Voltage-gated potassium (K_v) channels act as "electronically controlled valves" on the cell membrane regulating sophisticated bio-electrical activity. After neurons or cardiomyocytes fire, these channels can rapidly restore the membrane potential to a "resting" state, thereby ensuring the precise transmission of neural signals and the stability of cardiac rhythms. Once the function of these channels becomes abnormal, it may trigger diseases such as epilepsy, cardiac arrhythmias, and neuropathic pain.

In the central nervous system, the KCNQ (K_v7) family of channels is considered a crucial "voltage stabilizer".M-channel—a heteromeric tetramer composed of KCNQ2 and KCNQ3—is widely regarded as a "molecular brake" against neuronal hyperexcitability. Clinical studies have shown that loss of function in either subunit can induce early-life epilepsy; therefore, the M-channel has long been a significant target for antiepileptic drugs. However, most previous structural studies on KCNQ channels have focused on homomeric complexes. For the heteromeric M-channels that actually perform physiological functions, there has been a persistent lack of direct structural evidence regarding their specific assembly mode, how the subunits work synergistically, and how they are modulated by drugs.

On May 27, 2026 (Beijing Time), Jian Huang and Xiao Fan's team at the Shenzhen Medical Academy of Research and Translation (SMART) published a research paper titled "Structural basis for the assembly and modulation of human M-channels" online in Vita (Figure 1). This study resolved the first high-resolution cryo-electron microscopy (cryo-EM) structure of the human heteromeric M-channel, systematically revealing its heteromeric assembly mode, voltage-sensing mechanism, drug recognition pattern, and a novel PIP₂-mediated stepwise gating mechanism.

Figure 1. The research findings published online in Vita

Unrevealing the Heteromeric Assembly Rules of the M-Channel for the First Time

By co-expressing KCNQ2 and KCNQ3 proteins, the authors successfully reconstituted functional heteromeric M-channels with typical robust macroscopic currents, and verified the lower activation voltage using electrophysiological experiments. Cryo-EM structural analysis revealed that this heteromeric M-channel exhibits a unique stoichiometric distribution: it primarily assembles at a ratio of three KCNQ2 to one KCNQ3 (3:1), while a minor 2:2 assembly form was also observed. Notably, in the 2:2 assembly configuration, identical subunits are distributed diagonally (Figure 2), indicating that the M-channel possesses a highly ordered yet flexible heteromeric assembly mode. This discovery provides a critical structural basis for understanding the assembly rules of heteromeric ion channels.

Figure 2. Heteromeric assembly mode of the M-channel

KCNQ3 is the "Core Striker" Driving the Low-Threshold Activation of the Channel

The M-channel can be activated near the neuronal resting potential, thereby effectively inhibiting neuronal hyperexcitability. Structural comparisons revealed that compared to KCNQ2, the voltage-sensing domain (VSD) of KCNQ3 exhibits conformational features that make it easier to enter the activated state.

Figure 3. KCNQ3 decreases the voltage sensitivity of the M-channel

To verify this structural finding, the research team conducted functional experiments using whole-cell patch voltage clamp. The results demonstrated that when the VSDs of the M-channel are completely replaced with those of KCNQ3, the activation threshold of the channel is significantly lowered, allowing it to open earlier at more negative membrane potentials (Figure 3). This result directly proves that the KCNQ3 subunit plays a “striker” role in the heteromeric complex and is the core driving force conferring the low-threshold activation characteristic to the M-channel.

Selective Recognition and Synergistic Gating: Drug Modulation Mechanisms Empower Precise New Drug Design

In terms of drug recognition, the study thoroughly elucidated the distinctly different mechanisms of action of two representative M-channel modulators, providing precise templates for the development of subunit-specific targeted drugs:

The "subunit-specific" recognition mechanism of ICA-110381: This small-molecule agonist selectively binds to the VSD of the KCNQ2 subunit. The structure not only explains its high selectivity for KCNQ2 at the atomic level but also further corroborates the presence and arrangement of KCNQ2 subunits in the heteromeric complex (Figure 4).

