Silent Kv subunits as major players in cell membrane excitability

Introduction

Every thought, movement, and sensation begins with a simple physiological decision: whether a neuron will fire an action potential or not. That decision depends on the neuron’s membrane potential, the coordinated activity of its ion channels, and how the electrical signals propagate and influence their target tissues. For decades, efforts to understand neuronal excitability have focused on the ion channels themselves, the pore-forming proteins that mediate these ionic currents and allow that decision to be made.

Action potentials in excitable cells

Ion channels are the biological machinery responsible for the electrical excitability of cell membranes. They are membrane protein complexes that transduce cues from the environment into a variable that cells can handle, i.e., the membrane potential. Ion channel gating behavior is central to membrane potential in general, and most importantly to action potential. Ion channels are determinants of action potential waveform and firing frequency, and consequently they also determine the transmission of synaptic signals (Hille, 2001). They are conventionally classified by the stimulus that primarily controls their open probability and by the specific ions they selectively conduct. Ligand-gated channels open upon the binding of a chemical messenger, voltage-gated channels respond to changes in membrane potential, and mechanically gated channels respond to physical forces acting on the membrane, such as stretch or pressure (Anon, 2013; Zheng and Trudeau, 2015). Gating, the process by which channels interconvert between closed and open states, is a complex relationship involving multiple coordinated molecular mechanisms, and it is what we are trying to understand in our everyday work in our research labs. As science evolves by the day, new mechanisms are unveiled that show the marvelous versatility of these protein complexes that are the ion channels. Here in this perspective paper, we discuss an unprecedented, genetically controlled gating mechanism operating on voltage-gated potassium (Kv) channels, one that effectively creates biophysically distinct ion channels by heterotetramerization of their constituent subunits, with potentially important effects on Kv2-mediated currents and, consequently, on cellular excitability. We believe heteromeric Kv channels represent a new frontier for drug development, since some of their constituent protein subunits remain unexplored.

Voltage-gated ion channels are key elements to produce action potentials in excitable cell membranes (Hodgkin and Huxley, 1952). Starting from a resting, negative membrane potential, a depolarizing stimulus such as a synaptic input, receptor potential, or generator potential raises the open probability of voltage-gated sodium (Nav) channels. Because of their voltage dependence, these channels are the first to open during a depolarizing event. The influx of Na⁺ ions they mediate further depolarizes the membrane, which in turn recruits and activates more Nav channels. If the overall depolarizing currents surpass the hyperpolarizing currents that generate the resting potential, an action potential is fired. The process becomes auto regenerative, and a strong depolarization with overshoot (a reversal of the membrane potential to positive values) takes place. Inactivation processes develop next, as an intramolecular mechanism by which Nav channels render themselves non-conductive even while the membrane is still strongly depolarized, acting as a self-restraining brake against hyperactivity (Bezanilla and Armstrong, 1977). The depolarization produced by the Na⁺-mediated upstroke also increases the open probability of Kv channels, whose activation mediates an efflux of K⁺ that contributes to repolarizing the membrane back to resting levels. Repolarization and hyperpolarization currents conducted by voltage-gated potassium channels then reset these channels back into their closed states, ready to open again (Bean, 2007). This minimally described sequence of Na⁺ influx followed by a slightly delayed K⁺ efflux represents the simplest action potential. Action potentials serve to trigger action potentials in adjacent membranes of the same cell, in a process called regenerative propagation, that takes place in neuronal and muscle cells (Aidley, 1998). An action potential can also fire in the membrane of gland cells, such as those in the endocrine pancreas or the medulla of the adrenal gland. In these cells, action potentials do not propagate since the whole membrane fires instantly. In all cases, an action potential ultimately serves to depolarize the membrane potential to values that are enough to open voltage-gated Ca²⁺ channels. The Ca²⁺ influx mediated by these channels then triggers neurotransmitter or hormone release, or modifies the function of intracellular proteins that may act as second messengers, metabolic modifiers, or even downstream regulators of gene expression (Huang et al., 2021).

Kv channels

Kv channels are nearly symmetric radial assemblies of alpha Kv subunits in the plane of the membrane. About 40 different Kv subunits have been identified in mammals, each containing six transmembrane segments (S1–S6) (Long et al., 2005; Gutman et al., 2005). At the central axis of the assembly is the conduction pore domain (PD), formed by the S5 and S6 segments of all four Kv subunits, with each subunit contributing one-fourth of the pore. Voltage sensor domains (VSDs), one per subunit, sit peripherally and are formed by segments S1 through S4; these are the regions specialized in sensing the membrane potential and transducing it into ion conductance. The position of the VSDs is associated with the open probability of the channel: macroscopically, the more depolarized the membrane, the more the VSDs are driven outward by the changed electric field, the more time the channels spend open, and the larger the overall current. The S6 segments are widely considered the activation gates, and all four, each controlled by its own VSD, must be in the active position for the channel to conduct K⁺ ions (del Camino and Yellen, 2001). These gates are the subject of intense research, since they are controlled by multiple regions of the channel, even regions that do not belong to their own subunit. During persistent stimulation, Kv channels also undergo different types of inactivation that, as in Nav channels, shut off the conduction pathway. Inactivation in Kv channels is a more diverse and complex matter than in Nav channels. Inactivation in Kv channels is a crucial determinant of cell excitability, as it shapes the action potential and dictates the refractory periods for firing. Beta subunits may also be present in the assembly, but they are not necessary for ion conduction; their role is modulatory.

