A Single Amino Acid Replacement Boosts the Analgesic Activity of α-Conotoxin AuIB through the Inhibition of the GABA(B)R-Coupled N-Type Calcium Channel

α-conotoxin AuIB is the only one of the 4/6 type α-conotoxins (α-CTxs) that inhibits the γ-aminobutyric acid receptor B (GABA(B)R)-coupled N-type calcium channel (Ca(V)2.2). To improve its inhibitory activity, a series of variants were synthesized and evaluated according to the structure–activity relationships of 4/7 type α-CTxs targeting GABA(B)R-coupled Ca(V)2.2. Surprisingly, only the substitution of Pro7 with Arg results in a 2–3-fold increase in the inhibition of GABA(B)R-coupled Ca(V)2.2 (IC(50) is 0.74 nM); substitutions of position 9–12 with basic or hydrophobic amino acid and the addition of hydrophobic amino acid Leu or Ile at the second loop to mimic 4/7 type α-CTxs all failed to improve the inhibitory activity of AuIB against GABA(B)R-coupled Ca(V)2.2. Interestingly, the most potent form of AuIB[P7R] has disulfide bridges of “1–4, 2–3” (ribbon), which differs from the “1–3, 2–4” (globular) in the isoforms of wildtype AuIB. In addition, AuIB[P7R](globular) displays potent analgesic activity in the acetic acid writhing model and the partial sciatic nerve injury (PNL) model. Our study demonstrated that 4/6 type α-CTxs, with the disulfide bridge connectivity “1–4, 2–3,” are also potent inhibitors for GABA(B)R-coupled Ca(V)2.2, exhibiting potent analgesic activity.