Kv7 channels are heterotetramers comprised of Kv7.2 and Kv7.3 α-subunits. Kv7 channels generate the M-current, which stabilizes the resting membrane potential and prevents excessive neuronal firing (Shah et al., 2008). Kv7 channels were among the first potassium channels to be identified at AIS and nodes (Devaux et al., 2004; Figures 2A, B). At nodes, Kv7 channels underlie the slow nodal potassium current and stabilize membrane potential. As such, blocking nodal Kv7 channels leads to an increase in axonal excitability (Schwarz et al., 2006). Consistent with the notion that Kv7 channels are responsible for the slow nodal potassium current, patients with a point mutation in the Kv7.2 voltage sensor have myokymia (muscle rippling) and loss of frequency adaptation (Dedek et al., 2001).

Kv7 channel enrichment at the AIS and nodes is mediated by a conserved nine amino acid motif (IAEGES/TDTD) in their C-terminal region, that binds directly to AnkG (Figure 2E). This motif is homologous to the Nav channel AnkG-binding motif (Garrido et al., 2003; Lemaillet et al., 2003; Pan et al., 2006). Despite its high degree of similarity, the motif is thought to have evolved independently and represents a remarkable example of convergent molecular evolution (Hill et al., 2008). This motif is sufficient for its interaction with AnkG, and loss of AnkG disrupts Kv7 channel clustering at the AIS (Pan et al., 2006). As noted above, in the absence of nodal AnkG, Nav channel clustering may be rescued by nodal AnkR. However, loss of nodal AnkG is accompanied by a complete loss of nodal Kv7.2, suggesting that although its ankyrin-binding motif is homologous to Nav channels, AnkR cannot cluster Kv7 channels (Wang et al., 2018).

Kv7 channel interaction with AnkG, and presumably its localization at the AIS and nodes, is regulated by phosphorylation. Protein kinase CK2 phosphorylates the AnkG-binding motif of Kv7.2, enhancing its interaction with AnkG (Xu and Cooper, 2015). This phosphorylation dependent regulation suggests that Kv7.2 localization may be dynamic in response to neuronal activity or extracellular signals. Given that CK2 activity is known to be influenced by synaptic input and intracellular signaling, it is possible that Kv7 channel clustering at the AIS is plastic, allowing neurons to fine tune their excitability under different physiological conditions (Kimura and Matsuki, 2008). The identity of other kinases or the phosphatases that regulate the stability and function of Kv7 channels is unknown, but it is clear that phosphorylation is an additional layer of regulation.

Heterotetrameric Kv1 channels consisting of Kv1.1, Kv1.2, and Kv1.4 α-subunits are enriched at the AIS (Lorincz and Nusser, 2008; Ogawa et al., 2008). While AIS Kv7 channels regulate subthreshold excitability, AIS Kv1 channels modulate high-frequency firing and the shape of the action potential waveform (Kole et al., 2007; Rama et al., 2017). Immunostaining of AIS Kv1 channels showed that they are more highly enriched in the distal regions of the AIS (Van Wart et al., 2007; Lorincz and Nusser, 2008). In addition, Kv1 channels are not found at nodes of Ranvier. Instead, they are excluded from nodal and paranodal domains and are clustered beneath the myelin sheath on each side of the node at the juxtaparanodal domain (Wang et al., 1993). Together, these observations suggest the mechanisms of AIS Kv1 channel clustering are unique and may rely on scaffolding proteins or mechanisms other than AnkG.

Kv1 channel α-subunits have a conserved C-terminal PDZ binding motif (L/ETDV) (Figure 2E) and can be clustered on the cell surface by PDZ domain-containing proteins of the membrane-associated guanylate kinase (MAGUK) family (Kim et al., 1995). Among the MAGUKs, the postsynaptic density 93 (PSD93) scaffolding protein was found to be enriched at the AIS (Ogawa et al., 2008). In vitro knockdown experiments demonstrated that reducing PSD93 levels also reduced AIS Kv1 channel clustering. However, subsequent studies in PSD93 knockout mice showed that Kv1 channels remained clustered at the AIS, indicating that PSD93 is not essential for AIS Kv1 channel clustering and that other scaffolding proteins must also contribute to their localization (Ogawa et al., 2010).

