The discovery of Kv10.1 channel can be traced back to the study of voltage-gated potassium ion channels. Kv10.1 was first discovered by Kaplan et al (2) in 1969 in the mutant X chromosome of Drosophila. Its name 'ether-a-go-go' comes from the irregular leg tremor phenotype exhibited by Drosophila mutants under ether anesthesia, which is caused by a functional defect in voltage-gated potassium channels due to gene locus mutations (17). Studies have shown that molecular cloning technology confirms that the protein encoded by the Eag gene belongs to the Kv superfamily and reveals its direct association with the regulation of neural excitability in Drosophila (17). Kv10.1 is located in chromosome 1, band q32.1-32.3 and comprises 457,343 bases and 12 exons (18).

Kv10.1 is encoded by KCNH1 gene, which encodes a 989 amino acid protein with an estimated molecular weight of 111,423 Daltons. The overall structure of Kv10.1 channel is similar to these of the other Kvs. The core region contains six transmembrane domains (transmembrane helices S1-S6), including voltage sensor domains (transmembrane helices S1-S4) and potassium ion selective permeability channels (transmembrane helices S5, pore helices S6) and the N-terminal and C-terminal of this channel protein (Fig. 1). The single-hole channels of these six transmembrane regions (S1-S6) are activated by depolarization (19-21). The N-terminal contains Per-Arnt-Sim (PAS) domain. C-terminal contains cyclic nucleotide binding homology domain (CNBHD), C-linker, ciliary localization signal and tetrameric coiled helix domain (19,22).

Although the structure of Kv10.1 has a number of similarities with other Kvs, based on the three-dimensional structure of the rat Kv10.1 obtained by single-particle cryoelectron microscopy, it was found that Kv10.1 has a number of unique structural features compared with other Kv channels. The S2-S3 connector of Kv10.1 extending into cytoplasm is different from those of other voltage-gated potassium channels. This connector is a conservative feature of Kv family and the only other Kv subfamily member containing the similar connectors is Kv7 (19,23). In Kv10.1, S4 region is the main positive charge concentration region, usually with 5-7 positive charges distributed. As a voltage receptor, this region can respond to the electric field force when the membrane potential changes and drive the conformational change of the channel. In addition, other transmembrane regions (such as S1, S2 and S3) may also contain a small amount of positively charged amino acid residues, but the positive charge density is relatively low and the sensitivity to voltage change is not as significant as that of S4 region. S1-S4 constitute positively charged amino acids in the field of voltage sensors, which move membrane potential with the depolarization of cerebral cortex (24-26). The S4-S5 linker in Kv10.1 is a short loop composed of five amino acid residues, which contrasts with the helical structure formed by 15 residues parallel to the membrane in domain exchange potassium channels (such as Kv1.2) (19,27). The segments S5-S6 form the pore domain of the channel, while the ring P (between S5-S6) is responsible for the K⁺ selectivity. These local structural features determine that Kv10.1 has a different voltage gating process compared with other types of Kv channels. S4 in Kv10.1 directly interacts with the carbon terminal C-linker to close the channel, that is, the channel can realize voltage-dependent activation gating without covalent connection between S4 and S5 (27,28).

Kv10.1 also has unique N-terminal and C-terminal domains, which are necessary for subfamily-specific channel assembly (17,29,30). Kv10.1 has a large N-terminal and a C-terminal in cells, accounting for ~70% of its molecular weight. In addition, there are several regulatory domains at the ends of cells. N-terminal contains a PAS domain, which is usually involved in the detection of redox reaction. It is considered as an oxygen sensor, which can induce the production of hypoxia inducible factor hypoxia-inducible factor 1 (HIF-1) in hypoxic environment, thus causing the increase of glycolysis rate and angiogenesis, which is beneficial to the growth of tumor cells in hypoxic environment. The C-terminal contains a CNBHD, which is a calmodulin binding site (31). The fragment between CNBHD and S6 is called C-linker. S6 spirally extends to the intracellular region and is connected to C-linker, forming an intracellular ring above CNBHD. The C-linker couples the movements of S6 and CNBHD and CNBHD is connected to the pore region through C-linker (32). As with other ion channels, Kv10.1 undergoes nitrogen chain glycosylation at N388 and N406 sites, which is very important for the correct transport of the channel to the membrane. Three-dimensional structural analysis by cryo-electron microscopy showed that the PAS domain of Kv10.1 channel in rats was located in the intracellular region and mainly interacted with CNBHD, a neighboring subunit. In addition, Kv10.1 channel contain amino acid residues carrying glycosylation regions of polymers, among which glycosylation of N406 is a necessary condition to maintain the normal structure and function of Kv10.1 and deglycosylation will lead to weakening of channel current and slow activation (19,33).

