Evaluation of Small-Molecule Candidates as Modulators of M-Type K+ Currents: Impacts on Current Amplitude, Gating, and Voltage-Dependent Hysteresis

Abstract

The core subunits of the K_V7.2, K_V7.3, and K_V7.5 channels, encoded by the KCNQ2, KCNQ3, and KCNQ5 genes, are expressed across various cell types and play a key role in generating the M-type K⁺ current (I_K(M)). This current is characterized by an activation threshold at low voltages and displays slow activation and deactivation kinetics. Variations in the amplitude and gating kinetics of I_K(M) can significantly influence membrane excitability. Notably, I_K(M) demonstrates distinct voltage-dependent hysteresis when subjected to prolonged isosceles-triangular ramp pulses. In this review, we explore various small-molecule modulators that can either inhibit or enhance the amplitude of I_K(M), along with their perturbations on its gating kinetics and voltage-dependent hysteresis. The inhibitors of I_K(M) highlighted here include bisoprolol, brivaracetam, cannabidiol, nalbuphine, phenobarbital, and remdesivir. Conversely, compounds such as flupirtine, kynurenic acid, naringenin, QO-58, and solifenacin have been shown to enhance I_K(M). These modulators show potential as pharmacological or therapeutic strategies for treating certain disorders linked to gain-of-function or loss-of-function mutations in M-type K⁺ (K_V7x or KCNQx) channels.

Keywords: M-type (KV7x or KCNQx) channel; M-type K+ current; current kinetics; small-molecule modulator.

Figure 1

Biophysical characteristics of the M-type K⁺ current (I_K(M)) present in pituitary GH₃ cells. The cells were immersed in a high-K⁺ (145 mM) Ca²⁺-free solution with a reversal potential of around 0 mV, while we filled the recording pipette with a K⁺-enriched solution. The top diagram depicts a simplified schematic representation of a cell membrane (lipid bilayer) containing a K_M (K_V7x or KCNQx) K⁺ channel. The solid arrow in the top diagram points to the activation of I_K(M) in response to membrane depolarization, with K⁺ ions moving inward. The middle diagram shows the voltage-clamp protocol (indicated in black), where the holding potential was set to −50 mV, and a depolarizing pulse was applied from −50 to −10 mV for a duration of 1 s. The bottom diagram represents a schematic representation of the I_K(M) trace (indicated in purple). The asterisk (*) marks the activation phase of the current, while the double asterisk (**) indicates the deactivation phase. The dashed curve illustrates the activation and deactivation time courses of I_K(M) in response to membrane depolarization.

Figure 2

Steady-state activation curve of I_K(M) in GH₃ cells. The activation curve of I_K(M) was derived from GH₃ cells bathed in a high-K⁺ Ca²⁺-free solution. The relationship between membrane potential and normalized I_K(M) is depicted. The current amplitude was measured at the end of a 1 − s depolarizing pulse. The solid blue circles represent the normalized I_K(M) values recorded at each test voltage. The smooth sigmoidal red curve, generated by fitting the data points to the Boltzmann equation detailed in the text, highlights the voltage-dependent activation of I_K(M), particularly at low voltages.

Figure 3

Simplified graphic representation of voltage-dependent hysteresis (Hys_(V)) in I_K(M). The upper panel illustrates a schematic of a prolonged upright isosceles-triangular waveform, representing a double ramp voltage (V_ramp) protocol applied over 2 s. The lower panel shows the relationship between voltage and whole-cell I_K(M), emphasizing the Hys_(V) behavior, highlighted in purple. The dashed black lines in the upper and lower panels indicate the trajectories of the applied potential and the resulting current trace over time, respectively.

