Novel KCNJ10 Gene Variations Compromise Function of Inwardly Rectifying Potassium Channel 4.1

Abstract

TheKCNJ10gene encoding Kir4.1 contains numerous SNPs whose molecular effects remain unknown. We investigated the functional consequences of uncharacterized SNPs (Q212R, L166Q, and G83V) on homomeric (Kir4.1) and heteromeric (Kir4.1-Kir5.1) channel function. We compared these with previously characterized EAST/SeSAME mutants (G77R and A167V) in kidney-derived tsA201 cells and in glial cell-derived C6 glioma cells. The membrane potentials of tsA201 cells expressing G77R and G83V were significantly depolarized as compared with WTKir4.1, whereas cells expressing Q212R, L166Q, and A167V were less affected. Furthermore, macroscopic currents from cells expressing WTKir4.1 and Q212R channels did not differ, whereas currents from cells expressing L166Q, G83V, G77R, and A167V were reduced. Unexpectedly, L166Q current responses were rescued when co-expressed with Kir5.1. In addition, we observed notable differences in channel activity between C6 glioma cells and tsA201 cells expressing L166Q and A167V, suggesting that there are underlying differences between cell lines in terms of Kir4.1 protein synthesis, stability, or expression at the surface. Finally, we determined spermine (SPM) sensitivity of these uncharacterized SNPs and found that Q212R-containing channels displayed reduced block by 1 μmSPM. At 100 μmSPM, the block was equal to or greater than WT, suggesting that the greater driving force of SPM allowed achievement of steady state. In contrast, L166Q-Kir5.1 channels achieved a higher block than WT, suggesting a more stable interaction of SPM in the deep pore cavity. Overall, our data suggest that G83V, L166Q, and Q212R residues play a pivotal role in controlling Kir4.1 channel function.

Keywords: KCNJ10; Kir4.1; epilepsy; kinetics; molecular cell biology; patch clamp; potassium channel; potassium transport; single nucleotide polymorphism (SNP); spermine.

FIGURE 1.

Whole-cell currents measured from tsA201 (A1–A4 and B1–B4) and C6 glioma cells (C1–C4 and D1–D4) in response to a voltage step protocol from −100 to +100 mV in the presence and absence of 100 μm barium. Cells were clamped at V_h, which was equal to the resting V_m (V_h = V_m). V_h values were −28.6 ± 1.8 and −74.0 ± 0.6 mV for non-transfected (A1) and Kir4.1-transfected (B1) tsA201 cells and −38.9 ± 0.8 and −83.2 ± 1.7 mV for non-transfected (C1) and Kir4.1-transfected (D1) C6 glioma cells. Representative current traces within the −120 to +20-mV range from non-transfected tsA201 cells (A), Kir4.1-transfected tsA201 cells (B), non-transfected C6 glioma cells (C), and Kir4.1-transfected C6 glioma cells (D) are shown. Whole-cell currents were recorded from tsA201 cells (A1 and B1) and C6 glioma cells (C1 and D1) in response to a voltage step protocol from −100 to +100 mV from the holding potential. Note that 1) V_h is more hyperpolarized in Kir4.1-transfected cells and 2) where there is no error bar shown it is smaller than the size of the symbol. Whole-cell currents were recorded from tsA201 cells (A2 and B2) and C6 glioma cells (C2 and D2) in response to a voltage step protocol in the presence of 100 μm Ba²⁺ (a blocker of Kir channels). Barium-sensitive currents from tsA201 cells (A3 and B3) and C6 glioma cells (C3 and D3) are shown. The graph shows the subtraction of currents obtained in the presence of barium from total whole-cell currents (Control). Barium-sensitive currents reflect the contribution of Kir channels to the whole-cell currents, and in untransfected cells, there is virtually no barium-sensitive current. Summaries of inward current measured at −120 mV from tsA201 cells (A4 and B4) and C6 glioma cells (C4 and D4) are also shown. Data (A4–D4) were analyzed using central tendency measures (means), dispersion measures (standard deviations), and Kruskal-Wallis k independent group test with a Bonferroni correction (p < 0.05). Results (A4–D4) are expressed as mean ± S.E., and significant differences from control are shown (*). Error bars represent ±S.E.

FIGURE 2.