Kv10.1 is a voltage-gated potassium channel, which is highly expressed in nervous system and some cancer cells. It has unique electrophysiological characteristics and plays an important role in cell excitability regulation, tumor occurrence and development.

Kv10.1 channel is voltage-dependent and its activation degree changes with the membrane potential. This characteristic makes Kv10.1 channel play an important role in regulating cell excitability. In addition, the gating characteristics of Kv10.1 channel are also different from other potassium channels, with a slow activation and deactivation process. The activation characteristics of Kv10.1 can be associated with Cole-Moore shift observed in squid axons published by Cole KS and Moore JW in 1960 (57). This effect was originally used to describe the potassium current in squid axons, which is characterized by a significant delay in current activation when hyperpolarized resting membrane potential channels are activated (58,59). The Cole-Moore shift of Kv10.1 is ~10s of milliseconds, while that of other members of the voltage-gated potassium channel family is close to 1 msec and the Cole-Moore shift of Kv10.1 is steeper than that of Kv10.2 (60). This electrophysiological feature can be used as a marker to identify the expression of Kv10.1 in cells and ectopic expression. The activation of Kv10.1 depends on cell membrane resting potential and extracellular Mg²⁺ concentration and low extracellular pH can slow its activation (61,62). Cole-Moore shift can be enhanced by increasing extracellular Mg2+ concentration in physiological range (63). The Cole-Moore shift of Kv10.1 can be inhibited by using amiodarone and mibefradil (64,65).

Kv10.1 is mainly expressed in the nervous system and its electrophysiological characteristics are closely associated with the function of the nervous system. In adult rats, the mRNA of Kv10.1 can be mainly detected in olfactory bulb, cerebral cortex, hippocampus and cerebellum and it is consistent with its protein expression level (66). Further research found that Kv10.1 was mostly expressed on dopaminergic cells in physiological state, which may be associated with its electrophysiological function (67). In the nervous system, Kv10.1 can be used as a regulator of local action potential, especially when other potassium channels suffer from high-frequency bursts of cumulative inactivation. Part of the function adjustment of Kv10.1 channel involves its detergent resistant membrane part (also called fat raft) (68,69).

Kv10.1 channel is highly expressed in 70% of tumors and its expression level is closely associated with the malignant degree, metastatic ability and clinical prognosis of tumors. Kv10.1 is highly expressed in head and neck cancer, breast cancer and acute myeloid cells (54,70-72) and inhibiting or knocking out Kv10.1 can reduce tumor growth (73-76). Therefore, Kv10.1 is considered as a potential target for cancer treatment.

Kv10.1 channel affects the production of resting potential and action potential by regulating the potassium ion permeability of cell membrane. This electrophysiological change may further affect the intracellular signal transduction pathway, thus regulating the cell proliferation process. In tumor cells, the high expression of Kv10.1 channel may lead to uncontrolled cell proliferation and promote tumor growth. In addition to regulating cell proliferation, Kv10.1 channel may also participate in tumor metastasis by regulating cell migration and invasion.

Kv10.1 is very important for the proliferation of tumor cells. In 1997, Brüggemann et al first discovered the function of Kv10.1 in cell proliferation in Xenopus oocytes (77). Kv10.1 leads to abnormal proliferation of cells, including enhanced metabolic activity and reduced dependence of cells transfected with Kv10.1 on growth factors in the medium. Cells transfected with Kv10.1 can continue to grow in the medium with low concentration of serum, even without matrix indicating the loss of contact inhibition characteristics (17). In vitro, inhibiting the expression of Kv10.1 can reduce the cell proliferation, migration and invasion of cancer cells. In vitro experiments, injecting exogenous CHO cells expressing Kv10.1 into immunosuppressed mice induced the occurrence of invasive tumors (78), while using specific monoclonal antibodies to inhibit Kv10.1 can inhibit the growth of tumors in vitro (79) and similar effects were also observed in animal experiments (80-85). In recent years, the closed state wild-type channel model of cryoelectron microscope structure based on HERG and Kv10.1 channels has been constructed by using Rosetta software and cryoelectron microscope structure. These models were then used in molecular docking studies to explore the mechanism of drug channel interaction (86-89).

Generally, Kv10.1 channel plays an important role in the occurrence and development of cancer and its high expression is closely associated with the clinical prognosis of a number of tumors. In-depth study on the pathological function and regulation mechanism of Kv10.1 channel is expected to provide new theoretical basis and therapeutic strategies for cancer prevention and treatment.

In 2006, it was found that the expression activity of Kv10.1 was associated with the cell cycle (90). Cell cycle regulates the progress of mitosis. During mitosis, if progesterone or mitotic promoter exists, the activity of Kv10.1 channel will be inhibited, which also proves its cell cycle sensitivity (91).