Without a doubt, all functional aspects of ion channels are determined by structural aspects of their macromolecular assemblies. Kv channel assemblies are commonly homomeric tetramers, i.e., formed by four copies of the same Kv subunit. The nearly 40 Kv subunits are structurally diverse and give rise to functionally distinct channels with characteristic voltage dependence, kinetics, and inactivation patterns. These three aspects define ion channels biophysically. By amino acid sequence identity, Kv subunits are organized into 12 families, Kv1–Kv12. Families Kv1, Kv2, Kv3, Kv4, Kv7, Kv10, Kv11, and Kv12 form mostly homomeric structures, with some intra-family heterotetramerization between highly similar subunits, and each channel so formed has a distinct functional signature and tissue expression (Figure 1). But what about the subunits in families Kv5, Kv6, Kv8, and Kv9? Within these four families there are 10 Kv subunits that remain structurally and functionally not fully understood. What is known is that they co-assemble with Kv2 subunits to form new, distinct channels (Bocksteins, 2016).

Silent Kv subunits and heteromeric Kv channels

Kv subunits from families Kv5, Kv6, Kv8, and Kv9 are deemed “silent”. Structurally, they are analogous to other Kv subunits, with six transmembrane segments, the voltage-sensing S4 segment, and a cytoplasmic NH₂ terminus comprising a T1 domain (Bocksteins and Snyders, 2012) which are all conserved regions of Kv subunits (Gutman et al., 2005). The denomination “silent” (KvS) stems mainly from the belief that they do not form functional channels on their own: they fail to assemble into conducting homotetramers, so a putative homomeric KvS structure would be electrically silent. The field still does not know for a fact whether homomeric KvS structures reach the cell membrane as is or whether their folding is disrupted before that; the prevailing view is that KvS subunits are retained in the endoplasmic reticulum and kept from trafficking to the plasma membrane unless they co-assemble with Kv2 (Bocksteins and Snyders, 2012). Such kind of impairment for cell membrane expression could be explained if such homotetramers require specific post-translational modifications or cellular cofactors for stability that in vitro models have yet to replicate. So, then, what would be the function of these subunits in excitable cells? Factually, genetic data show KvS-encoding genes in association with disease, suggesting a role in homeostasis, as we detail in the next section.

KvS can form cross-family heterotetramers with other subunits. Their assembly with members of the Kv2 family has been detected by many independent groups. In a breakthrough, a recent study has reported cross-family association between KvS and Kv7 (Renigunta et al., 2024). Cross-family associations in a common channel has been proposed to be mediated by T1 domains, in the N-terminal part of the proteins, from different subunits. This proposition was based on an expected uncommon tolerance in the association between T1 domains from different Kv subunits. In isolation, the T1 domains from Kv2 subunits forms pentameric structures denoting flexibility. This aspect led the authors to suggest the heterotetramerization of Kv2 subunits with KvS subunits could be thermodynamically allowed (Chiara et al., 1999; Dementieva et al., 2009; Bocksteins et al., 2011; Xu et al., 2022). From controlled studies using co-expression of Kv2.1 subunits and various KvS exclusively, we have learned that the heteromeric channel is substantially different from homomeric channels formed by Kv2 or Kv7 alone. KvS in association with Kv2 (Kv2.1–KvS channels) is the heteromeric structure most scrutinized to date, and the functional differences reported are all over the place in the voltage- and time-dependence of both the activation and inactivation gating processes.

Homomeric channels formed by Kv2 subunits alone (homomeric Kv2 channels) are classically known as delayed rectifiers, because their voltage dependence is shifted toward more depolarized potentials and they activate and inactivate slowly. These properties make Kv2 channels a regulator of neuronal excitability, but only after an action potential has fired; they do not set the threshold for the first one. Their contribution comes later. For a neuron to fire repeatedly, it must produce one action potential after another while resetting the channels that have inactivated, and much of that work falls to delayed rectifier potassium currents. Kv2 channels act as a brake against excessive excitation while still allowing the neuron to sustain firing (Long et al., 2005). These homomeric channels are thought to be key determinants of a neuron’s firing frequency, and by extension of synaptic strength where that neuron connects to another cell. Because they activate comparatively slowly, Kv2 channels matter most during this repetitive activity: they restore the repolarizing current between successive spikes and let voltage-gated channels recover from inactivation, so the neuron can keep firing at high frequencies (Liu and Bean, 2014). When Kv2 function is impaired, that recovery is less effective, since the membrane does not fully repolarize in time, and firing is disrupted. Conversely, enhanced Kv2 activity produces exaggerated hyperpolarization that suppresses subsequent spike generation, disrupting firing as well. In both cases, the membrane potential is driven away from the optimal range required for continued spike generation, resulting in impaired sustained firing (Blaine and Ribera, 2001).

Kv2 channels are constitutively expressed in cell types from various excitable tissues such as nervous, muscular and glandular. In these tissues, Kv2 channels mediate the repolarizing phase of the action potential. In this sense Kv2 channels are not tissue-specific (Johnson et al., 2019). It appears that the genes encoding Kv2 subunits in humans, KCNB1 and KCNB2, are both under a less restrictive transcriptional control in these tissues as noted by (Stas et al., 2015). This breadth suggests Kv2 channels are not under especially tight genetic control. Instead, they are heavily shaped by post-translational modifications such as SUMOylation, phosphorylation, and glycosylation, which diversify the channel’s function and provide fine adjustments to cell excitability (Mohapatra et al., 2007; Plant et al., 2011).