For more than a decade, these contradictory in vitro and in vivo results remained unresolved. Recently, our lab used immunoproximity biotinylation to perform a deep proteomic analysis of the AIS (Zhang et al., 2023). This was followed by CRISPR-mediated genome editing to endogenously tag all PDZ domain-containing proteins found in the AIS proteome. Among the 18 candidates examined, this knock-in strategy revealed two PDZ domain-containing scaffolding proteins enriched at the AIS: PSD93 and SCRIB. SCRIB was previously reported to be a regulator of dendrite development, spine morphology, and synaptic plasticity (Moreau et al., 2010; Szczurkowska et al., 2020). Additional experiments showed that SCRIB co-immunoprecipitates with Kv1 channels and co-expression of SCRIB with Kv1 channels is sufficient to induce their surface clustering with Kv1 channels. In contrast, loss of SCRIB significantly reduces Kv1 channel clustering at the AIS. AnkG is the master organizer of the AIS, and while loss of PSD93 reduces AIS Kv1 channels in vitro, loss of AnkG completely abolishes Kv1 channel clustering. So how is SCRIB connected to AnkG and PSD93? SCRIB can not only bind to and cluster Kv1 channels, but it can also recruit AnkG into those surface clusters and it co-immunoprecipitates with SCRIB. Loss of both SCRIB and PSD93 in vitro abolishes Kv1 channel clustering. Thus, SCRIB functions as a bridge between AnkG and Kv1 channels, and acts synergistically with PSD93 to cluster Kv1 channels (Zhang et al., 2025). Although AIS Kv1 channel clustering depends on AnkG, Kv1 channels do not interact directly with AnkG. Intriguingly, although it binds directly to AnkG, SCRIB is found neither at nodes nor juxtaparanodes (Zhang et al., 2025). The mechanisms that restrict SCRIB to the AIS and exclude them from distal axons are unknown. AIS localization of Kv1 channels may also rely on posttranslational modifications. For example, some evidence supports the idea that the palmitoyl acyltransferase ZDHHC14 palmitoylates both Kv1 channels and PSD93; loss of ZDHHC14 eliminates the AIS clustering of both Kv1 channels and PSD93 (Sanders et al., 2020).

The delayed rectifier Kv2.1 channel, found primarily in somatodendritic domains of cortical and hippocampal neurons (Antonucci et al., 2001) may also be found at the AIS (Sarmiere et al., 2008). However, the physiological functions of AIS Kv2.1 remain poorly understood. In addition, Kv2.1 has not been reported at nodes of Ranvier, and unlike Kv7, Kv1, and K2P channels, it is not uniformly distributed along AIS, but rather it is found in clusters, at endoplasmic reticulum-plasma membrane (ER-PM) junctions that form in “holes” in the AnkG-dependent cytoskeleton. Unlike other Kv channels, AIS clustering of Kv2.1 channels does not occur through an Ankyrin-directed protein complex. Interestingly, the vesicle-associated membrane protein-associated proteins isoforms A and B (VAPA and VAPB) colocalize with and bind to Kv2.1, and loss of VAPA reduces the amount of Kv2.1 at ER-PM junctions. However, Kv2.1 appears to recruit VAPs to ER-PM junctions (Kirmiz et al., 2018). Nevertheless, Jensen et al. (2017) identified a 20 amino acid region (residues 720–745) in the C-terminus of Kv2.1 that is necessary for AIS localization. In addition, within this domain they found two phosphorylation sites (T728 and S732) that when mutated to alanine abolish Kv2.1’s AIS localization. Thus, like Kv7 and K2P channels (see below) the AIS localization of Kv2 channels depends on phosphorylation. Future studies should focus on the putative scaffolding proteins responsible for AIS Kv2.1 clustering at ER-PM junctions.