The research after 2016 gave more specific results. That is, Kv10.1 is located in centrosome and primary cilia (92) and promotes the decomposition of primary cilia in G₂/M phase, which is beneficial to the progress of cell cycle (93). Following Kv10.1 knock-out, ciliary decomposition is inhibited and proliferation is delayed. Therefore, the regulation of Kv10.1 on ciliary development can explain the influence of Kv10.1 expression on normal cell proliferation and may be a main mechanism of its tumorigenesis (94). Kv10.1 affects Ca²⁺ entry through hyperpolarization and Ca²⁺ inhibits EAG1 function through calmodulin (3). Ca²⁺ is associated with the cell cycle and cell replication. Calcium influx is considered necessary for the transition from G₁ phase to S phase in the cell cycle. This hyperpolarization also facilitates the entry of a large amount of nutrients into the cell, thereby promoting cell proliferation (10,95).

Similar research also support the aforementioned view. Urrego et al (96) reported that Kv10.1 could be detected only in G₂/M phase. As the E2F transcription factor 1 (E2F1) coordinates cell division and induces the expression of Kv10.1 in G₂ phase, Kv10.1 can only be detected in G₂/M phase and downregulation of Kv10.1 will prolong the duration of G₂/M phase. However, Kv10.1 will be diluted in G₀/G₁ phase, so it cannot be detected in other stages of cell cycle (97). Some studies also hypothesize that the strong influence of Kv10.1 channel in cell cycle progress is also associated with ciliary disintegration, which usually provides the signal transduction needed to start proliferation (96,97). Cyclic expression of Kv10.1 has been proved to exist at the base of primary cilia of hTERRPE-1 cells, which may lead to significant disposability of microtubules in the subsequent process (10).

Regarding microtubule activity, the upregulation of Kv10.1 is closely associated with a significant increase in microtubule dynamics, characterized by assembly and disassembly rates. Therefore, there is an important association between Kv10.1 overexpression and certain processes including cellular variability (96,97). It has been proved that the mobility of MDA-MB-231 cells decreased markedly when Kv10.1 was exposed to a specific blocking agent such as astemizole. Some hypotheses suggest that cell migration caused by Kv10.1 channel is mainly due to the change of microtubule dynamics caused by hyperpolarization of action potential, which has been proved to induce calcium to enter through Ca²⁺ release and activates ORAI calcium release-activated calcium modulator 1(ORAI1). ORAI1 association with Ca²⁺ ATPase seems to be associated with the proliferation and survival of cancer cells (98).

The outflow of K⁺ through Kv10.1 channel makes cells hyperpolarized, thus promoting the influx of Ca²⁺, leading to the proliferation of non-invasive cells (10,99,100). However, the relationship between Kv10.1 and intracellular Ca²⁺ is complicated. Although Kv10.1 is beneficial to Ca²⁺ influx under hyperpolarization, high intracellular Ca²⁺ reversibly inhibits the overexpression of Kv10.1 in a number of tumors (101,102). Overexpression of Kv10.1 can lead to membrane potential hyperpolarization and affect cell cycle progression. Potential changes in the cell cycle are much slower, smoother and less pronounced than rapid action potentials. Due to membrane hyperpolarization, the driving force for calcium influx is enhanced. Ca²⁺ is a major second messenger involved in cell proliferation, migration, survival and apoptosis (103). Depletion of extracellular calcium ions can arrest cells in the early G₁ and G₁/S phase transition. Normal cell proliferation relies on intracellular and extracellular calcium ions, especially during the G₁ phase. However, cancer cells can bypass this requirement and continue to proliferate in calcium-deficient media (104,105). This mechanism exhibits high similarity across various types of cancer. Calcineurin is one of the main regulators of intracellular Ca²⁺ signaling. Calcineurin inhibits the degradation of cyclin D1 through dephosphorylation of T286 residue, promoting cell cycle progression (106). Ca²⁺ can also form a dynamic interaction network with the Hippo pathway, jointly regulating cell proliferation, differentiation and tissue homeostasis (107). Although there are currently no specific studies directly elucidating the role of Kv10.1 in the interaction with Ca²⁺ regulation of cyclin D1 and the Hippo pathway, based on the existing understanding of the complex regulatory relationship between Ca²⁺ and cyclin D1 and the Hippo pathway, it is reasonable to speculate that Kv10.1 may play a role in this regulatory network. Cell hyperpolarization is very sensitive to the activation of Kv10.1, which can regulate cell hyperpolarization through the interaction between N-terminal of PAS domain and S4-S5 connector (28).