Phosphorylation has been studied as an effective post-translation functional modification of Kv channels. Under conditions of elevated neuronal firing, Kv2.1 channels undergo rapid calcineurin-dependent dephosphorylation, producing a hyperpolarizing shift in voltage-dependent activation and dispersal of somatodendritic clusters (Misonou et al., 2005; Aras et al., 2009). Collectively these changes limit firing frequency and increase the refractory period, both of which are homeostatic effects that suppress hyperexcitability and may reduce susceptibility to seizure activity. Current research supports that the calcineurin dependent hyperpolarizing shift in activation arises from the cumulative dephosphorylation of several C-terminal serine residues and that the magnitude of this shift scales as more sites are dephosphorylated (Park et al., 2006). Given that phosphorylation occurs at the C-terminal domain of Kv2.1 subunits and these sequences are highly divergent even between closely related channel families such as Kv2.1 and Kv2.2, it is plausible that heteromeric Kv2/KvS channels cannot mount the same coordinated phosphorylation response as homomeric Kv2.1 channels. Because the magnitude of the gating shift depends on the total number of de-phosphorylatable C terminal sites contributed across all four subunits, the incorporation of even a single KvS, which almost certainly lacks an equivalent C terminal site, would reduce that pool and blunt the gating shift relative to a Kv2.1 homotetramer. In this way, KvS expression and phosphorylation state may function as orthogonal rather than redundant mechanisms of excitability control, with phosphorylation dynamically tuning the homotetramer pool while KvS expression sets a baseline gating shift insensitive to that same signal.

KvS subunits may also be expressed constitutively though variably across many cell types in excitable tissues, where they are assumed to reside in the membrane alongside Kv2 subunits forming heteromeric Kv channels (Figure 2). Their expression and stability in fact appear to depend on Kv2.1 and Kv2.2, which may be a further way KvS shape excitability: simply by tracking Kv2 levels. Kv5.1 protein, for instance, was reduced by 70% in Kv2.1 knockout mouse brains and by 95% in Kv2.1/Kv2.2 double-knockout brains, indicating that KvS stability in the brain depends on successful assembly with Kv2 (Ferns et al., 2025). Whether this dependence acts before membrane expression or after it, for example through faster recycling of the channel from the membrane or lowering its density where it works, for instance in the axon hillock, remains unclear. The genes encoding KvS subunits are thought to be under more restrictive transcriptional control than those for Kv2. KvS expressions in different tissues are more specific than that of Kv2.1. KvS subunit expressions are also highly regulated by physiological conditions and pathologies as pointed out by independent studies (Patel et al., 1997; Lee et al., 2015; Lacey et al., 2025). Little to nothing is known about KvS subunits post-translational modifications. Together with the genetic data in humans, we propose that KvS subunits are a genetically controlled reserve for expanding the dynamic range of variables (voltage-dependence and kinetics) associated with Kv2 channels and reflected in membrane excitability when physiological or pathological conditions demand it. This also makes KvS selectively expressed according to the homeostatic status of the body, producing patterns of cell excitability appropriate to circumstantial body states.

KvS subunits seem to be better expressed natively than in the membrane of heterologous systems like HEK cells. This aspect has been suggested by many as immunofluorescent labeling using highly specific nanobodies against KvS subunits suggest a higher level of expression than those recorded in HEK cells using electrophysiology (Flood and Carvalho-de-Souza, 2023; Lacey et al., 2025). It appears that there are required components in native cells, like neurons and other electrically excitable cells, that are not present, or not active, in cell lines. Nevertheless, KvS subunits seem to be well expressed in the membrane of Xenopus oocytes, a feature that might stem from the more permissive embryonic status of these haploid cells.

One further aspect of heteromeric Kv channels concerns the stoichiometry of the assemblies. Only a few studies have been devoted to counting different types of subunits in heteromeric Kv2-KvS channels. A larger group of studies using electrophysiology, statistics, immunofluorescence and computational work bet in a dominant 3:1 spontaneous stoichiometry: heteromeric channels would have three Kv2 subunits and only one KvS subunit (Kerschensteiner et al., 2005; Bocksteins et al., 2017; Pisupati et al., 2018; 2020). Another study, also using electrophysiology, immunofluorescence, and statistics, sides with a 2:2 spontaneous stoichiometry as the dominant form of assembly (Möller et al., 2020).

