Structural and functional mapping of ion access pathways in the human K ⁺ -dependent Na ⁺ /Ca ²⁺ exchanger NCKX2 using cysteine scanning mutagenesis, thiol-modifying reagents, and homology modelling

Figures

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  Figure 1.. Cartoon representation of the effect of modifying a substituted cysteine residue by MTSET.

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  Figure 2.. Schematic representation of the human NCKX2 topology highlighting the different residue groups that showed shifts in Ca2+ transport activity when treated with MTSET and compared with WT NCKX2.

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  Figure 3.. Examples of the different effects of MTSET on Ca2+ transport activity of mutant human NCKX2 proteins carrying single residue cysteine substitutions and expressed in HEK293 cells.

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  Figure 4.. Bar graph representing the effect of MTSET on the Ca2+ transport activity of the different NCKX2 single residue cysteine substitutions of the WT-NCKX2 clone.

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  Figure 5.. Homology model of OF human NCKX2 based on the X-ray crystal structure of the archaebacterial NCX_Mj protein, illustrating the results from the MTSET measurements.

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  Figure 6.. Schematic representation of the human NCKX2 topology highlighting the different residue groups that showed shifts in Ca2+ transport activity when treated with MTSEA and compared with WT NCKX2.

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  Figure 7.. Bar graph representing the effect of MTSEA on the Ca2+ transport activity of the different single residue cysteine substitutions of the WT-NCKX2 clone. The bars represent the effect of MTSEA on transport activity (normalized to WT NCKX2 transport activity) of WT NCKX2 and 18 different cysteine substitution mutants. Experiments were replicated between 3 and 6 times, 11 times for the WT NCKX2. Comparison against the WT-NCKX2 activity was carried out using a two-tailed t-test; values represent the means ± SEM.

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  Figure 8.. Homology model of IF human NCKX2 based on the cryoEM structure of the human Ca2+ bound activated NCX1 protein, illustrating the results from the MTSEA measurements.

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  Figure 9.. The effect of MTSET and MTSEA on the transport activity of the NCKX2 A181C substitution and cysteine substitutions of immediate neighboring residues.