When the cell membrane is hyperpolarized, the Kv10.1 channel can sense this potential change and open, allowing potassium ions to flow from the inside of the cell to the outside (17). Therefore, drug development for Kv10.1 channel has become a potential cancer treatment strategy, aiming at inhibiting the growth and spread of tumor by blocking its hyperpolarization mechanism.

Epigenetic regulation mechanism is a mechanism that changes biological phenotype without involving DNA sequence changes, but including DNA methylation, histone modification and non-coding RNA-mediated regulation. These modifications can affect the transcription activity of genes, thus regulating gene expression (108). For example, the DNA methylation status in the promoter region of Kv10.1 gene may affect its transcription activity, thus regulating the expression level of Kv10.1. In addition, histone modification may also regulate the transcription process of Kv10.1 gene by affecting the position and arrangement of nucleosomes. Studies have shown that several abnormally methylated genes, including Kv10.1, have been found in gastric cancer tissue and the methylation level of Kv10.1 in the samples is more than three times higher, suggesting that the hypermethylation of Kv10.1 may play a role in the occurrence and development of gastric cancer (17,35). Some have suggested that epigenetic changes in early life may alter lung cell function and lead to asthma risk (109). In mouse models, early exposure to house dust mite allergens alters DNA methylation and the expression of different genes detected, up to three consecutive generations and is associated with airway hyperresponsiveness and inflammation (109). In mice exposed to allergens, the Kcnh1 gene was hydroxymethylated and upregulated, indicating a susceptibility to asthma (109). In head and neck squamous cell carcinoma (HNSCC), histone acetylation, rather than DNA methylation, is hypothesized to be involved in the regulation of Kv10.1 (40).

Simultaneous drug resistance to multiple drugs with different chemical structures and targets is the main obstacle to effective cancer treatment (110-114). Multidrug resistance (MDR) is an acquired resistance of microorganisms and tumor cells to chemotherapeutic drugs, which is characterized by different chemical structures and mechanisms of action. MDR is the result of overexpression of a number of protein and these protein drugs squeeze chemotherapy drugs out of cells to make their concentration lower than the effective concentration (115). MDR in cancer treatment causes tens of thousands of deaths every year and it can be endowed by a number of transporters that pump drugs out of cells. They can transport various substrates, including amino acids, peptides, ions, sugars, toxins, lipids and drugs and are associated with several serious human diseases (116). First-line therapy is usually followed by the proliferation of a small number of surviving cancer cells, which leads to the development of secondary tumors, which are insensitive to initial drugs. This may lead to tumor progression after stabilization or significant regression, because successful chemotherapy in the first stage becomes ineffective (117,118).

Platinum drugs (such as cisplatin and carboplatin) are one of the commonly used chemotherapy drugs in clinic and are widely used to treat various tumors. However, the resistance of tumor cells to platinum drugs limits their clinical application. As aforementioned, the ectopic expression of Kv10.1 is not only associated with the proliferation of tumor cells, but also associated with the phenotype of chemotherapy-resistant cells. In vitro experiments in ovarian cancer cells showed that the expression of Kv10.1 was associated with the chemotherapy resistance of cisplatin. Compared with the cells treated with cisplatin alone, the downregulation of Kv10.1 combined with cisplatin increased the apoptosis of ovarian cancer cells (16). Similar results were also observed in the chemotherapy-resistant glioblastoma cell line U251AR, which showed a high level of Kv10.1 (mRNA and protein). Notably, when the expression of Kv10.1 is downregulated, U251AR cells are more sensitive to chemotherapy drugs (119). Therefore, the inhibition of Kv10.1 channel can improve the response of cells to commonly used therapeutic drugs in chemotherapy.

The tumorigenic mechanism of Kv10.1 is associated with the fact that calcium-dependent calmodulin kinase-Ⅱ is always activated, which can cause imbalance of cell proliferation and apoptosis even at low calcium ion concentration (120). Kv10.1 also promotes tumor progression by increasing the activity of hypoxia sensor hypoxia-inducible factor-1 alpha (HIF-1α), stimulating the secretion of vascular endothelial growth factor (VEGF) and increasing angiogenesis under hypoxia (78). The tumor microenvironment usually shows changes in extracellular matrix (ECM). ECM products, such as type 1 collagen and fibronectin, promote tumor progression by increasing cell movement (121).

Ion channels are highly fine-tuned protein necessary for cell physiology. Therefore, the synthesis and degradation of ion channels should be a quality control process, which is necessary for correct cell function. However, the knowledge about the synthesis and degradation of Kv10.1 is limited. A study has shown that protein degradation of Kv10.1 depends on the protein ligase cullin 7 (Cul 7) (122). Cul7 degrades Kv10.1 protein on plasma membrane through proteasome and lysosomal pathways respectively (122).