The stoichiometry of heteromeric Kv channels assemblies affects the channel’s biophysical properties, and therefore the channel functional aspects in many ways. One is by the quality and the number of Kv subunits that form the channels. Different Kv subunits, e.g., Kv2.1 and for instance Kv5.1, are expected to move differently, because they are different proteins when the membrane potential changes. Moving differently is another way to say the channel will have different voltage- and time-dependence of the activation, inactivation, and the deactivation processes. Another way that the stoichiometry may be crucial for the function of heteromeric Kv channels is related to the interfaces between the different Kv subunits in the assembly. We have studied these inter-subunit interfaces in Kv channels in the past, and we demonstrated that they are active in fine-tuning the gating processes in Kv channels (Flood and Carvalho-de-Souza, 2023). For example, a 3:1 stoichiometry will have three types of interfaces, while a 2:2 stoichiometry will only have two types of interfaces. Because the interfaces are formed by amino acids from different Kv subunits, each interface configuration is expected to work differently. Yet another way in which stoichiometry can influence heteromeric Kv channel function is by determining how genetic variants are represented within the tetrameric assembly. Homomeric Kv channels have non-conservative variants of their respective gene codons represented four times in the final channel tetrameric assembly in case of homozygosis. In heteromeric Kv channels, those variants can be represented according to the stoichiometry of the assembly. Finally, the stoichiometry of heteromeric Kv channels can affect the function by the number of interfaces that are directly or indirectly affected by the different amino acid residues stemming from the genetic variant. In all cases, different stoichiometries will bring about a different overall structure; that in turn will relate to unique channel biophysical properties.

Despite these efforts to reveal the stoichiometry of the heteromeric Kv channel assemblies, a question remains unanswered: what are the physiologically relevant stoichiometries? All these methods suffer from relative problems such as, heterologous expression that may change the efficiency of expression of the silent Kv subunits, protein super-expression that may change the forces that drive the spontaneous stoichiometry of the heteromeric assemblies, light diffraction limit that prevents unbiased direct detection of single Kv subunits in a channel assembly using fluorescence microscopy, and others. We propose that both may be physiologically relevant, with one or the other becoming more prominent as a function of the given KvS gene’s transcription intensity. Settling this is key both for studies aiming to discover the gating mechanisms that govern heteromeric Kv channel function and for developing new, currently nonexistent modulators that could become tissue-specific therapeutics for conditions associated with cell excitability.

Ion channels can be studied by directly observing the ionic currents mediated by them in a membrane under voltage clamp. The currents are fed to a model that assumes the channels exist in mainly three different conformations, closed, open and inactivated, that undergo interconversions governed by the membrane potential and time. Closed and inactivated states are not conductive conformations. The open state is conductive. Simply speaking, the interconversion between closed and open states is reversible and controlled by the membrane potential. The process from closed to open is called activation, and the reverse direction process is called deactivation. These steady states are modeled by the Boltzmann formalism, from which the voltage-dependence V50 (in mV) and voltage sensitivity (k, in e-fold/mV) are derived. The V50 denotes the membrane potential that is associated with equal distribution between closed and open states. The V50 corresponds to the activation of 50% of the maximal conductance of the system, while k tells how sensitive are changes in conductance per unit of the membrane potential. The transition between the open and the inactivated state is called inactivation, and it is mostly controlled by time after channels reach the open conformation due to membrane depolarization. Therefore, membrane depolarizations will determine the level of inactivation. This relationship is also described by Boltzmann formalism. Likewise, a V50 is associated with the membrane potential that inactivates 50% of the maximal conductance, after the channels open, and k is the voltage sensitivity of the process. Activation, deactivation, and inactivation are all probabilistic processes that are described by exponentials.

Ion channels analysis and current knowledge about heteromeric Kv2.1/KvS channels

Ion selectivity, and the time- and the voltage-dependence of the activation and the inactivation processes describe voltage-gated ion channels’ function. The time course of ionic currents themselves is a direct representation of the channel’s kinetics, and the activation and inactivation curves can be built to represent the steady states of open and inactivated channels at various membrane potentials. Heteromeric Kv channels are highly selective to potassium just like their homomeric counterparts. However, what sets heteromeric Kv channels apart from homomeric assemblies is the time- and the voltage-dependence of their activation and inactivation processes.

Figure 3 shows typical activation and inactivation curves from homomeric Kv2.1 channels and many heteromeric Kv2.1/KvS channels. The plots are simulations using Boltzmann equations (N o r m G = 1 / 1 + exp V 50 − V m / k, where NormG is the normalized conductance, V50 is the voltage dependence parameter, and k is the voltage sensitivity of the process) with typical values for V50 and k for both the activation and the inactivation processes from each channel. The parameters V50 and k are listed in Table 1. As the data shows, heteromeric Kv2.1/KvS channels represent a great diversification of the biophysical properties of the channels that mediate the delayed rectifying currents in any excitable cell. This diversity is expected to contribute to differences in cellular excitability in the tissues where these channels are expressed. Note that these channels, homomeric and heteromeric, might coexist in the same cell membrane at a given time, driven by different cues from the environment that are related to diseases or simply to homeostatic adjustments.

TABLE 1. Typical values of V50 and k for the activation and the inactivation of homomeric Kv2.1 channels and various heteromeric Kv2.1/KvS channels.

References cited in this table:

Kramer et al., 1998.

Bocksteins and Snyders, 2012.

Salinas et al., 1997a.

Mederos et al., 2009.

Zhu et al., 1999.

Ottschytsch et al., 2002.

Ottschytsch et al., 2005.

Sano et al., 2002.

Bocksteins et al., 2017.

Lee et al., 2020.

Flood and Carvalho-de-Souza, 2023.

Castellano et al., 1997.

Salinas et al., 1997b.

Czirják et al., 2007.

Smith et al., 2012.

Richardson and Kaczmarek, 2000.

Shepard and Rae, 1999.

Zhong et al., 2010.

Kerschensteiner and Stocker, 1999.

Patel et al., 1997.

