Lin et al.
Appendix
Fig.1 ATP response of WT and PNDM mutant channels. ATP dose response curves in the absence of Mg2+. Currents in different ATP concentrations were normalized to the current observed in the absence of ATP. Data points (n = 5-9) were fitted by the Hill equation: Irel = 1/(1+([ATP]/IC50)H.
TableI. Comparison of IC50 ATP from this study and previous studies
Mutation / IC50 ATP from this study (M); H = Hill coefficient / IC50 ATP from other studies (M) / PhenotypeWT / 15 (H = 1.5) / 7 (1); 6 (2) / none
Q52R / 889 (H = 2.8) / 84 (1); 125 (2) / DEND
V59G / >10,000 (H = 3.7) / 7,400 (1) / DEND
V59M / 176 (H = 1.7) / 58 (3); 16 (2) / Intermediate DEND
R201C / 415 (H = 1.6) / 106 (1) / PNDM/some DEND
R201H / 296 (H = 1.7) / 299 (4) / PNDM
I296L / 4,200 (H =2.1) / 3,400 (5); 771 (2) / DEND
The IC50 of ATP inhibition for the various homomeric PNDM mutant channels obtained in this study are compared to those obtained from previous studies (see reference cited following each value). In general, the values we obtained are somewhat higher than those reported previously, especially for the Q52R mutation. A notable difference between our experiments and those of others is that 1 mM EDTA was included in the bath solution during our inside-out patch-clamp recording. EDTA chelates residual Mg2+ and has been previously shown to eliminate channel rundown, thereby allowing for more stable current and accurate measurement of ATP sensitivity (6). Since channel rundown is known to lead to an increase in the apparent channel ATP sensitivity (7), the higher IC50 values in the absence of rundown, as is the case in this study, are therefore expected. With the exception of V59M (see main text discussion on the role of channel expression in disease phenotype), the IC50 ATP from this study correlates quite well with the disease phenotype (also see MgATP sensitivity comparison in Appendix Figure 2).
Fig.2 Activity of WT and homomeric or heteromeric PNDM mutant channels in 1 mM ATP or 1 mM MgATP. Currents were normalized to that seen in the absence of ATP (K-INT solution).
Table II. Correlation between disease phenotype and combined effect of mutation on channel gating and expression
Mutation / %Iat 1 mMMgATP (Po1) / %I at 5 mM
MgATP (Po2) / % Expression (N)a / N * Po1 / N * Po2 / Disease phenotype
WT / 0.9 / 0.6 / 100 / 90 / 60 / None
Q52R / 93.2 / 45.4 / 19.2 / 1790 / 908 / DEND
V59G / 100.0 / 103.7 / 19.1 / 1910 / 1659 / DEND
V59M / 25.4 / 4.2 / 47.0 / 1194 / 197 / Int DEND
R201C / 96.5 / 30.0 / 11.3 / 1090 / 362 / PNDM/DEND
R201H / 72.7 / 8.0 / 15.0 / 1090 / 120 / PNDM
I296L / 102.7 / 86.6 / 17.7 / 1805 / 1533 / DEND
a Data from chemiluminescence assays (Fig.1D of the main manuscript).
Relative channel open probability (homomeric mutant channels) in 1 mM MgATP (Po1) or 5 mM MgATP (Po2) was assessed using inside-out patch clamp recording (normalized to activity in the absence of MgATP). Relative channel open probability was then multiplied by channel expression level (N*Po) to estimate the impact of combined effect of mutation on both gating and expression on the overall channel activity. The estimated N*Po values correlate well with disease phenotypes. Consideration of the contribution from mutant expression level helps explain why the V59M mutation (highlighted in red) exhibits severe phenotype even though it has the highest MgATP sensitivity.
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