Studies have shown that the tumor suppressor gene regulatory network includes the p53-miR-34 pathway and E2F1 transcription factor, which can regulate the expression of Kv10.1 (96,123). The expression of Kv10.1 may be regulated by the negative feedback of this signal pathway (124,125). This negative regulation may be achieved through two mechanisms: direct inhibition of Kv10.1 at the post-transcriptional level and negative regulation of Kv10.1 through negative feedback mechanism through the p53-miR-34-E2F1 pathway. Studies have also shown that estrogen and human papillomavirus (HPV) regulate the level of Kv10.1 protein (126). As estrogen receptor interacts with p53 (125), HPV inhibits p53 and activates E2F1 (127), suggesting that EAG1 may affect downstream gene transcription by regulating E2F1. In addition, insulin-like growth factor 1 can also increase the expression of EAG1 by activating kinase AKT1 (128). Studies have shown that AKT1 can overcome the apoptosis-inducing effect of p53 (129), suggesting that p53 and Kv10.1 can act in opposite directions along the axis of apoptosis and proliferation (130).

The influence mechanism of silent Kv10.1 on HIF-1α/STAT3/VEGF pathway is a multi-level and multi-step process. As a potassium channel, the abnormal expression of Kv10.1 may regulate the activity of intracellular signal transduction pathway by affecting the balance of potassium concentration inside and outside the cell. Kv10.1 can upregulate the expression of HIF-1α by interacting with intracellular hypoxia homeostasis system. HIF-1α is a key transcription factor to regulate the expression of VEGF, which promotes the transcription of VEGF under hypoxia, thus inducing angiogenesis (131). In osteosarcoma cells, the phosphorylation level of STAT3 decreased markedly after Kv10.1 silencing, which indicated that Kv10.1 might regulate the expression of STAT3-VEGF downstream by influencing its activation state (84,132).

The PI3K/AKT signaling pathway is an important signal transduction pathway with multiple biological functions mediated by enzyme-linked receptors in mammals. The PI3K/AKT pathway plays an important role in the occurrence and development of tumors. When stimulated by upstream signals, PI3K activates AKT, which further activates downstream signal molecules and regulates cancer cell proliferation, invasion and metastasis, angiogenesis and carbohydrate metabolism. In osteosarcoma, tumor growth and angiogenesis in osteosarcoma can be inhibited by downregulating VEGF/PI3K/AKT signaling pathway to silence Kv10.1 (133). The Kv10.1 channel and PI3K/AKT signaling pathway form a cancer-promoting signal axis through functional correlation and Kv10.1 is the key target of Nutlin-3 regulating PI3K/AKT through p53 (81). Therefore, it is of great significance to study PI3K/AKT signaling pathway and develop targeted drugs for tumor treatment.

The nuclear localization signal at the C-terminal of Kv10.1 potassium channel can activate the MAPK pathway to cause changes in cell morphology (134). The Kv10.1 channel plays the role of oncogene, such as promoting the proliferation of human osteosarcoma cells. The high expression of Kv10.1 in osteosarcoma cells is regulated by p38MAPK/p53 pathway (135). Inhibition of p38MAPK activation by p38 MAPK inhibitor SB203580 or short interfering RNA also decreased the level of Kv10.1 protein, but increased the level of p53 protein. In addition, the activation of p53 will lead to the growth stagnation of osteosarcoma cells and decrease the level of Kv10.1 protein, while the inactivation of p53 will promote the cell growth and increase the expression of Kv10.1 protein. This suggests that Kv10.1 may affect the proliferation of osteosarcoma cells by regulating p53 (136).

Although Kv10.1 exhibits mechanistic similarities across various malignancies, namely, influencing tumor proliferation through hyperpolarization, exerting effects via the PI3K/AKT signaling pathway and inhibiting apoptosis (81), its downstream signaling network still demonstrates significant cancer type specificity. For instance, the expression of Kv10.1 in endometrial cancer tissues is markedly higher than that in normal endometrial tissues. Kv10.1 can be regulated by cyclin D1, p53 and other factors, thereby affecting the proliferation, migration and invasion of tumor cells (137). Kv10.1 may form different complexes or interaction networks with other molecules in various cancers, thereby influencing its function. For example, in osteosarcoma and hepatocellular carcinoma, Kv10.1 enhances tumor angiogenesis by upregulating the expression of hypoxia-inducible factor HIF-1α and promoting the secretion of VEGF. This effect is not dependent on its ion transport function but is achieved through interaction with the cellular hypoxia homeostasis system (133,138). Different mechanisms predominate in various cancers; for instance, in breast cancer, the PI3K/AKT pathway is central, driving cell proliferation and metabolic reprogramming; in hepatocellular carcinoma, the HIF-1α/VEGF pathway is predominant, promoting angiogenesis and tumor progression; in endometrial cancer and head and neck cancer, it may affect tumor behavior through cell cycle regulation and cytoskeleton dynamics (137-139). Drug targets are critical in drug discovery and therapy, as their identification is essential for achieving precision treatment (140). Therefore, Kv10.1 is considered a potential therapeutic target in various cancers.