The molecular basis of the mechanisms of gating that operate in heteromeric channels that are mediated by the presence of silent KvS subunits are currently unknown. Efforts to unveil such mechanisms are currently being labored by many groups, including our own. In general, in heteromeric channels, the KvS VSDs would have only a slight effect on activation, since all four VSDs, from KvS subunits or not, must move outward to their active position for the channel to activate its conductance (Bocksteins and Snyders, 2012). Inactivation, however, is more affected by the presence of a destabilized VSD like that of KvS since there is a well-known pathway for channel inactivation from those closed states. This type of inactivation may not require all four VSDs to be in active position. Table 2 shows what is currently known about how each type of KvS subunit alters the time- and the voltage-dependence of both the activation and the inactivation processes in the heteromeric Kv channels they take part.

TABLE 2. Current knowledge about the time- and voltage- dependence on heteromeric Kv2.1/KvS channels

→, right shift; ←, left shift, —: no change; i.d., insufficient data.

A right shift and a left shift in voltage dependence means that, in the heteromeric Kv2.1/KvS channels containing the indicated KvS (leftmost column), the process takes place at more depolarized, or hyperpolarized membrane potentials, respectively, than the same process in homomeric Kv2.1 channels.

References cited in this table:

Kramer et al., 1998.

Bocksteins and Snyders, 2012.

Salinas et al., 1997a.

Mederos et al., 2009.

Zhu et al., 1999.

Ottschytsch et al., 2002.

Ottschytsch et al., 2005.

Sano et al., 2002.

Bocksteins et al., 2017.

Lee et al., 2020.

Flood and Carvalho-de-Souza, 2023.

Castellano et al., 1997.

Salinas et al., 1997b.

Czirják et al., 2007.

Smith et al., 2012.

Richardson and Kaczmarek, 2000.

Shepard and Rae, 1999.

Zhong et al., 2010.

Kerschensteiner and Stocker, 1999.

Patel et al., 1997.

High quality data available from previous studies on heteromeric Kv2.1/Kv6.4 channels sheds light on the inactivation curve shift in heteromeric Kv2.1/Kv6.4 channels. The study suggests that the VSD from Kv6.4 subunits moves at substantially more hyperpolarized, negative membrane potentials, than the VSDs from Kv2.1 subunits of the same heteromeric channel. The difference between the putative V50 for the Kv6.4 subunits in the channel, and the ones attributable to Kv2.1 subunits in the channel was enough to distinguish two independent processes. From there the authors suggested the 3:1 stoichiometry of the assembly (Kv2.1:Kv6.4). This Kv6.4 VSD movement at a more hyperpolarized membrane potential is interpreted as the cause of the displaced in the inactivation curve (V50 for inactivation from Boltzmann analysis) and the sped-up inactivation process verified with these channels (Bocksteins et al., 2017; Flood and Carvalho-de-Souza, 2023). Similar experiments involving the recording of gating currents and of voltage-dependent fluorescent as single subunit readout in the same channel are currently due.

The overlapping area between the inactivation and activation curves as seen in Figure 3 is generically called window current. This measurement is roughly proportional to the contribution of a given type of channel to cell excitability in general. Because KvS subunits preferentially shift the inactivation V50 in the hyperpolarizing direction while leaving the activation curve little changed or unchanged, the window current is decreased when KvS is expressed, and it is part of Kv channels together with Kv2. Subunits. Physiologically, a smaller window current, the case for most heterotetramers, means fewer channels are available to provide repolarizing current during and immediately after depolarization, slowing the membrane’s return to rest. It is noteworthy to recall that voltage activated potassium currents are not monotonically directly proportional to lack of membrane excitability.

Besides regulating the voltage dependence of Kv channels, another important way KvS subunits influence the function in heteromeric Kv2/KvS channels is by changing the channel’s gating kinetics. Kinetics matters because it determines how much theoretical availability is realized within the brief periods of membrane potential changes during an action potential. In heteromeric Kv2/KvS channels the kinetics of the activation, deactivation, and the inactivation processes are distinct from those of homomeric Kv2 channels. To illustrate, we simulated voltage activated potassium currents with realistic values for the inactivation time constants (Figure 4). We used the Hodgkin and Huxley description of potassium conductance development (activation and inactivation) upon membrane depolarization, after adding an inactivation particle to the model to make it a n 4 h model, where n are activation particles and h is an inactivation particles (Hodgkin and Huxley, 1952). The equation used to simulate the data was I K = 1 − exp − t / t a u n × exp − t / t a u h . The deactivation process was described with a single exponential as I K = − exp − t / t a u d e a c t. On both equations, t is the time and the various t a u x are time constants of different processes. KvS subunits effectively and diversely change channels’ activation, inactivation, and deactivation kinetics as shown in Table 2.

Yet another example of functional diversity added by KvS comes from a study about Kv9.3. The incorporation of Kv9.3 with Kv2.1 to form heteromeric channels produces a conductance that possesses substantially altered state-dependent inactivation compared to that of homomeric Kv2.1 channels. Whereas homomeric Kv2.1 channels preferentially, but incompletely inactivate from the open state, kv2.1/Kv9.3 heterotetramers inactivate rapidly and completely from intermediate closed states but slowly and incompletely from open states (Kerschensteiner and Stocker, 1999). This difference is functionally important because in Kv2.1 homotetramers, inactivation is tied to each individual spike depolarization, and in the Kv2.1/Kv9.3 heterotetramers, inactivation will depend on the entire interspike period. Therefore, with increased firing rate, the window for recovery shrinks, making the neuron progressively harder to fire as firing rate increases. In essence Kv9.3 incorporation creates a use-dependent brake on excitability that does not occur in Kv2.1 homotetramers alone.

