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. 2016 Mar 8;110(5):1089–1098. doi: 10.1016/j.bpj.2016.01.006

Figure 1.

Figure 1

PIP2 regulates the voltage-activation properties of neuronal KCNQ channels. (a) Representative current traces at different membrane potentials from HEK293T cells expressing (i) KCNQ2/3, (ii) KCNQ3/5, (iii) KCNQ5, or (iv) KCNQ3A315T alone (top panel) or in the presence of PIPKIγ90 (bottom panels). For these experiments, KCNQ currents were measured at various test potentials elicited by a 1 s depolarization from −85 mV, followed by a return step to −70 mV. Test potentials ranged from −115 mV to +15 mV in 10-mV increments. (b) Normalized G-V relationship to the maximum tail current at −70 mV for (i) KCNQ2/3 (n = 8, +PIPKIγ90 n = 6), (ii) KCNQ3/5 (n = 7, +PIPKIγ90 n = 6), (iii) KCNQ5 (n = 10, +PIPKIγ90 n = 5), or (iv) KCNQ3A315T (n = 5, +PIPKIγ90 n = 5). Data represent mean ± SEM. Note that there was a large increase in the current responses in KCNQ5-containing cells when coexpressed with PIPKIγ90. For instance, in the absence of PIPKIγ90 the KCNQ3/5 and KCNQ5 current responses at +15 mV were 921 ± 238 pA (n = 7) and 510 ± 38 pA (n = 10), respectively, whereas in the presence of PIPKIγ90 they were 8141 ± 1088 pA (n = 6) and 3416 ± 747 pA (n = 5). In addition to shifting the voltage activation of KCNQ3A315T channels to the left, PIPKIγ90 induced constitutive voltage-independent activity of KCNQ3A315T (∼15%) (iv). We verified that this basal activity occurred through KCNQ3A315T channels, as application of the selective KCNQ blocker XE991 (20 μM) completely eliminated it. Coexpression of KCNQ3/5 seemed to give rise to a small (∼10%) voltage-independent current. However, and in contrast to the data obtained for KCNQ3A315T, we could not verify that this current flows through KCNQ3/5 channels, as application of 40 μM XE991 did not affect its amplitude at hyperpolarized membrane potentials (at −75 mV: 17 ± 1 pA; +XE991 16 ± 2 pA, n = 4). Elevated PIP2 levels led to slower deactivation of the tail current in the presence of PIPKIγ90. For instance, after a step from −35 mV, the deactivation time constant of KCNQ2/3 at −70 mV was 51 ± 7 ms (n = 8) and 167 ± 44 ms (n = 6; p < 0.05) in the presence and absence of PIPKIγ90, respectively. We found similar changes for KCNQ3/5 channels (KCNQ3/5, τ = 127 ± 29 ms, n = 7 τPIPKIγ = 231 ± 17 ms, n = 6, p < 0.05 Student’s t-test), but not KCNQ5 channels (τ = 140 ± 26 ms, n = 10, τPIPKIγ = 166 ± 7 ms, n = 5; p = 0.5 Student’s t-test). To see this figure in color, go online.