Rheumatoid arthritis (RA) is a systemic autoimmune disorder, clinically presenting as symmetric polyarthritis mainly affecting small-to-medium-sized joints (Figus et al., 2021). A hallmark pathological feature is the formation of invasive pannus from synovial lining layer hyperplasia. This pannus promotes cartilage degradation and subchondral bone erosion via the continuous secretion of matrix metalloproteinases (MMPs) and pro-inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6), culminating in irreversible joint deformity and functional decline (Huang et al., 2021). Synovial macrophages are crucial effector cells in RA pathogenesis, polarized into pro-inflammatory M1 and anti-inflammatory M2 phenotypes. The dysregulated immune microenvironment in RA drives metabolic reprogramming, disrupting the M1/M2 balance and skewing it towards a pro-inflammatory state, thereby exacerbating inflammation and tissue destruction (Chou et al., 2008; Cutolo et al., 2022).

The intermediate-conductance calcium-activated potassium channel (KCa3.1, encoded by KCNN4 gene) is activated by intracellular Ca2+ (>100 nM) via calmodulin binding, triggering K+ efflux, membrane hyperpolarization, and subsequent store-operated Ca2+ entry (Chou et al., 2008; Ghanshani et al., 1998; Wang and Xiang, 2013). In general, abnormal expression or dysregulation of ion channels is associated with synovial hyperplasia, cartilage destruction, endochondral ossification, inflammatory response and pain (Kelly et al., 2015; Lin et al., 2024; Nakamoto et al., 2021; Obeidat et al., 2023; Savadipour et al., 2023; Zhou et al., 2024). Clinical studies have shown that the KCa3.1 channel is abnormally activated and upregulated in patients with RA compared with healthy individuals (Toldi et al., 2013). Additionally, Ca2+-activated K+ currents characteristic of the KCa3.1 channel have been detected in synovial fibroblasts isolated from RA patients (Friebel et al., 2015). Mechanistically, KCa3.1 regulates multiple macrophage functions associated with RA, including proliferation, migration, reactive oxygen species (ROS) production and cytokine production (Hanley et al., 2004; Toyama et al., 2008). KCNN4-mediated macrophage fusion represents an essential step in inflammatory osteoclastogenesis (Kang et al., 2014). It has been reported that KCa3.1 maintains Ca2+ influx and membrane hyperpolarization in macrophages (Zhu et al., 2019). Moreover, most studies have demonstrated that the role of KCa3.1 in macrophages is closely associated with the NF-κB and STAT signaling pathways (Xu et al., 2014, 2017; Zheng et al., 2021). Therefore, it is necessary to clarify the role of KCa3.1 and its underlying molecular mechanisms in M1 macrophage polarization during RA.

Inflammasome activation represents a central inflammatory pathway, characterized by the oligomerization of sensor proteins to form a pro-Caspase-1 activation platform in response to damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs) (Franchi et al., 2009). Among all inflammasomes, the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is the most extensively studied in RA research. Clinical studies have demonstrated elevated NLRP3 protein expression in peripheral blood, monocytes, macrophages and dendritic cells from RA patients, implicating NLRP3 activation in both systemic and local inflammatory responses in RA (Choulaki et al., 2015; Murakami et al., 2022; Paramel et al., 2015). The key activating signals of NLRP3 include K+ efflux, mitochondrial dysfunction, ROS generation, and Ca2+ signal dysregulation (Lee et al., 2012; Murakami et al., 2012; Nakahira et al., 2011; Petrilli et al., 2007; Zhou et al., 2011). Notably, two of these critical signals—K+ efflux and Ca2+ signal dysregulation—are processes directly regulated by the KCa3.1 channel, establishing a clear mechanistic link to its function in RA. However, whether KCa3.1 is involved in M1 macrophage polarization by affecting NLRP3 activation remains to be explored.

Transient receptor potential (TRP) channels are a superfamily of ion channels and characterized by a unique domain structure containing a cation channel and a protein kinase (Li et al., 2007). Activation of transient receptor potential melastatin-subfamily member 7 (TRPM7) induces a rapid increase in intracellular Ca2+ levels, which is crucial for cell proliferation, migration, invasion, inflammation, and other pathophysiological processes (Lin et al., 2024; Schappe et al., 2018; Schilling et al., 2014). Given the potential role of TRPM7-mediated Ca2+ influx in KCa3.1- and NLRP3-dependent inflammation, we aimed to investigate whether TRPM7 affects the regulatory effect of KCa3.1 on NLRP3 inflammasome activation. This study aimed to investigate the role of pharmacological inhibition or genetic knockout of KCa3.1 in M1 macrophage polarization during RA progression, which is characterized by the description of KCa3.1 participating in M1 synovial macrophage polarization by influencing NLRP3 activation. Furthermore, we explored whether TRPM7 affects the regulatory effect of KCa3.1 on NLRP3 inflammasome activation via pharmacological activation of TRPM7. Additionally, in RA mouse models, we employed both pharmacological inhibition of KCa3.1 or genetic knockout of KCNN4 to investigate its impact on M1 macrophage polarization, the NLRP3 inflammasome, synovial inflammation, hyperplasia, and cartilage damage. Our findings identify novel therapeutic targets for RA prevention and treatment, and provide a mechanistic basis for the clinical application of the KCa3.1 inhibitor TRAM-34 in RA management.