Expression of KvS subunits finely controls cell excitability

If the firing pattern of a neuron depends on which KvS subunit it expresses, the question is what determines that choice? The answer is the control of gene expression. KvS subunit expressions modulate cell excitability. KvS subunits, under a gene expression control, modify neuron, muscular and gland cell excitability to comply with homeostasis and during pathologies. In Figure 5 we propose a generalist action potential firing pattern modified by KvS subunits expressed in each cell. KvS expression is thought to interfere with the formation of homomeric Kv2 channels in favor of heteromeric Kv2/KvS channels that in turn modify the voltage dependence and the kinetics of the voltage-activated potassium currents that directly determine the action potential firing pattern of the cell. This perspective also reframes the study of these KvS subunits long dismissed as “silent”. Since natural variants of the genes encoding KvS subunits are associated with diseases, i.e., genetic variants are associated with diseases, we conclude these subunits have important biological functions in the body. We exemplify this aspect next with some examples.

In the brain, a common variant near and associated with KCNG1 gene was recently identified as non-coding and protective locus for Alzheimer’s disease, highlighting the potential relevance between excitability disorders and neurodegeneration (Willett et al., 2024). In the heart, a nucleotide deletion in KCNG2 gene that encodes Kv6.2 subunit deletion has been reported in multiple members of a family with sick sinus syndrome. This disease is characterized by a sinus node that is unable to properly control the cardiac rhythm in various ways. As pointed out by the authors in this study, it is possible that a disruption in the normal formation of heteromeric Kv2/Kv6.2 channels in the membranes of automatic sinus node cells disrupt the cardiac rhythm and perturbates autonomic adjustments as needed for homeostasis (Sun et al., 2022). In peripheral nerve cells, nerve injury downregulates the expression of KCNS1 gene that encodes the Kv9.1 silent subunit that contributes to neuronal excitability through Kv2/Kv9.1 heteromeric channels. Consequently, the resulting neurons adopt a hyperexcitable phenotype, firing spontaneously and responding excessively to sensory input, changes that directly drive chronic neuropathic pain (Tsantoulas et al., 2012). Consistent with this, a common amino-acid-changing variant in human KCNS1 gene is associated with increased susceptibility to chronic pain across multiple independent cohorts, reemphasizing the importance of the silent subunits on neuronal excitability (Costigan et al., 2010). It is noteworthy to mention that heteromeric Kv2.1/Kv9.1 channels activate and inactivate slower and at a more negative membrane potential than homomeric Kv2.1 channels. These biophysical properties are reflected on the voltage-activated potassium currents mediated by these channels, consistent with increased excitability.

Kv6.4 subunits, encoded by the KCNG4 gene in humans, have been found in association with the threshold for pain during labor. Patients noted to be remarkably tolerant to labor pain, unusually not requesting gas and air, or epidural for pain relief, were found as carriers of a genetic variant of the KCNG4 gene. Kv6.4 subunits are functional as they co-assemble Kv2 subunits to form heteromeric Kv2.1/Kv6.4 channels. These channels display faster inactivation than Kv2.1 channels, the reason why cells expressing those heteromeric channels are more active. Note that promoted inactivation of Kv channels are associated with fast firing neurons, and this notion has been proven by fast-inactivating Kv3 channels expressed in the inner ear, in the cerebellum, and in sonar organs of bats (Lien and Jonas, 2003; Labro et al., 2015). Upon investigation, it was found that a mutation in the KCNG4 gene was found prevalent in these pain tolerant patients. The genetic mutation predicts the replacement of a key amino acid residue in the Kv6.4 subunit: V419M. This amino acid sits in the selectivity filter contributing region in the Kv6.4 subunit. In vitro studies concluded that V419M mutation interferes with the traffic of heteromeric Kv2.1/Kv6.4 channels to the membrane, basically eliminating Kv6.4 contributions to the excitability of the membrane in the sensory neurons (Lee et al., 2020). Consequently, the increased neuronal excitability normally conferred by Kv6.4-containing heterotetramers is lost, suggesting a plausible mechanistic explanation for the higher pain tolerance observed in carriers of the V419M variant. It is still an open question why a mutation in the selectivity filter would interfere with channel traffic to the membrane. In addition, KCNG4 gene has also been found with a higher than the average number of missenses in migraine-diagnosed patients. This study evaluated 150 genes encoding brain-expressed ion channels and other proteins (Lafrenière and Rouleau, 2012).

Still in the central nervous system, Kv8.2 subunit that is encoded by KCNV2 gene in humans was found in association with epilepsy susceptibility (Jorge et al., 2011). The research identified the Kcnv2 gene in mice, that encode homolog Kv8.2 subunit as associated with epilepsy severity as it is threefold more expressed in the hippocampus. In humans, a screening involving 209 pediatric epilepsy patients supported the association between KCNV2 gene and epilepsy. Two specific variants of KCNV2 gene found in epilepsy patients but not in the 368-patient control group, R7K and M285R, were tested in vitro and demonstrated to be likely causative of changes in the heteromeric Kv2.1/Kv8.2 channel abundance, voltage-dependence and kinetics.

