Sudden Death-Associated KCNH2 Variants Have Opposing Effects on hERG1 NP Function

Figures

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  Figure 1.. hERG1NP decreases hERG1a current and protein. (A) Sample IhERG traces elicited by the protocol in the inset from HEK293-hERG1a cells transfected with either GFP (green circles) or hERG1NP (blue squares). Scale bar indicates 2 s by 5 pA/pF. (B) Steady-state hERG1a current plotted as a function of test potential from cells transfected with either GFP (green circles) or hERG1NP (blue squares). (C) Peak hERG1a tail current density plotted as a function of pre-pulse potential and fitted with a Boltzmann equation (Equation 1) from HEK293-hERG1a cells transfected with either GFP (green circles) or hERG1NP (blue squares). (D) Sample western blot of HEK293-hERG1a cells transfected with either GFP or hERG1NP displaying bands for GAPDH, hERG1NP, core-glycosylated hERG1a and fully glycosylated hERG1a protein. (E) Total hERG1a protein (core-glycosylated + fully glycosylated) normalized to GAPDH from cells transfected with either GFP (green circles) or hERG1NP (blue squares). (F) Fully glycosylated hERG1a protein (155 kDa) normalized to GAPDH from cells transfected with either GFP (green circles) or hERG1NP (blue squares). (G) Core-glycosylated hERG1a protein (135 kDa) normalized to GAPDH from…

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  Figure 2.. hERG1NP variants differentially modulate hERG1a current. (A) Linear schematic depicting the relative location of KCNH2 variants within hERG1NP. (B) Sample IhERG traces from HEK293-hERG1a cells transfected with either GFP (green) or hERG1NP (blue). (C-H) Patch clamp data from HEK293-hERG1a cells expressing GFP (green circles), hERG1NP (blue squares), or hERG1NP carrying one of the following KCNH2 variants (red triangles): (C) R885CNP, (D) R1035WNP, (E) G1036DNP, (F) R1047LNP, (G) Q1068RNP, or (H) R1069SNP. Each panel displays a sample IhERG trace recorded in the presence of the variant (left, red), steady-state current/voltage plot (middle), and peak tail current/voltage plot fitted with a Boltzmann equation (right). All variant data are compared to the same GFP and hERG1NP groups but visually separated for clarity. Data in C-H were compared using a two-way ANOVA and post-hoc Dunnet multiple comparisons test with hERG1NP set as the control group. Error bars represent the mean ± SD. N ≥ 3. n ≥ 7. * indicates p < 0.05 for GFP compared to hERG1NP. # indicates p < 0.05 for KCNH2 variant compared to hERG1NP. See Table 1 for specific experimental values.

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  Figure 3.. G1036DNP and Q1068RNP depolarize the voltage dependence of hERG1a activation. (A-F) Peak tail current/voltage relationships normalized to the maximum peak tail IhERG for HEK293-hERG1a cells transfected with GFP (green circles), hERG1NP (blue squares), or hERG1NP carrying one of the six KCNH2 variants (red triangles): (A) R885CNP, (B) R1035WNP, (C) G1036DNP, (D) R1047LNP, (E) Q1068RNP, and (F) R1069SNP. Data plotted as a function of pre-pulse potential and fitted with a Boltzmann equation (Equation 1). All variant data are compared to the same GFP and hERG1NP groups but visually separated for clarity. Data in A-F were compared using a two-way ANOVA and post-hoc Dunnet multiple comparisons test with hERG1NP set as the control group. Error bars represent the mean ± SD. N ≥ 3. n ≥ 7. * indicates p < 0.05 for GFP compared to hERG1NP. # indicates p < 0.05 for any given KCNH2 variant compared to hERG1NP. See Table 1 for specific experimental values.

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  Figure 4.. G1036DNP and Q1068RNP accelerate hERG1a’s time course of deactivation. (A) Sample IhERG traces recorded from HEK293-hERG1a cells transfected with GFP (green), hERG1NP (blue), G1036DNP (red), or Q1068RNP (purple) elicited by the displayed voltage-step protocol and scaled as indicated. Only sample traces for statistically significant variants are represented for clarity. (B) Fast and (C) slow deactivation time constants recorded from HEK293-hERG1a cells transfected with GFP (green circles), hERG1NP (blue squares), R885CNP (yellow upward triangles), R1035WNP (aqua downward triangles), G1036DNP (red diamonds), Q1068RNP (purple circles) or R1069SNP (grey squares). Data in B and C were compared using a nonparametric Kruskal-Wallis test with a post-hoc Dunn multiple comparisons correction. Comparisons included GFP vs hERG1NP and all variants vs both GFP and hERG1NP. Error bars represent the mean ± SD. N ≥ 3. n ≥ 7. **** indicates p < 0.0001, *** indicates p < 0.0002, ** indicates p < 0.0021, and * indicates p < 0.0332. See Table 2 for specific experimental values.

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  Figure 5.. G1036DNP and Q1068RNP accelerate the time course of hERG1a inactivation recovery. (A) Sample IhERG traces recorded from HEK293-hERG1a cells transfected with GFP (green), hERG1NP (blue), G1036DNP (red), or Q1068RNP (purple) elicited by the displayed voltage-step protocol and scaled as indicated. (B) Time constants of inactivation recovery recorded from HEK293-hERG1a cells transfected with GFP (green circles), hERG1NP (blue squares), G1036DNP (red upward triangles), or Q1068RNP (purple downward triangles). Data in B were compared using a two-way ANOVA and post-hoc Tukey multiple comparisons test. Error bars represent the mean ± SD. N = 3. n ≥ 12. **** indicates p < 0.0001, *** indicates p < 0.0002, ** indicates p < 0.0021, and * indicates p < 0.0332.

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  Figure 6.. KCNH2 variants alter hERG1NP nuclear localization. (A) Sample images of un-transfected HEK293-hERG1a cells as well as HEK293-hERG1a cells transfected with GFP, hERG1NP, R885CNP, R1035WNP, G1036DNP, R1047LNP, Q1068RNP, or R1069SNP. Un-transfected cells are immunolabeled for the hERG1 distal C-terminal domain (green) and DAPI to delineate the nuclear (blue). Transfected groups display GFP or mCitrine fluorescence (green) and DAPI (blue). Scale bar indicates 20 μm. (B) Quantification of nuclear fluorescence intensity relative to cytoplasmic fluorescence intensity (FNucleus/FCytoplasm) for cells depicted in A. Data in B were compared using a nonparametric Kruskal-Wallis test with a post-hoc Dunn multiple comparisons correction and hERG1NP set as the control group. Black bars represent the median. N = 3. n ≥ 15. **** indicates p < 0.0001, *** indicates p < 0.0002, ** indicates p < 0.0021, and * indicates p < 0.0332.