Membrane potential of tsA201 and C6 glioma cells expressing WT, different SNPs, and EAST/SeSAME mutant Kir4.1 channels. A, average membrane potential of tsA201 cells expressing homomeric Kir4.1 channels. B, average membrane potential of tsA201 cells expressing heteromeric Kir4.1-Kir5.1 channels. C, average membrane potential of C6 glioma cells expressing homomeric Kir4.1 channels. D, average membrane potential of C6 glioma cells expressing heteromeric Kir4.1-Kir5.1 channels. Data were analyzed using central tendency measures (medians), dispersion measures (standard deviations), and a k independent group one-way ANOVA test with Tukey-Kramer post hoc tests (p < 0.05). Statistical differences from Mock or Kir5.1 control (*) or from WTKir4.1- or Kir4.1-Kir5.1-transfected cells (¤) are shown. Error bars represent ±S.E.

FIGURE 3.

Whole-cell currents measured in tsA201 cells using a voltage step protocol. A step protocol with a 10-mV step increment from −100 to 100 mV from the holding potential (V_h = V_m) was applied. A, representative current traces from cells expressing homomeric Kir4.1 channels. B, representative current traces from cells expressing heteromeric Kir4.1-Kir5.1 channels. C, summary of the results within the range of −120 to 30 mV. D, summary of the results within the range of −120 to 30 mV. Error bars represent ±S.E.

FIGURE 4.

Whole-cell currents measured in C6 glioma cells using a voltage step protocol. A step protocol with a 10-mV step increment from −100 to 100 mV from the holding potential (V_h = V_m) was applied. A, representative current traces from cells expressing homomeric Kir4.1 channels. B, representative current traces from cells expressing heteromeric Kir4.1-Kir5.1 channels. C, summary of the results within the range of −140 to 20 mV. D, summary of the results within the range of −140 to 20 mV. Error bars represent ±S.E.

FIGURE 5.

Summary of the inward currents measured in response to a step from 3 to 10 mm extracellular K⁺ for tsA201 cells expressing homomeric Kir4. 1 channels (A), tsA201 cells expressing heteromeric Kir4.1-Kir5.1 channels (B), C6 glioma cells expressing homomeric Kir4.1 channels (C), and C6 glioma cells heteromeric Kir4.1-Kir5.1 channels (D). Data were analyzed using central tendency measures (medians), dispersion measures (standard deviations), and a k independent group one-way ANOVA test with Tukey-Kramer post hoc tests (p < 0.05) for tsA201 cells. For C6 glioma cells, data were analyzed by frequencies and a Kruskal-Wallis k independent group test. Results are expressed as mean ± S.E., and significant differences (p < 0.05) from mock- or Kir5.1 control- (*) or from WTKir4.1- or WTKir4.1-Kir5.1-transfected cells (¤) are shown. V_h = V_m. Error bars represent ±S.E.

FIGURE 6.

Intracellular SPM sensitivity experiments on tsA201 cells expressing homomeric Kir4.1 and Q212R mutant channels. Representative inside-out patch clamp recordings of currents recorded in response to voltage steps from −100 and +100 mV in the presence of 0, 1, 10, and 100 μm SPM or in extracellular solution at pH 5.5 (A1 and B1) are shown. A2 and B2, steady state current-voltage relationships from A1 and B1. A3 and B3, mean I_rel-voltage relationships from WTKir4.1 (n = 7) and Q212R (n = 6) experiments as in A1 and B1.

FIGURE 7.

Intracellular SPM sensitivity experiments on tsA201 cells expressing heteromeric Kir4.1-Kir5.1, Q212R-Kir5.1, and L166Q-Kir5.1 mutant channels. A1, B1, and C1, representative inside-out patch clamp recordings of currents recorded in response to voltage steps from −100 and +100 mV in the presence of 0, 1, 10, and 100 μm SPM or in extracellular solution at pH 5.5. A2, B2, and C2, steady state current-voltage relationships from A1, B1, and C1. A3, B3, and C3, mean I_rel-voltage relationships from heteromeric Kir4.1-Kir5.1 (n = 9), Q212R-Kir5.1 (n = 6) and L166Q-Kir5.1 (n = 7) experiments as in A1, B1, and C1.

FIGURE 8.

Proposed model of the Kir4.1 channel protein and location of Gln-212, Leu-166, and Gly-83 amino acid changes.