Figure 4. ICA-110381 selectively targets the voltage-sensing domain of the KCNQ2 subunit

"Cooperative Stepwise Activation" by XEN1101 and PIP₂: As a crucial antiepileptic drug candidate currently in Phase III clinical trials, XEN1101 binds to a conserved "fenestration" site on the side of the channel's pore domain (PD). More importantly, the research revealed for the first time that this drug can synergistically interact with endogenous phospholipid PIP₂ to mediate the gradual opening of the channel in a novel "stepwise activation" mode (Figure 5).

Figure 5. Stepwise activation mode of the heteromeric M-channel synergistically modulated by XEN1101 and PIP₂

This study not only fills a critical gap in the structural biology field of heteromeric M-channels, revealing subunit-specific drug recognition sites and the unique "stepwise activation" mode, but also provides an accurate molecular blueprint for the development of next-generation antiepileptic drugs with high selectivity, high efficacy, and low side effects. This bears significant implications for the treatment of neurological disorders such as developmental and epileptic encephalopathies (DEE).

Notably, during the same period, Huaizong Shen's team from Westlake University and Huaiyu Yang's team from East China Normal University also published a research paper titled "Structural basis for heteromeric assembly and subthreshold activation of human M-channel" online in Vita. This study similarly focused on the heteromeric assembly and subthreshold activation mechanisms of the human M-channel, resolving high-resolution structures of the M-channel in various functional states. Also, a letter to editor in Cell Research reported similar result and modulation mechanisms of M-channel from Jin Zhang's team from Nanchang University. These back-to-back studies approached the subject from different research paths, comprehensively utilizing multidisciplinary techniques such as high-resolution cryo-EM, medicinal chemistry, and electrophysiological experiments. Together, they formed a highly complementary and mutually corroborating systematic research framework, further deepening the understanding of the assembly rules, gating mechanisms, and pharmacological modulation modes of the M-channel.

Furthermore, all of these research findings were initially posted on the "LTS Preprint Server" in December 2025. Following rigorous peer review, they were formally published in Vita, a high-level international journal in the life sciences and biomedical fields jointly established by the Open Life Science Alliance. This complete dissemination chain—from rapid public disclosure on a preprint platform to formal publication in a high-level journal—fully reflects Vita's publishing philosophy of "zero OA fees, returning to the essence of academia". Meanwhile, relying on the "LTS Preprint Server" to achieve real-time open sharing of scientific research results also demonstrates an innovative academic communication model of "free real-time global preview + rigorous peer review + formal journal publication."

Jian Huang and Xiao Fan, Junior PIs at SMART, are the co-corresponding authors of the paper. Fangzhou Lu, a Ph.D. student from the inaugural Class of 2024 SMART-Westlake University joint program; Xiaoshuang Huang, an Associate Investigator at SMART; and Guanxing Cai, an engineer at the Electrophysiology Core Facility of SMART, are the co-first authors. Yuzhen Xie, a research assistant in Jian Huang's lab, as well as Ph.D. students Xianglong Shen, Pei Huang, and Feifan Yu, also made significant contributions to this research. Kun Wu, Deputy Director of the Rare Disease Center of SMART and Shenzhen Bay Laboratory (SZBL), provided crucial support in clinical information related to channel mutations for this project. This study was strongly supported by the Computing Labware for Electron-microscopy Visualization and Experimental Research (CLEVER) at the Biomedical Data Center, and received funding from SMART and the National Natural Science Foundation of China and the Guangdong Pearl River Talent Program.

Reference:

1. Wulff, H., Castle, N.A. & Pardo, L.A. Voltage-gated potassium channels as therapeutic targets. Nat Rev Drug Discov 8, 982-1001 (2009).

2. Huang, J., Pan, X. & Yan, N. Structural biology and molecular pharmacology of voltage-gated ion channels. Nat Rev Mol Cell Biol 25, 904-925 (2024).

Translation: Yang Shen

Proofreading: Fangzhou Lu