The Kv10.1 channel is formed by assembling tetramer subunits and each subunit contains N-terminal PAS domain and C-terminal cyclic nucleotide binding homology domain. Small molecular ligands can inhibit Kv10.1 channels by binding to their PAS domains. Deleting PAS domain can cancel the inhibitory effect of chlorpromazine (141). At low voltage, chlorpromazine can inhibit mouse Eag1 by binding with PAS domain, but when the channel is open at high voltage, it will be inhibited by blocking the channel (142). Chlorpromazine, a small molecule binding agent of PAS domain, changes the interaction between PAS and CNBH domain and reduces the coupling between tetramer ring and channel hole in cells, while the coupling between PAS and voltage sensor domain (VSD) domain has little effect. In addition, the combination of chlorpromazine and PAS domain does not change the Cole-Moore shift characteristics of Kv10.1 channel, which further indicates that chlorpromazine has no effect on the movement of VSD from deep closed state to open state (143). Chlorpromazine inhibits the proliferation of oral cancer cells by regulating the PI3K/AKT/mTOR signaling pathway and effectively suppresses tumor growth in zebrafish and mouse models. Given that chlorpromazine can act on Kv10.1, it is hypothesized that it regulates tumor proliferation by modulating the PI3K/AKT signaling pathway through Kv10.1. Based on the mechanism of action of chlorpromazine on oral cancer cells and its potential association with Kv10.1, it is reasonable to hypothesize that chlorpromazine may regulate the PI3K/AKT signaling pathway through Kv10.1, thereby affecting tumor proliferation (144).

VSD has a small molecule binding site as a gating regulator and extracellular VSD allows molecules to regulate channels without transmembrane, as with toxins. Most of the known small molecules that regulate Kv10.1 activity first cross the cell membrane and bind to the inner side of the channel. Most of these compounds are physically closed pore blocker ion permeation pathways, such as mibefradil (65,145), Purpurealidin analog 5 (146), 20 (s)-Ginsenoside Rg3 (147,148), APETx4 (149) and Corydaline (150).

Mibefradil has been reported to alter the gating of Kv10.1 by binding to the VSD (65,145). When the hyperpolarizing potential acts outside the cell, mibefradil induces obvious open inactivation, but not from inside the cell. In addition, mibefradil also inhibited the Cole-Moore shift. It is also not used as a pore size blocker because it does not compete with the known pore size blocker, quinidine. Mibefradil 1 also inhibits other potassium channels and L-type and T-type calcium channels (65).

Purpurealidin is considered to be a gating modifier, which binds to the mibefradil binding site near the voltage sensor Kv10.1. Mibefradil is a Ca²⁺ channel antagonist and a Kv10.1 door control regulator. As with purpurealin, mibefradil moves the activation curve to the left. Studies have shown that the binding site of purpurealidin on Kv10.1 overlaps with that of mibefradil (65,145,146).

Ginsenoside is a steroidal glycoside that inhibits the KCNH family at sub µM concentrations. Its function is not limited to Kv channels. Studies have shown that ginsenosides can markedly inhibit the voltage dependent activation curves of Kv10.1 and HERG channels and increase their opening probability at more negative potentials (147,148). When combined with cisplatin, it can enhance chemotherapy sensitivity and reverse drug resistance by blocking the PI3K/AKT pathway (151).

Apetx4 is a new toxin isolated from sea anemones, which can inhibit the Kv10.1 channel. APETx4 inhibits its current by binding to Kv10.1 channel and the inhibitory effect is concentration dependent. APETx4 is a gating modifier that presumably binds to the S3b-E2-S4 region (voltage sensor paddle) of Kv10.1 (149). Corydaline is a novel natural product that selectively inhibits Kv10.1 channels, while being insensitive to other KCNH channels. Corydaline can inhibit the proliferation and migration of liver cancer cells by targeting Kv10.1. This previously unidentified new site can specifically bind to the medicinal pocket of Corydaline, providing possibilities for drug screening against diseases associated with abnormal Kv10.1 channels (150).

The selectivity filter of Kv10.1 consists of a highly conserved amino acid sequence, forming a structure similar to the 'potassium channel signature sequence' (152). Through the spatial arrangement of carbonyl oxygen atoms and threonine hydroxyl oxygen atoms, this region mimics the hydration layer mechanism of water molecules surrounding K⁺, forming a series of K⁺ binding sites (153,154).

