Characterization of a Novel α-Conotoxin TxID from Conus textile That Potently Blocks Rat α3β4 Nicotinic Acetylcholine Receptors

Abstract

The α3β4 nAChRs are implicated in pain sensation in the PNS and addiction to nicotine in the CNS. We identified an α-4/6-conotoxin (CTx) TxID from Conus textile. The new toxin consists of 15 amino acid residues with two disulfide bonds. TxID was synthesized using solid phase methods and the synthetic peptide was functionally tested on nAChRs heterologously expressed in Xenopus laevis oocytes. TxID blocked rat α3β4 nAChRs with a 12.5 nM IC50, which places it amongst the most potent α3β4 nAChR antagonists. TxID also blocked the closely related α6/α3β4 with a 94 nM IC50 but showed little activity on other nAChR subtypes. NMR analysis showed that two major structural isomers exist in solution, one of which adopts a regular α-CTx fold but with different surface charge distribution to other 4/6 family members. α-CTx TxID is a novel tool with which to probe the structure and function of α3β4 nAChRs.

α-Conotoxins Identify the α3β4* Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells

“…For example, a-Ctx TxIB is highly selective for rat a6/a3b2b3 nAChRs and shows very little activity on other subtypes (Luo et al, 2013). a-Ctx PnIA is a peptide similar in sequence to TxIB and is selective for rat a3b2 and a6b2 nAChRs over the a3b4 and a6/a3b4 subtypes (Luo et al, 1999;Hone et al, 2012a).…”

Alanine Scan of α-Conotoxin RegIIA Reveals a Selective α3β4 Nicotinic Acetylcholine Receptor Antagonist

“…6C and are also marked with asterisks in Fig. 7 55 ) and ␤2-Phe 119 (␤4-Gln 117 ) at the complementary face. These residues may be essential for RegIIA inhibition of ␣3␤2.…”

“…Recent studies in development of selective and potent ␣3␤4 antagonists led to the discovery and synthesis of two novel ␣-conotoxins: TxID, a novel ␣4/6-conotoxin from Conus textile that potently blocks ␣3␤4 (IC 50 ϭ 12.5 nM) (55) and TP-2212-59, a synthetic analog of ␣4/4-conotoxin BuIA (IC 50 ϭ 2.3 nM) (56). Although both peptides are potent inhibitors of the ␣3␤4 nAChR subtype, TxID also inhibits ␣6/␣3␤4 with only 7.5-fold less potency (IC 50 ϭ 94.1 nM) (55) and the activity of TP-2212-59 at other nAChR subtypes such as ␣6-containing receptors is yet to be tested (56).…”

Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins

“…Another ␣4/6-conotoxin, TxID from Conus textile, was recently described as the most potent ␣3␤4* nAChR-inhibiting ␣-conotoxin, however, besides ␣3␤4 it also inhibits ␣6/␣3␤4 with 7.5-fold less potency (14).…”