The 3/4- and 3/6-Subfamily Variants of α-Conotoxins GI and MI Exhibit Potent Inhibitory Activity against Muscular Nicotinic Acetylcholine Receptors

α-Conotoxins GI and MI belong to the 3/5 subfamily of α-conotoxins and potently inhibit muscular nicotinic acetylcholine receptors (nAChRs). To date, no 3/4- or 3/6-subfamily α-conotoxins have been reported to inhibit muscular nAChRs. In the present study, a series of new 3/4-, 3/6-, and 3/7-subfamily GI and MI variants were synthesized and functionally characterized by modifications of loop2. The results show that the 3/4-subfamily GI variant GI∆8G]-II and the 3/6-subfamily variants GI+13A], GI+13R], and GI+13K] displayed potent inhibition of muscular nAChRs expressed in Xenopus oocytes, with an IC₅₀ of 45.4–73.4 nM, similar to or slightly lower than that of wild-type GI (42.0 nM). The toxicity of these GI variants in mice appeared to be about a half to a quarter of that of wild-type GI. At the same time, the 3/7-subfamily GI variants showed significantly lower in vitro potency and toxicity. On the other hand, similar to the 3/6-subfamily GI variants, the 3/6-subfamily MI variants MI+14R] and MI+14K] were also active after the addition of a basic amino acid, Arg or Lys, in loop2, but the activity was not maintained for the 3/4-subfamily MI variant MI∆9G]. Interestingly, the disulfide bond connectivity “C1–C4, C2–C3” in the 3/4-subfamily variant GI∆8G]-II was significantly more potent than the “C1–C3, C2–C4” connectivity found in wild-type GI and MI, suggesting that disulfide bond connectivity is easily affected in the rigid 3/4-subfamily α-conotoxins and that the disulfide bonds significantly impact the variants’ function. This work is the first to demonstrate that 3/4- and 3/6-subfamily α-conotoxins potently inhibit muscular nAChRs, expanding our knowledge of α-conotoxins and providing new motifs for their further modifications.