Tetrandrine, a natural compound used in traditional Chinese medicine, can inhibit Kv10.1 in a concentration dependent manner with an IC₅₀ of 69.97±5.2 µM (155). Tetrandrine specifically inhibited Kv10.1 channel, blocked potassium outflow, induced cell cycle arrest in G₁/S phase and enhanced cisplatin sensitivity by downregulating PI3K/AKT signaling pathway (44). Tetrandrine may also have the same binding site as procyanidin B1, a natural compound present in grape seeds, which inhibits Kv10.1 in a concentration dependent manner with an IC₅₀ value of 10.4±0.9 µM (13). Procyanidin B1 directly inhibits channel activity and blocks tumor cell proliferation by hydrogen bonding to i550, t552 and q557 amino acids in the filter. Similar to tetrandrine, 100 µM procyanidin B1 did not markedly inhibit Kv7.1, Kir2.1, or HERG (13).

The pore region of Kv10.1 is composed of S5 and S6 transmembrane helices and intermediate pore rings, which is the core domain of ion selective permeation. In recent years, in the development of targeted drugs for the Kv10.1 channel, the pore domain has become the core target in the design of small molecular inhibitors because of its unique structural characteristics and functional importance. Tetraethylammonium is a commonly used potassium channel blocker, which can physically block ion passage (156). Tetraethylammonium can combine with aromatic amino acid residues on the inner wall of the channel, which hinders the selective filtration function of potassium ions, thus inhibiting the channel current. The mechanism of quinidine and tetraethylammonium is similar and does not depend on the voltage gating state of the channel, so it belongs to non-competitive inhibition (60). The blocking effect of quinidine on Kv10.1 channel is concentration-dependent and the inhibitory effect is more obvious at high concentration. Astemizole competitively binds to Kv10.1 channel with tricyclic antidepressants imipramine and tetraethylammonium (157). The blocking effect of imipramine is voltage-dependent and can be antagonized by intracellular tetraethylammonium. Amiodarone and dronedarone both act through the pore region, but their blocking effects on Kv10.1 are different and dronedarone cannot inhibit the Cole-Molar displacement characteristics of Kv10.1 channel (76). Haloperidol also inhibited the Kv10.1 channel through the pore region and the inhibition was associated with the drug concentration and membrane potential (67). κ-Hefutuxin 1, as the first peptide inhibitor of Kv10.1 channel, inhibits its activity by binding to Kv10.1 channel (158). As the first peptide inhibitor of Kv10.1 channel, κ-hefutuxin 1 inhibits its activity by binding to Kv10.1 channel and may act on or near the pore area of Kv10.1 channel. Channel residues met397 and asp398 may be the anchors to stabilize its binding (158).

Studies have investigated the effects of Haloperidol on the PI3K/AKT signaling pathway in PC12 cells (a neuroblastoma cell line) (159). The research found that Haloperidol can induce the nuclear translocation of PI3K, generating phosphatidylinositol-3,4,5-trisphosphate (PIP3) in the nucleus, effectively inhibiting the phosphorylation of AKT, leading to a decrease in AKT activity. Furthermore, Haloperidol can effectively suppress the expression of Kv10.1 (159,160). Although no studies have yet explored the direct relationship between these factors, it is possible that Haloperidol exerts its anticancer effects by indirectly influencing the PI3K/AKT signaling pathway through the inhibition of Kv10.1 expression (159).

At present, the mechanism of action of Kv10.1 inhibitors is complex and diverse and the binding sites of some inhibitors are not yet clear. Heme is an endogenous regulator of the Kv10.1 channel, which inhibits its activity by binding to the CxHxH motif in the C-Linker region (161). The aforementioned results are listed in Table II.

Inhibitors with clear binding sites are beneficial for the development of new drugs for treating diseases, while inhibitors with unknown binding sites reveal the complexity and diversity of channel regulation. Future research should further strengthen the exploration of the mechanism of action of inhibitors with unknown binding sites and reveal the molecular mysteries of their interaction with Kv10.1 channels. Meanwhile, based on the mechanism of action of inhibitors, more efficient and specific Kv10.1 inhibitors have been developed, providing new strategies and means for the treatment of related diseases.

