membrane phosphoinositides, we investigated the influence of phosphatidylinositol-4,5-bisphosphate (PIP2) availability on SGN electrophysiology. Reducing PIP2 production using wortmannin, or sequestration of PIP2 using a palmitoylated peptide (PIP2-PP), slowed adaptation rate in SGN populations. PIP2-PP specifically inhibited the LVA current in SGNs, an effect reduced by intracellular dialysis of a nonhydrolysable analog of PIP2. PIP2-PP also inhibited heterologously expressed Kv1.1/Kv1.2 channels, recapitulating its effect in SGNs. Collectively, the data identify Kv1.1/Kv1.2 heteromeric channels as key regulators of action potential initiation and propagation in the auditory nerve, and suggest that modulation of these channels by endogenous phosphoinositides provides local control of membrane excitability.Significance statementRapid spike adaptation is an important feature of auditory neurons that preserves temporal precision. In spiral ganglion neurons, the primary afferents in the cochlea, adaptation is regulated by heteromeric ion channels composed of Kv1.1 and Kv1.2 subunits. These subunits colocalize to common functional microdomains, such as juxtaparanodes and the somatic membrane. Activity of the heteromeric channels is controlled by cellular availability of PIP2, a membrane phospholipid. This mechanism provides an intrinsic regulation of output from the auditory nerve, which could be targeted for therapeutic adjustment of hearing sensitivity.

Dataset Information

Conotoxin ?M-RIIIJ, a tool targeting asymmetric heteromeric Kv1 channels.

Publications

Conotoxin κM-RIIIJ, a tool targeting asymmetric heteromeric K<sub>v</sub>1 channels.

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