Figure 4

Effect of BIS on the deactivating tail I_K(M) during repolarizing phases with varying durations (109–280 ms) to simulate different repolarizing slopes of bursting action potential patterns. (A) Representative current traces recorded in response to the voltage protocol shown in the topmost panel, obtained in the absence (upper) and presence (lower) of 10 μM BIS. The topmost panel indicates the applied voltage protocol. The different colors in current traces correspond with those in the voltage-clamp protocols shown at the topmost panel. (B) Effect of BIS on the peak amplitude of deactivating I_K(M) upon return to −50 mV with different falling phase durations (mean ± SEM; n = 11 for each point). The peak amplitude indicated at each point was measured at various falling phase durations. ■: control (in the absence of BIS); □: in the presence of 3 μM BIS. This figure is adapted from So et al. [56] and published under the terms and conditions of the Creative Commons Attribution (CC BY) license.

Figure 5

Effect of CBD on the activation of I_K(M) through pulse-train stimulation in GH₃ cells. To perform the experiment, cells were placed in a high-K⁺, Ca²⁺-free solution, and the pulse-train stimulation protocol involved a series of 40 depolarizing pulses lasting 20 ms each, applied at −10 mV with 5 ms intervals, for a total duration of 1 s. (A) Representative current traces acquired during the control period (upper trace in black) and in the presence of 3 μM CBD (lower trace in red). The top portion of the figure shows the voltage-clamp protocol applied. The * symbol in the middle portion of the figure indicates the activating I_K(M), while ** represents the deactivating (or tail) component of I_K(M) obtained after returning to −50 mV. Summary bar graphs in (B,C) display the activating and deactivating densities of I_K(M), respectively, in the absence and presence of 1 or 3 μM CBD. The values are presented as mean ± SEM, with each bar representing data from seven independent experiments. The activating density of I_K(M) was measured at the end of the pulse-train stimuli from −50 to −10 mV, while the deactivating density was measured following the return to −50 mV. The * symbol indicates statistical significance when compared to the control group (p < 0.05), while ** denotes statistical significance when compared to the CBD (1 μM) alone group (p < 0.05). This figure is adapted from Liu et al. [76] and published under the terms and conditions of the Creative Commons Attribution (CC BY) license.

Figure 6

Effect of RDV on I_K(M) in GH₃ cells. The experiments were conducted in cells immersed in high-K⁺, Ca²⁺-free solution, and the pipette used was filled with K⁺-containing solution. (A) Reprsentative I_K(M) traces elicited by 1 − s step depolarization from −50 to −10 mV (indicated in inset). The current trace labeled 1 is the control, and that labeled 2, 3, or 4 was obtained after the addition of 0.3 μM RDV, 1 μM RDV, or 3 μM RDV, respectively. (B) Concentration-dependent inhibition of RDV on I_K(M) amplitude in GH₃ cells (mean ± SEM; n = 9). Current amplitude was measured at the end of the 1 − s depolarizing pulse. The continuous line was fitted by a Hill function. The IC₅₀ value (as indicated in the vertical dashed line) needed for an RDV-induced decrease in I_K(M) was 2.5 μM. This figure is adapted from Chang et al. [101] and published under the terms and conditions of the Creative Commons Attribution (CC BY) license.

Figure 7

Stimulatory effect of SOL on voltage-dependent hysteresis (Hys_(V)) of I_K(M) in GH₃ cells. This series of experiments was conducted with an isosceles-triangular V_ramp. (A) Representative current traces evoked by isosceles-triangular V_ramp for a duration of 2 s obtained in the control period (a, black) and during the exposure to 0.3 μM SOL (b, blue) or 1 μM SOL (c, red). The dashed arrows indicate the distinctive patterns of current trajectory by which time passes as V_ramp is applied. The voltage-clamp pulse is illustrated in inset at the left upper corner. (B) Hysteretic area (i.e., Δarea of I_K(M)’s Hys_(V) obtained in control period (i.e., SOL was not present) or during exposure to SOL and SOL plus linopirdine (Lino). The area encircled by current amplitudes activated in the ascending and descending limbs at the voltages between −45 and 0 mV was calculated. Each bar indicates the mean ± SEM (n = 7 for each bar). The * symbol indicates statistical significance when compared to the control group (p < 0.05), while ** denotes statistical significance when compared to the SOL (0.3 μM) alone group (p < 0.05). This figure is adapted from Cho et al. [21] and published under the terms and conditions of the Creative Commons Attribution (CC BY) license.