Kv10.1 is expressed in a variety of malignant tumors, including clear cell renal cell carcinoma, breast cancer, cervical cancer, gastric cancer, colon cancer and cervical intraepithelial neoplasia (14,42,43,162-165). According to clinical data analysis reported in relevant literature, a total of 68 clinical samples were tested in the study of esophageal squamous cell carcinoma. The results showed that 51 cases showed positive expression, with a positive rate of 75%. Further analysis showed that the positive expression was closely associated with the depth of tumor infiltration and the survival time of positive patients was generally shorter (166). In clinical data of ovarian cancer, there were 336 samples, of which 249 showed positive results, with a positivity rate of 65%. The expression of Kv10.1 is associated with tumor size, differentiation degree, staging and metastasis (167). In the clinical data of head and neck squamous cell carcinoma, there were a total of 54 clinical samples, of which 45 samples showed positive results, with a positive rate as high as 83.00%. It is worth noting that Kv10.1 plays a key role in this head and neck squamous cell carcinoma and has been proven to be a tumor marker and potential therapeutic target. The expression of Kv10.1 is associated with important clinical pathological features such as tumor size, differentiation degree, staging and metastasis (40).

A research group has conducted in-depth predictive analysis of the clinical correlation between cancer-related genes and renal cell carcinoma cell lines using the Cancer Genome Atlas database. The research results indicate that Kv10.1 is a key biomarker and potential therapeutic target in clear cell renal cell carcinoma (ccRCC) (162). The widespread abnormal expression of Kv10.1 in numerous tumors suggests that it may be intrinsically associated with the occurrence and development of tumors and has potential value as a tumor marker for tumor screening and diagnosis. However, most of the data on Kv10.1 inhibitors to date have been derived from in vitro cell lines or simple animal models and there is no systematic patient-derived xenografts (PDX) model data to clearly demonstrate the quantitative indicators of tumor inhibition rate and survival extension effect of representative inhibitors. Due to the lack of such data, it is difficult to directly support the conclusion that Kv10.1 inhibitors have significant clinical translational value in PDX models. Kv10.1 is highly expressed in brain tissue and involved in regulating neuronal excitability, neurotransmitter release and synaptic plasticity. When treating brain tumors, Kv10.1 inhibitors need to penetrate the blood-brain barrier (BBB) to target tumor cells. At this time, increased permeability of the BBB (such as through hyperosmotic solutions or nanocarrier technology) may facilitate drug entry into the brain, but it also increases the risk of exposure to normal brain tissue. When treating non-brain tumors, the BBB can prevent Kv10.1 inhibitors from entering the brain tissue, thereby avoiding neurotoxicity. Studies have shown that knocking out Kv10.1 did not result in significant abnormalities in multiple behavioral tests in mice and its function may have a redundant compensation mechanism with other channels. Therefore, Kv10.1 inhibitors have less interference with normal tissue, especially brain tissue function (67). Therefore, at present, it can only be concluded that Kv10.1 is a potential therapeutic target.

The BBB is a selective barrier between the central nervous system and the circulatory system. Its unique structural characteristics, such as tight junction, stability of cytoskeleton and active efflux of transmembrane proteins, together constitute the physical and chemical barrier for drugs to enter the brain tissue. Kv10.1 is highly expressed in brain tumors (such as glioma), but the existence of the blood-brain barrier makes it difficult for most drugs to penetrate effectively, resulting in low treatment efficiency of brain tumors. Even if drugs can break through the blood-brain barrier for a short time, its action time is often not enough to play a lasting effect (168,169).

The molecular weight of peptide inhibitors is relatively small and the structure is relatively simple. It is generally considered to have low immunogenicity potential and it is easy to be degraded and excreted in the body, reducing the contact time with the immune system, thus reducing the immunogenicity. In addition, in the production process of peptide inhibitors, some impurities may be introduced, such as pollutants, solvents, enzymes, impurities formed by amino acid side chain modification, truncation, duplication, oxidation, insertion or deletion in the active pharmaceutical ingredient sequence. These impurities may have potential immunogenicity and can trigger the immune response of the body, thus affecting the safety and effectiveness of drugs.

Kv10.1 inhibitors combined with chemotherapeutic drugs can act on tumor cells simultaneously through different mechanisms to produce synergistic killing effect. This combined strategy is expected to improve the therapeutic effect and prolong the survival time of patients. For example, Kv10.1 can participate in metabolic adaptation to cancer cells by regulating mitochondrial dynamics and inhibit Kv10.1 expression or function, resulting in mitochondrial fragmentation, increased reactive oxygen species and increased autophagy. Kv10.1 endogenous overexpression cells were also more sensitive to mitochondrial metabolic inhibitors than low expression cells, indicating that they were more dependent on mitochondrial function. Therefore, the combined treatment of Kv10.1 functional monoclonal antibody and mitochondrial metabolism inhibitor leads to the enhanced efficacy of the inhibitor (31). Kv10.1 specific scFv was fused with soluble TNF-related apoptosis inducing ligand. The combination of ligands and different chemotherapeutic drugs can overcome drug resistance and selectively induce apoptosis (170).