Figure 2

The structural and functional properties of the quadruple W67 KcsA mutant are similar to the ones presented by the wild-type channel. (A) SDS-PAGE (13.5%) analysis of the tetramer integrity of the detergent-solubilized protein in the presence of 20 mM Hepes, pH 7 buffer with 5 mM DDM and 200 mM Cs⁺, Rb⁺, K⁺ or 10 mM TBA⁺ or 10 mM succinic acid, pH 4 buffer with equivalent amounts of DDM and salts. In the particular case of Na⁺, 200 mM NaCl was added to the W67 KcsA samples and 1 M to the E71A W67 batches, respectively. pH 7 and pH 4 samples represent the closed and open states of the channel, respectively. (B) Protein thermal stability assay (monitoring changes in fluorescence emission at 340 nm) of 1 µM protein solubilized in 20 mM Hepes, pH 7 buffer with 5 mM DDM and 100 mM NaCl indicates that the introduced mutations induced only a 5 °C decrease in stability (t_m: 66 °C) when compared to WT channel (t_m: 70 °C). The inset reveals that in the presence of 100 mM K⁺, KcsA W67 is as highly resistant to thermal denaturation as the WT channel. (C) Patch-clamp inside-out patches recordings in continuous mode at + 150 mV of WT, W67, E71A and W67 E71A KcsA channels reconstituted in asolectin liposomes. The dashed lines indicate the closed channel state. Channel openings appear as upward deflections over the closed state line. Bar graphic illustrates open probability (NPo) determined at +150 mV. Data is presented as average ± SD of at least three independent experiments. (D) Normalized fluorescence emission spectra (λ_ex = 295 nm) of W67 KcsA mutant channel at pH 7 in the presence of different blocking and permeant cations. The inset compares the fluorescence emission spectra of WT and W67 KcsA mutant in the presence of 200 mM K⁺.