Overall, our complementary results using point mutations to abrogate individual hydrogen bonds suggested by a homology model of human ATP1A3 support the proposed CS‐6‐binding mode, provide insights into the critical residues for ATP1A3 channel activation and, more importantly, identify mechanisms clarifying how the negative feedback loop connecting ATP1A3 expression and the AQP4‐p38 pathway influences the synergistic treatment effect of CS‐6 and TMZ in GBM cells. The gene discussed is AQP4; the disease is glioblastoma.