The Kv3 subfamily of Kv channels functions mainly in neurons that engage in high-frequency firing. Among the Kv3 channels, Kv3.1b and Kv3.3 have been reported at subsets of mammalian nodes of Ranvier in the CNS, but not at the AIS (Devaux et al., 2003). In contrast, Kuba et al. (2015) reported Kv3.1-containing channels at the AIS in chick brainstem auditory nuclei. Unlike Kv7.2, Kv7.3 and K2P channels, which regulate resting membrane potential and subthreshold excitability, Kv3.1b and Kv3.3 are activated at more depolarized voltages, possibly contributing to the rapid repolarization of nodes (Hernandez-Pineda et al., 1999; Rudy and McBain, 2001). As described above, Nav, Kv7, and K2P channels are clustered at nodes by AnkG. However, Kv3.1b and Kv3.3 are not clustered by AnkG but instead rely on AnkR (Stevens et al., 2021; Stevens et al., 2022). The binding between AnkR, Kv3.1b, and Kv3.3 occurs through a conserved 6-amino acid AnkR-binding motif (EDCPHI) located in the C-terminus of the channel subunits (Figure 2E). Thus, nodes that have AnkG, but no AnkR, have clustered Kv7 and K2P channels. In contrast, nodes that have AnkR, but no AnkG, have only Kv3 channels (Stevens et al., 2021), and nodes with both AnkR and AnkG have Kv7, Kv3, and K2P channels. This remarkable diversity, complexity, and clustering of nodal potassium channels is dictated by the types of ankyrin scaffolding proteins found at mammalian nodes of Ranvier. The striking observation of Kv3.1 channels at the AIS of neurons in avian brainstem auditory nuclei suggests that avians may have evolved distinct Kv3.1 channel clustering mechanisms at the AIS (Kuba et al., 2015). In mammals, we never observed AIS AnkR (Liu et al., 2020a), which may explain the absence of AIS Kv3 channels.

TWIK-related arachadonic acid activated K⁺ channel (TRAAK) and TREK-1 are closely related members of the K2P channel family that display both thermal and mechanical sensitivity (Maingret et al., 1999a; Maingret et al., 1999b). K2P channels are heterodimers, expressed at low levels in neurons, and underlie leak potassium currents (Talley et al., 2001). Unlike Kv channels that are gated by membrane potential changes, K2P channels provide voltage-independent background leak potassium currents, influencing neuronal excitability by stabilizing the resting membrane potential (Enyedi and Czirjak, 2010). Although a nodal leak potassium current was proposed decades ago (Chiu et al., 1979), only recently were TRAAK and TREK-1 identified as the molecular basis of the leak current at nodes of Ranvier (Brohawn et al., 2019; Kanda et al., 2019). The shift from Kv channel mediated repolarization in invertebrates and unmyelinated axons to K2P-mediated repolarization in mammals represents a critical evolutionary adaptation that optimizes conduction velocity (Tonomura and Gu, 2022).

Immunostaining experiments indicated that K2P channels were not found at the AIS, suggesting that they may be clustered through an AnkG-independent mechanism. However, subsequent studies using glyoxal as a fixative (Konno et al., 2023), rather than paraformaldehyde, revealed AIS TRAAK and TREK-1 (Escobedo et al., 2024). Thus, like Kv7 channels, K2P channels are found at both AIS and nodes of Ranvier (Figures 2C, D). Although the function of AIS K2P channels has not been investigated, it is reasonable to assume they are primary drivers of action potential repolarization as they are at nodes of Ranvier.

The AIS and nodal clustering of TRAAK and TREK-1 depends on AnkG. A structure-function analysis revealed that TRAAK has a highly conserved AnkG-binding motif in its C-terminal region (YPSENLAFIDESSDT), that is both necessary and sufficient for its clustering at the AIS (Escobedo et al., 2024; Luque-Fernandez et al., 2024). This sequence has little similarity to that of Nav and Kv7 channels (Figure 2E), suggesting that TRAAK’s AnkG-binding motif evolved independently. In support of the notion that AIS ion channel densities are plastic and can be dynamically modulated, TRAAK’s affinity for AnkG depends on phosphorylation, just like Kv7 and Nav channels. In contrast, TREK-1 does not have an AnkG-binding motif and does not independently localize to the AIS. Instead, TREK-1 clustering requires its co-assembly with TRAAK (Escobedo et al., 2024). Thus, heterodimerization between TRAAK and TREK-1 is required for TREK-1 localization at excitable domains such as the AIS. AnkG conditional knockout mice have normal Nav channel clustering due to compensation by AnkR. However, these AnkG-deficient nodes of Ranvier lack both Kv7 and K2P channels, indicating that the complement of nodal potassium channels has a high degree of specificity for their binding to ankyrin scaffolding proteins (Wang et al., 2018; Escobedo et al., 2024).