An integrative approach to the facile functional classification of dorsal root ganglion neuronal subclasses

Abstract

Somatosensory neurons have historically been classified by a variety of approaches, including structural, anatomical, and genetic markers; electrophysiological properties; pharmacological sensitivities; and more recently, transcriptional profile differentiation. These methodologies, used separately, have yielded inconsistent classification schemes. Here, we describe phenotypic differences in response to pharmacological agents as measured by changes in cytosolic calcium concentration for the rapid classification of neurons in vitro; further analysis with genetic markers, whole-cell recordings, and single-cell transcriptomics validated these findings in a functional context. Using this general approach, which we refer to as tripartite constellation analysis (TCA), we focused on large-diameter dorsal-root ganglion (L-DRG) neurons with myelinated axons. Divergent responses to the K-channel antagonist, κM-conopeptide RIIIJ (RIIIJ), reliably identified six discrete functional cell classes. In two neuronal subclasses (L1 and L2), block with RIIIJ led to an increase in [Ca]i. Simultaneous electrophysiology and calcium imaging showed that the RIIIJ-elicited increase in [Ca]i corresponded to different patterns of action potentials (APs), a train of APs in L1 neurons, and sporadic firing in L2 neurons. Genetically labeled mice established that L1 neurons are proprioceptors. The single-cell transcriptomes of L1 and L2 neurons showed that L2 neurons are Aδ–low-threshold mechanoreceptors. RIIIJ effects were replicated by application of the Kv1.1 selective antagonist, Dendrotoxin-K, in several L-DRG subclasses (L1, L2, L3, and L5), suggesting the presence of functional Kv1.1/Kv1.2 heteromeric channels. Using this approach on other neuronal subclasses should ultimately accelerate the comprehensive classification and characterization of individual somatosensory neuronal subclasses within a mixed population.

Marine Toxins Targeting Kv1 Channels: Pharmacological Tools and Therapeutic Scaffolds

“…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 approach that can be exploited to accelerate the classification and study of other cell types within mixed population.…”

“…Recently, the discerning sensitivity of RIIIJ to its heteromeric Kv1 channel target was exploited to comprehensively classify and characterize individual somatosensory neuronal subclasses within heterogenous populations of dorsal root ganglion (DRG) neurons [83]. RIIIJ's selectivity was used to distinguish two functional Kv1 complexes in mouse dorsal root ganglion (DRG) neurons.…”

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

“…The peptide has also been characterized using calcium imaging combined with a pharmacological separation of different neuronal subtypes within the lumbar dorsal root ganglion of mice, a platform called Constellation Pharmacology [20]. The results of the constellation pharmacology analysis revealed that the peptide is surprisingly potent in mammalian systems, with nanomolar affinity for its targets.…”

“…This method was recently successfully employed to obtain conopeptide AtVIA (V28L:T30S) [15]. 20), and ribbon (Cys3-20, Cys9-15) forms of cdg14a were synthesized following a two-step oxidative folding: first in the presence of reduced and oxidized glutathione to form the first disulfide bridge, followed by folding in the presence of iodine to facilitate the second disulfide bond formation. Based on the co-elution experiments with the peptide obtained in a one-step, glutathione-assisted folding experiment (which co-eluted with the native peptide) and the product of a two-step directed folding ( Figure S5, Supplementary Information), it was established that the native peptide has the globular (Cys3-15, Cys9-20) fold, and the kinetically favored form, cdg14a_pk2, has the bead (Cys3-9, Cys15-20) fold ( Figure 5D).…”

K+ Channels in Primary Afferents and Their Role in Nerve Injury-Induced Pain

“…Although DRG neurons are heterogeneous and highly specialized (Zheng et al, 2019;Giacobassi et al, 2020) the simple answer to the question of which neuronal types are affected by nerve injury is "probably all of them depending on the nature of the injury" (see Table 2).…”