A Single Amino Acid Replacement Boosts the Analgesic Activity of α-Conotoxin AuIB through the Inhibition of the GABAR-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 (GABAR)-coupled N-type calcium channel (Ca2.2). 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 GABAR-coupled Ca2.2. Surprisingly, only the substitution of Pro7 with Arg results in a 2-3-fold increase in the inhibition of GABAR-coupled Ca2.2 (IC 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 GABAR-coupled Ca2.2. 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, AuIBP7R 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 GABAR-coupled Ca2.2, exhibiting potent analgesic activity.