Tamoxifen inhibition of kv7.2/kv7.3 channels

Abstract

KCNQ genes encode five Kv7 K(+) channel subunits (Kv7.1-Kv7.5). Four of these (Kv7.2-Kv7.5) are expressed in the nervous system. Kv7.2 and Kv7.3 are the principal molecular components of the slow voltage-gated M-channel, which regulates neuronal excitability. In this study, we demonstrate that tamoxifen, an estrogen receptor antagonist used in the treatment of breast cancer, inhibits Kv7.2/Kv7.3 currents heterologously expressed in human embryonic kidney HEK-293 cells. Current inhibition by tamoxifen was voltage independent but concentration-dependent. The IC50 for current inhibition was 1.68 ± 0.44 µM. The voltage-dependent activation of the channel was not modified. Tamoxifen inhibited Kv7.2 homomeric channels with a higher potency (IC50 = 0.74 ± 0.16 µM). The mutation Kv7.2 R463E increases phosphatidylinositol- 4,5-bisphosphate (PIP2) - channel interaction and diminished dramatically the inhibitory effect of tamoxifen compared with that for wild type Kv7.2. Conversely, the mutation Kv7.2 R463Q, which decreases PIP2 -channel interaction, increased tamoxifen potency. Similar results were obtained on the heteromeric Kv7.2 R463Q/Kv7.3 and Kv7.2 R463E/Kv7.3 channels, compared to Kv7.2/Kv7.3 WT. Overexpression of type 2A PI(4)P5-kinase (PIP5K 2A) significantly reduced tamoxifen inhibition of Kv7.2/Kv7.3 and Kv7.2 R463Q channels. Our results suggest that tamoxifen inhibited Kv7.2/Kv7.3 channels by interfering with PIP2-channel interaction because of its documented interaction with PIP2 and the similar effect of tamoxifen on various PIP2 sensitive channels.

Figure 1. Tamoxifen inhibited Kv7.2/Kv7.3 channels expressed in HEK-293 cells.

A and B, ionic currents recorded in control condition (A) and in presence of 1 µM tamoxifen (B) using voltage protocol shown in the inset. C, Current-voltage relationship in control (square, n = 6) and in presence of 1 µM tamoxifen (circles, n = 6). D, Kv7.2/Kv7.3 activation curves in control (squares, n = 5) and in presence of 1 µM tamoxifen (circles, n = 6).

Figure 2. Kv7.2/Kv7.3 current inhibition by tamoxifen is voltage independent but concentration-dependent.

A, Time course of the inhibitory effect of 1 µM tamoxifen on Kv7.2/Kv7.3 current (n = 6). B, Concentration–effect relationship for block of Kv7.2/Kv7.3 channels by tamoxifen. IC₅₀ = 1.68 ± 0.44 µM, n_H = 0.86 ± 0.12(n = 6). The line represents the fit of the experimental data by a Hill equation with the values given in the text. C, Fractional block of Kv7.2/Kv7.3 current produced by 1 µM of tamoxifen as voltage function (n=5).

Figure 3. Homomeric Kv7.2 channels are inhibited by tamoxifen.

A, Current traces of Kv7.2 in presence and absence of tamoxifen 1 µM. B, Current-voltage relationship in control (squares, n = 5) and in presence of 1µM tamoxifen (circles, n = 5). C, Kv7.2 activation curves in control (squares, n = 5) and in presence of 1 µM tamoxifen (circles, n = 5). D, Concentration–effect relationship for block of Kv7.2 channels by tamoxifen. IC₅₀ = 0.74 ± 0.16 µM, n_H = 0.4 ± 0.05(n = 5). The line represents the fit of the experimental data by a Hill equation with the values given in the text.

Figure 4. Tamoxifen inhibition of Kv7.2 channels is related to PIP₂-channel affinity.

A and B, Superimposed representative Kv7.2 R463Q and Kv7.2 R463E current traces recorded in control (black) and in presence of 10 µM tamoxifen (gray). Currents were evoked depolarizing the membrane to +40 mV for 3 s and then repolarizing to -60 mV. C, Concentration–effect relationship for the inhibitory effect of tamoxifen on Kv7.2 WT channels (dashed line) and the mutants Kv7.2 R463Q (triangles) and Kv7.2 R463E (diamonds). The line represents the fit of the experimental data by a Hill equation with the values given in the text. Each point represents the mean ± SEM from n = 5 to 6 experiments. D, Activation curves for Kv7.2 WT (dashed line, n = 5), Kv7.2 R463Q (triangles, n = 6) and Kv7.2 R463E (diamonds, n = 6). E, Deactivation time constants resulting from the monoexponential fit of the tail current decay (τ_deact) at -60 mV after a test pulse to +40 mV, are shown for Kv7.2 WT (n = 5), Kv7.2 R463Q (n = 6) and Kv7.2 R463E (n = 6).

Figure 5. The affinity of the heteromeric mutant channels Kv7.2 R463Q/Kv7.3 and Kv7.2 R463E/Kv7.3 for PIP₂ correlates with the degree of current inhibition by tamoxifen.

A and B, Superimposed representative Kv7.2 R463Q/Kv7.3 and Kv7.2 R463E/Kv7.3 current traces recorded in control (black) and in presence of 10 µM tamoxifen (gray). Currents were evoked depolarizing the membrane to +40 mV for 3 s and then repolarizing to -60 mV. C, Concentration–effect relationship for the inhibitory effect of tamoxifen on Kv7.2/Kv7.3 WT channels (dashed line) and the mutants Kv7.2 R463Q/Kv7.3 and Kv7.2 R463E/Kv7.3 (triangles and diamonds respectively). The line represents the fit of the experimental data by a Hill equation with the values given in the text. Each point represents the mean ± SEM from n = 5 to 6 experiments.

Figure 6. Overexpression of PIP5K-2A diminished the effect of tamoxifen on Kv7.2/Kv7.3 and Kv7.2 R463Q channels.

Inhibition of the currents recorded at 40 mV by tamoxifen 1 µM when Kv7.2/Kv7.3 and Kv7.2 R463Q channels were expressed alone compared to the effect of tamoxifen 10 µM when those channels were co-expressed with the kinase.