Conotoxin κM-RIIIJ, a tool targeting asymmetric heteromeric K _v 1 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 κM-conotoxin RIIIJ (κM-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 κM-RIIIJ has high affinity, and a putative Kv1.2 homomer, toward which κM-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 κM-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 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 Kv channel complexes that operate in native tissues.

Marine Toxins Targeting Kv1 Channels: Pharmacological Tools and Therapeutic Scaffolds

“…For example, testing of conotoxin κM-RIIIJ over 12 different heterodimers containing Kv1.2 subunits, Cordeiro et al showed that RIIIJ was most potent against Kv1.1/Kv1.2 heterodimers without apparent regard for their arrangement, showing significant discrimination against other heterodimeric constructs including those formed by association of Kv1.2 with Kv1.5 or Kv1.6. Further functional analyses showed that RIIIJ was indeed~100 more potent against hetero-tetramers made of three copies of Kv1.2 α-subunits and one of either Kv1.1 or Kv1.6 in a 3:1 stoichiometry [82]. This detailed functional and biochemical characterization then enabled the use of RIIIJ as a molecular tool for the classification of live large DRG neurons into six discrete functional populations [83].…”

“…An in-depth evaluation of RIIIJ was performed against heteromeric channels generated by a covalent linkage composed of Kv1.2 and all other Kv1s subunits (except for Kv1.8) at different stoichiometries and arrangements [82]. This work revealed that RIIIJ exquisitely targets asymmetric Kv channels composed of three Kv1.2 subunits and one Kv1.1 or Kv1.6 subunit.…”

“…RIIIJ's selectivity was used to distinguish two functional Kv1 complexes in mouse dorsal root ganglion (DRG) neurons. One being RIIIJ's high-affinity target (3 × Kv1.2 + Kv1.1 or Kv1.6), and the second component characterized by inhibition at higher RIIIJ concentrations arguably composed of homo-tetrameric Kv1.2 subunits [82]. The functional behavior of large DRG (L-DRG) neurons exposed to RIIIJ was used to classify L-DRGs in six discrete neuronal subpopulations (L1-L6).…”

Purification and Characterization of the Pink-Floyd Drillipeptide, a Bioactive Venom Peptide from Clavus davidgilmouri (Gastropoda: Conoidea: Drilliidae)

“…After 5 min, a solution of 20 mM K + plus 100 nM cdg14a was again applied to the neurons for 10 s. The cells were allowed to recover for 12 min, and then 20 mM K+ was applied to determine the reversibility of the cdg14a effect. The cells were then incubated for 5 min in a DRG observation solution containing 1 μM of the conopeptide κM-RIIIJ to identify cells expressing Kv1.1 and/or Kv1.2 [ 28 ]. Direct response to κM-RIIIJ, as well as response to 20 mM K+ after κM-RIIIJ incubation, indicating an indirect response, characterizes classes of DRG neurons [ 4 ].…”

Curses or Cures: A Review of the Numerous Benefits Versus the Biosecurity Concerns of Conotoxin Research

“…Voltage-sensitive binding/blocking of voltage-gated K-channels [15] Cancer [93], cardioprotection in ischemia [94] κM-RIIIJ Voltage-gated K + channels Subtype selectivity [95,96] Neuronal profiling [5,6,97,98], channel subtype expression profiling [96,99] ω-GVIA Voltage-gated Ca 2+ channels Subtype selective [37,99] Neurotransmission [100][101][102], pain [103], cardiology [55], epilepsy [104], renal function [105], nuclear signaling [106] ω-MVIIC Voltage-gated Ca 2+ channels Inhibits various subtypes broadly [107,108] Epilepsy [109], long-term depression [110], pain [111,112]…”