Pharmacological Modulation of Kir3.1 and Kir3.4 Cell Surface Expression
Authors: Reiser Michelle , Alhadidy Mohammed M , Szekeres Charles , Noujaim Sami F. 
Published: November 2023

BackgroundSmall molecules have been shown to pharmacologically modulate the cell surface expression of ion channel proteins. We thus investigated the effects of the antimalarial aminoquinolines Chloroquine and Primaquine on the cell surface density of Kir3.1/Kir3.4 proteins which mediate the acetylcholine activated inward rectifier potassium current (IKACh) in the heart. We compared the effects of Chloroquine and Primaquine to those of known lysosomal inhibitors‐ ammonium chloride (NH4Cl) and Leupeptin‐ on the surface density of the IKACh proteins. We hypothesized that long term Chloroquine treatment increases the surface expression of Kir3.1/Kir3.4 independently of the lysosomal pathway.MethodsExperiments were performed in human embryonic kidney (HEK293) cells stably transfected with Kir3.1 and Kir3.4. ATTO‐488 conjugated TertiapinQ (a potent blocker of IKACh) was used to label the IKACh proteins in live cells, and flow cytometry was used to quantify their expression on the cell surface after 24 hours treatment with 1, 10 and 50 μM Chloroquine, 10 μM Primaquine, 10 mM NH4Cl, and 300 μM Leupeptin. Endosomal/lysosomal pH was also measured in flow cytometry, using the pH‐sensitive pHrodo Green Dextran 10,000 MW dye conjugate. Western blotting was done after overnight treatment with Chloroquine and Leupeptin to determine the total levels of Kir3.1 and Kir3.4, and of MAP LC3α/β, a protein known to undergo lysosomal degradation. Results are reported as averages ± SD.ResultsIn flow cytometry experiments, Chloroquine at 1 μM, 10 μM and 50 μM showed a dose dependent increase in Kir3.1/Kir3.4 surface expression (5.6 % ± 6 vs. control, n.s.; 31.7% ± 18, p<0.01 vs. control, and 54.4% ± 18, p<0.01 vs. control). Moreover, treatment with 10 μM Primaquine, a compound structurally similar to Chloroquine, did not show a significant increase compared to vehicle treatment. The lysosomal inhibitor Leupeptin (300 μM) did not significantly increase cell surface IKACh channels expression compared to control. On the other hand, disruption of lysosomal pH with 10 mM NH4Cl significantly increased cell surface expression of Kir3.1/Kir3.4, but the increase was less pronounced compared to that of Chloroquine (24% ± 18 vs 32.7% ± 11.7, p<0.05). We subsequently examined the influence of Chloroquine, Primaquine, NH4Cl and Leupeptin on the endosomal/lysosomal pH. Endosomal/lysosomal pH was significantly increased (indicated by a decrease in pH‐sensitive pHrodo Green Dextran fluorescence) by NH4Cl and Primaquine versus control (1003 A.U. ± 133 and 1063 A.U. ± 115, vs 1368 A.U. ± 165, p<0.05) but not by Chloroquine and Leupeptin (1520 A.U. ± 199 and 1424 A.U. ± 151, vs 1368 A.U. ± 165, n.s.). Finally, in western blots, Chloroquine and Leupeptin did not significantly affect Kir3.1 and Kir3.4 total protein levels, however, Chloroquine significantly increased MAP LC3α/β levels.ConclusionLong term Chloroquine treatment promotes cell surface expression of Kir3.1 and Kir3.4. Accumulation of the IKACh mediating proteins at the cell surface appears to be independent of the possible effects of Chloroquine on lysosomal function.Support or Funding InformationFunded in part by NIH grant R01HL129136 to SFN