Comparing the effect of S367 and M368 mutations on Pomax. (A) Examples of single-channel recordings illustrating the effect on Pomax coming from the substitution of S367 by nonpolar residues of smaller (S367A) or larger side-chain surface areas. These results demonstrate a correlation between the channel Pomax and the free energy of transfer to water for the residue at position 367 for nonpolar amino acids. Also shown is the effect of the substitution S367E, confirming that electrostatic contribution to solvation energy can account in this case for a low Pomax (0.24 ± 0.08; n = 4) despite a side-chain surface area equivalent to Leu (Pomax of 0.88 ± 0.05; n = 3). (B) Bar graph summarizing the effect of mutating S367 on the channel Pomax value. (C) Single-channel recordings illustrating the effects on Pomax of mutating M368 described as being an anchoring point for CaM binding to KCa3.1. Mutating M368 by residues of equivalent or larger side-chain surface areas (M368L and M368W) had a small impact on Pomax, with values of 0.25 ± 0.07 (n = 4) for M368L and 0.27 ± 0.06 (n = 5) for M368W compared with 0.22 ± 0.07 (n = 8) for WT. The mutation M368A resulted in a Pomax of 0.13 ± 0.01 (n = 3), suggesting that decreasing the residue 368 free energy of transfer to water tends to impair the channel activity. Similarly, the mutation to a polar residue M368R led to a lower Pomax value of 0.15 ± 0.02 (n = 6). (D) Bar graph illustrating the effect of mutating M368 on Pomax. A decrease in hydrophobicity tends to result in lower Pomax, as observed for S367, but with a much smaller impact on the Pomax value. Experiments performed in symmetrical 200-mM K2SO4 conditions at 25 µM of internal Ca2+ for a pipette potential of 100 mV. Bars, 2 pA and 0.5 s.