A study that examined Cone Dystrophy with Supernormal Rod Response (CDSRR) found that Kv8.2 silent subunit also plays a critical role in allowing maximal light sensitivity of retinas. Photoreceptors contain both homomeric Kv2.1 channels as well as heteromeric Kv2.1/Kv8.2 channels. In these cells, homomeric Kv2.1 channels mediate a high voltage-activated potassium current, whereas heteromeric Kv2.1/Kv8.2 channels mediate a low voltage-activated potassium current that is active at the photoreceptor’s dark resting membrane potential. This is a great example of how silent KvS subunits can expand the dynamic range of Kv channels regarding the voltage region they operate. Under normal conditions, this low voltage current stabilizes the dark resting potential, maintaining the sustained glutamate release that signals downstream to bipolar cells the absence of light. Without their proper expression, this balance between low and high voltage-activated currents is disrupted and photoreceptors operate in a partially light-adapted state, the result being that CDSRR patients have a smaller functional range over which they can interpret dark and light settings (Jiang et al., 2021; Sakti et al., 2024).

The silent subunit Kv9.3, encoded in humans by the KCNS3 gene, was found associated with cell death and the proliferation of cancer cells (Lee et al., 2015). This study found heteromeric Kv2.1/Kv9.3 channels to be the major mediators of voltage-activated potassium currents in cervical adenocarcinoma cells. The study tested and found that the use of interference RNA caused the G0/G1 cell cycle arrest in carcinoma cell lines.

The molecular and clinical evidence reviewed above collectively demonstrates that KvS subunits are active regulators of Kv2-mediated ionic current across multiple tissues. While we focused on biophysical modulation of Kv2 current in this paper, there is strong evidence that KvS subunits are also involved in other cellular functions. Kv2 channels are known to cluster in the membrane, and some researchers argue that in clustered conditions Kv2 channels are non-conductive (Deutsch et al., 2012; Vierra et al., 2021). In this respect, KvS subunits would have a role in this Kv2 function since KvS subunits can interfere with the expression and the function of Kv2 subunits (Lacey et al., 2025).

GWAS studies and the rational about populational data

Various genome-wide association studies (GWAS) have identified genes encoding KvS subunits in association with human diseases. A GWAS compares the genomic data of a large population of diagnosed patients (with a trait or disease) to identify genetic variants or single-nucleotide polymorphisms (SNPs) that are in association with those trait or disease when compared to unaffected controls. Variants that cross a genome-wide significance threshold (p < 5 × 10⁻⁸), highlight genomic loci associated with a phenotype (Visscher et al., 2017). It is important to mention that these variants themselves do not establish causality alone and they highlight important associations. Using the NHGRI-EBI GWAS catalog (NHGRI-EBI database, United Kingdom), curated and publicly available results (Sollis et al., 2023), we surveyed all 10 genes encoding for KvS subunits and cataloged significant genome-wide associations. We found 427 GWAS associating KvS subunits with traits and diseases. We created arbitrary classes of phenotypes and DNA modifications for all those studies, as shown in Figure 6. Notably, KCNS2 gene did not show any significant genome-wide hits, suggesting that the variants associated at this locus either lack the statistical significance to show a hit or have not yet been adequately studied.

From our analysis, two notable features stood out. First, the spectrum of associated phenotypes differed markedly between KvS subunit genes. Our neuro/psychiatric/cognitive category was present for all KvS subunit genes, while renal/kidney diseases were concentrated for KCNG4, KCNV1 and KCNS3 genes. Second, the associated variants were mostly non-coding rather than the protein altering changes. These non-coding variants suggest the efficiency of transcription is the most affected parameter in those cases.

The phenotypes that were associated with each gene were considerably diverse. Neurological, psychiatric, and cognitive traits made up most associations for KCNG2, and represented a substantial portion for KCNG1, KCNS1, and KCNF1, consistent with the established roles for these subunits in neuronal excitability. KCNF1 stood out for its strong association with immune and inflammatory traits, while KCNG3 and KCNG4 showed broader profiles that extended into anthropometric and renal phenotypes. KCNV1 and KCNV2, on the other hand, were dominated by associations with quantitative molecular and protein level traits rather than discrete disease phenotypes. KCNS3, which yielded by far the greatest number of associations with 206 significant hits, spanned the broadest spectrum overall, including significant neurologic, respiratory, renal, and cardiometabolic contributions. Rather than pointing to a single conserved physiological role of these subunits, we argue that each silent subunit gene carries its own distinct signature of associations that influence distinct physiological processes that are expressed in a tissue-specific manner (Bocksteins, 2016). It is important to note that these profiles should be read with some caution, as the number of associations varied considerably between genes, and the traits represented reflect the phenotypes that have been most studied to date, further research may present some variability. KCNG1 locus that was linked to Alzheimer’s disease showed this same pattern, the associated variant itself was a non-coding one (Willett et al., 2024).

Most of the significant associations fell vastly within non-coding regions of the genome, predominantly intronic and intergenic, with smaller contributions from regular, untranslated, and non-coding exomic regions. This pattern was consistent even for the most heavily associated genes: KCNG2 gene showed associations that were almost entirely intronic, and KCNS3 gene showed associations that were spread across intronic, intergenic, and regulatory categories. Across the family of these subunits, most associated variants lie within non-coding regions, suggesting that genetic influences may operate through regulation of gene expression rather than alterations to protein sequence. This observation suggests that regulation of KvS gene expression may be an important contributor to the physiological effects associated with these loci, and that aspect also highlights the most relevant stoichiometry of the heteromeric Kv2/KvS assemblies that must be governed, among other features, by mass action.

