Examples of inside-out current recordings (A) and calculated Pomax (B) for KCa3.1 channels after the substitution of F248 by residues differing in volume size and/or hydrophobicity. Recordings were performed in symmetrical 200-mM K2SO4 conditions in saturating internal Ca2+ (25 µM) at a pipette potential of 100 mV. Low Pomax values compared with wild type were obtained with the F248T mutant only with a mean value of 0.19 ± 0.05 (n = 3). The substitution of F248 by either a smaller (F248A) or larger (F248W) residue led systematically to a Pomax increase. Also shown is an example of inside-out current trace obtained in conditions where F248 was replaced by the unnatural amino acid pMpa, a tyrosine analogue obtained by substituting the hydroxyl moiety of Tyr by an O-CH3 group. This substitution led to a drastic increase in Pomax with a mean value of 0.94 ± 0.03 (n = 4), compared with 0.22 ± 0.07 (n = 8) for wild type. (B) Bar graph summarizing the effect of substituting F248 by nonpolar residues differing in volume size and/or hydrophobicity. The resulting Pomax ranking reads: T ≈ F < H < Y < S < A < V < L ≈ W < pMpa, confirming the absence of correlation between Pomax and the volume size and/or hydrophobicity (A < S < T < H < V < Y < L < F < W) (Wimley et al., 1996) of the substituting residue. Also shown are all-point histograms computed from the entire recording. Bars, 2 pA and 0.5 s.
