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. 2018 Jan 8;8:45. doi: 10.1038/s41598-017-18499-0

Figure 6.

Figure 6

Cysteine scanning mutagenesis in S6 and C-linker and S-S bridges between S6 and the P-helix in the open state. (A,B) Web logo analysis of S6 (A) and P-helix (B) in CNG channels based on the alignment given in Supplementary Table S1. (C) Mapping CNGA1 residues on TAX-4 structure (PDB ID: 5H3O) shows the location of mutated residues in S6 and the P-helix. (D) Collected data of the effect of 2.5 mM MTSETi in the open (red) and closed (black) state in the S6 transmembrane domain and in the C-linker. (E) left: current recordings obtained in the absence (red) and in the presence of 1 mM cGMP (black) at ± 60 mV for mutant channels F380C_L356C. cGMP was added some seconds after setting the voltage command at ± 60 mV. The double mutant does not inactivate and it is gated by cGMP. Right: current recording obtained in the presence of 1 mM cGMP for mutant channels F380C_L356C, elicited by voltage steps from −200 to +200 mV. (F) as in (E) but in the presence of 2 mM DTT present in the patch pipette. (G) Macroscopic I–V relationships of F380C_L356C mutant channels in the presence (n = 3) and in the absence (n = 4) of DTT. The number (n) of experiments for different mutant channels in different states varies between 3 and 8 (for details see also Supplementary Table 2). (n.e.) represents mutant channels without expression, (n.a.) represents mutant channels with no experiments where the expression was too low. (f.r.) represents mutant channels with no experiments where the run-down was too fast. Significativity is shown only for mutants where the MTSET effect was >30%. Unpaired two–tail T-test in d for F380C and V391C: t10 = −7,407, P < 0.001; t4 = 25,158. Further statistical tests for these mutant channels and for all other tested mutants are reported in Supplementary Table 2e.