(a) Pooled data show 10 μM neomycin-induced rundown of WT (●) and R333K (○) KCNQ2 currents. KCNQ2 currents were normalized to KCNQ2 current at t = 0. (b) Dose-response curves for neomycin measured at 10 min after rupturing the plasma membrane. Indicated concentration of neomycin was included in the patch pipette. Solid lines are Hill fits to the mean data for WT, R333K, R345K, R353K, and R435K giving IC50 values of 59.1 ± 8.3, 9.2 ± 4.2, 5.2 ± 0.2, 5.4 ± 3 and 6.2 ± 8 μM and slopes of 0.8 ± 0.05, 0.8 ± 0.3, 0.6 ± 0.1, 0.6 ± 0.2, and 0.5 ± 0.2, respectively. (c) Representative traces showing a significant increase in R333K KCNQ2 mutant by 200 μM diC8-PIP2 addition to patch pipette (red line) compared to controls (black line). KCNQ2 (WT) did not show apparent augmentations. Currents were elicited by voltage steps from -60 mV to +40 mV. (d) from data such as shown in c current densities for 0, 20, and 200 μM diC8-PIP2 in the recording pipette were determined and plotted as bars with S.E.M. Currents were measured at >20 min after rupturing the plasma membrane. The numbers in parentheses indicate the number of cells. ANOVA Tukey test. **p<0.01 versus corresponding concentration to WT. #p<0.05; ##p<0.01; ###p<0.001. (e–g) Effects of Prmt1 (e-f) or 4RK mutation (g) on the binding of KCNQ2 and PIP2. Different amounts (10–500 pmol) of PIP2 were spotted onto nitrocellulose membranes and analyzed by a protein-lipid overlay procedure using cell lysate prepared from HEK293T cells transfected with indicated expression vectors or the control pcDNA vector (left panels). The control western blots are shown in right panels. (h) Left, representative traces show augmentation of M-current by 200 μM diC8-PIP2 in the patch pipette. Right, summary of M-current density from WT neurons and Prmt1+/- neurons with 0, 20, and 200 μM diC8-PIP2 in the recording pipette. Mean ± S.E.M. ANOVA Tukey test, ***p<0.001. (i–j) the mean value of input resistance (i), and threshold current for AP generation (100 ms duration; j) from WT (+/+) neurons, mutants (+/-), and mutants loaded with 20 μM diC8-PIP2 (+/-; PIP2). (k) the mean number of APs in response to 1-s depolarizing current injection (200 pA) from a WT (+/+) neuron, a mutant (+/-), and a mutant loaded with 20 μM diC8-PIP2 (+/-; PIP2).