Fig. 4: Restricting extracellular SF flexibility reduces ion selectivity of NaK2K.

A Cartoon representation of the proposed crosslink location between T73C residues (top of TM2) in NaK2K using a 6 nm crosslinker to restrict the extracellular SF flexibility. B NaK2K T73C single-channel recordings in symmetrical 200 mM K⁺ (blue) and symmetrical 200 mM Na⁺ (red) without and with the crosslinker (light and dark color, respectively). C NaK2K single-channel K⁺ conductance is reduced after crosslinking (blue bars), whereas Na⁺ conductance is increased (red bars). NaK2K open probability in K is reduced after crosslinking (blue bars). Crosslinked samples compared with Control (K⁺ conductance p = 0.0033; Na⁺ conductance p = 0.0035; Po in K⁺ p = 0.0194; Po in Na⁺ p = 0.3688). D Current-voltage relationships for multi-channel recordings in asymmetrical 200 mM K⁺ (bath) and 200 mM Na⁺ (pipette) without and with the 6 nm crosslinker shows significant left shift of reversal potential with the crosslinker, reflecting reduced ion selectivity in NaK2K. Error bars indicate s.e.m. in (C, D). In (C), **p < 0.01 vs. Control. *p < 0.05 vs. Control. ns (not significant), n patches are indicated in each condition. Comparisons between control and crosslink conditions analyzed by unpaired t-test. Source data are provided as a Source Data file.