3.4 MQ1-K39A is specific for human V2R.
We deepen the study of the variant MQ1-K39A as the substitution of a lysine residue by an alanine one significantly improves MQ1 affinity for hV2R. MQ1 and MQ1-K39A shifted to the right the AVP-dependent cAMP production on CHO cell lines expressing the hV2R (Fig. 5A-B). Arunlakshana–Schild plots show a purely competitive behavior between vasopressin and the toxins as IC50 regression can be linearized with slope close to unit (0.84 ± 0.04 for MQ1 and 0.99 ± 0.06 for MQ1-K39A, Fig. 5C). MQ1-K39A is 5.4 times more potent than MQ1 with a pA2 of 8.28 ± 0.08 (Kinact of 5.6 ± 0.9 nM) compared to a pA2 of 7.6 ± 0.2 for MQ1 (Kinact of 30 ± 7 nM). When 3 nmol/kg BW of toxins were injected in rats (i.p. route), 24h diuresis increased from 1.8 ml/kg/h (controls) to 5.0 ml/kg/h for MQ1 or 5.5 ml/kg/h for MQ1-K39A. Unexpectedly, MQ1-K39A did not induce stronger diuresis. In addition, this variant showed equivalent pharmacodynamics compared to MQ1 (Fig. 5E). To explain this discrepancy between human and rat V2R, we measured the affinity of MQ1 and MQ1-K39A for rat V2R. As published, MQ1 displays the same affinity for both species (Ki of 5.3 and 6.13 nM for human and rat V2R, Droctové et al., 2020). On its side, MQ1-K39A displayed similar affinity for rat V2R (Ki = 7.73 nM, pKi = 8.37 ± 0.74, Fig. 5F) than MQ1 for hV2R (Ki = 4.7 nM), explaining why MQ1-K39A never induced higher diuresis on rat than MQ1.