Alanine scan of the KCa3.1 pore helix region. (A) Inside-out current recordings obtained in symmetrical 200-mM K₂SO₄ conditions at a pipette potential of 100 mV (T240A, I244A, and F248A) or 150 mV (T250A) from KCa3.1 mutants expressed in Xenopus oocytes. The label “c” refers to zero current level. The internal Ca²⁺ concentration was 25 µM throughout. The mutation F248A caused a highly significant increase of the channel open probability at saturating internal Ca²⁺ concentration (Pomax) relative to wild type (P < 0.0005) compared with T240A (P < 0.01). The substitution I244A caused a nonsignificant variation in Pomax relative to wild type. Because of its strategic position at the N-terminal end of the selectivity filter, the mutation of T250 to Ala resulted in an important decrease in channel conductance (9 pS compared with 30 pS), with Pomax being not significantly different from wild type. These results are summarized in B with Pomax values of 0.42 ± 0.08 (n = 4), 0.10 ± 0.04, 0.75 ± 0.01 (n = 3), and 0.16 ± 0.01 (n = 2) for the T240A, I244A, F248A, and T250A channels, respectively. Our results point toward the interactions involving F248 with the S5 and S6 helices as being determinant in setting Pomax. Also shown are all-point histograms computed from the entire recording.