Calcins is a novel family of scorpion peptides that bind with high affinity and specificity to ryanodine receptors (RyRs) and increase their open probability by inducing the appearance of a long-lasting subconductance state. Here we report two newly-identified calcins (Vejocalcin and Urocalcin) and provide a comprehensive analysis of the structure-function relationship of the eight calcins known to date, based on primary sequence examination, 3D structure modeling, and their effect on RyR1. [3H]Ryanodine binding assays, used as index of the open probability (Po) of RyRs, revealed that all eight calcins (Opicalcin1, Opicalcin2, Imperacalcin, Maurocalcin, Hadrucalcin, Hemicalcin, Vejocalcin, and Urocalcin) activate RyR1 dose-dependently, with EC50s (in nM) 0.35, 5, 8, 12, 37, 71, 91, and 523, respectively. At 1 µM, calcins significantly augmented the bell-shaped [Ca2+]-activity curve of RyR1 with potency Opicalcin1>Opicalcin2>Vejocalcin>Urocalcin. In single channel recordings, the heretofore uncharacterized calcins Opicalcin1, Opicalcin2 and Vejocalcin increased the Po of RyR1 significantly and the fractional conductance was ∼0.45, 0.30, and 0.65, respectively, of the full conductance state. Opicalcin1, Maurocalcin, Hadrucalcin and Vejocalcin induced Ca2+ release from rabbit skeletal SR with ED50s (in nM) 2.8±0.02, 8.3±0.25, 16±0.4, 36±0.5, respectively. A clear Ca2+ release-[3H]ryanodine binding activity correlation (r2=0.99) was obtained. Primary sequence alignment and evolutionary analysis (ClustalW and MEGA5.2) showed high homology among all calcins. An inhibitor cysteine-knot (ICK) motif with βαββ domains stabilized by three disulfide bridges characterizes this peptide family, although Vejocalcin lacks the β2 sheet. Positively-charged residue mutations in the high variable N-terminal region (G1-N14) and negatively-charged residue variations in the conserved C-terminal region (D15-R33) greatly decrease the effect of calcins on RyR1. In conclusion, natural variation in calcin peptides offers a diversified set of RyR ligands with capacity to modulate RyRs with high dynamic range and potency.