The significance of GWAS data

All these observations above return us to the central theme of this perspective. The diversity of phenotypes across genes is what we would expect if each silent subunit tunes Kv2 in a tissue specific manner, rather than serving a single universal role. Furthermore, the concentration of variation within non-coding, regulatory sequence is more telling, as it suggests that the influence of these subunits on human traits is exerted largely through their expression, by setting how much subunit is made and where, rather than through the structure of the protein itself. These observations are consistent with, but do not by themselves establish, the hypothesis that silent Kv subunits function as a transcriptionally regulated layer of control over excitability.

While GWAS is informative in providing a broad overview of the disease landscape associated with KvS genetic variants, the data here is purely associative. In fact, to date, no variants in the gene encoding another KvS subunit than Kv8.2, as described earlier in this manuscript in the case of CDSRR, have been directly linked to a disease or pathology (Bocksteins, 2016). To further explore how KvS subunit mutations can contribute to the formation of disease states, we conducted an expression quantitative trait locus (eQTL) data analysis using the GTEx portal (Anon, n.d.b). We wanted to see if indeed changes in transcription of KvS subunit encoding genes in disease relevant tissues could be linked to the same genomic loci identified in our GWAS analysis. This would suggest that the genetic risk of these conditions potentially operates through altered KvS expression rather than through a neighboring gene. Therefore, as an example, we present a focused analysis of KCNG2 and KCNS3 genes, which together make up the broadest range of KvS associated disease phenotypes showed in Figure 5, and returned the highest number of genome-wide significant associations in our dataset, together with their corresponding GWAS and eQTL findings.

To carry out the eQTL analysis, we first identified the most significant disease-associated SNPs for KCNG2 and KCNS3 from our GWAS dataset and used the GTEx Portal Locus Browser to identify cis-eQTLs for each gene in disease relevant tissues. For the cis eQTL’s, we prioritized variants near the transcription start site with the lowest reported p-values. For each eQTL, we obtained the corresponding rsID via dbSNP and assessed linkage disequilibrium with the lead GWAS variant using LDlink (LDpair, EUR population). To directly test whether the GWAS variants themselves regulate gene expression, we additionally queried each lead GWAS SNP against KCNG2 and KCNS3 expression across all available GTEx tissues using the eQTL Dashboard.

KCNG2, the gene that encodes Kv6.2 subunit, is predominantly expressed in the astrocyte end feet of the hippocampus and cerebral cortex as shows in the Human Protein Atlas (Thul and Lindskog, 2018). GTEx expression analysis confirmed near-exclusive brain expression, consistent with the neurological and psychiatric phenotypes identified at this locus. KCNG2 returned to 74 significant genome-wide associations spanning psychiatric, pain, cognitive, and substance use phenotypes. The strongest individual hits included schizophrenia (p = 2 × 10⁻²⁰), pain intensity (p = 3 × 10⁻⁹), major depressive disorder, and educational attainment. To assess whether these associations operate through altered Kv6.2 expression, we identified significant cis-eQTLs for KCNG2 across three basal ganglia structures including the caudate (rs4021230, p = 3.02 × 10⁻⁵), nucleus accumbens (rs62096705, p = 1.27 × 10^-5), and putamen (p = 1.95 × 10⁻⁶) wherein the minor allele consistently decreased expression across all three regions, suggesting coordinated genetic regulation of Kv6.2 throughout corticostriatal circuitry. However, linkage disequilibrium (LD) analysis revealed that the disease-associated GWAS variants and the eQTL variants at this locus are in linkage equilibrium across all tissues tested (R ² < 0.3). Additionally, direct testing confirmed that neither the schizophrenia hit rs7238071 nor the pain hit rs151286028 significantly regulated KCNG2 expression across any of the 13 brain regions surveyed in GTEx (minimum p = 0.018 and p = 0.033 respectively, non-significant after correction for multiple testing).

KCNS3, encoding Kv9.3, was first identified in pulmonary artery smooth muscle cells where Kv2.1/Kv9.3 heterotetramers were seen opening at the resting membrane potential and are reversibly inhibited by hypoxia. This made them strong candidates for channel isoforms that initiate hypoxic pulmonary vasoconstriction (Patel et al., 1997; Hulme et al., 1999; Conforti et al., 2000). While they are also believed to play an important role in kidney and cardiovascular tissue, there is limited research describing their exact roles there as compared to in respiratory tissues. From the GWAS data, we found a significant variant associated with altered lung function as measured via FEV1/FVC (rs55884799, p = 7 × 10⁻⁵¹, n = 370,000). A significant cis-eQTL for KCNS3 was identified for lung tissue (rs4832509, p = 2.25 × 10⁻⁵), again demonstrating that Kv9.3 expression in respiratory tissues is subject to genetic regulation. However, LD analysis revealed that GWAS hit rs55884799 and the lung eQTL variant are in linkage equilibrium (R ² = 0.0006, EUR population). Lastly, direct testing confirmed that rs55884799 does not significantly regulate KCNS3 expression in bulk lung tissue (p = 0.631).

Our analyses suggest that bulk tissue expression changes in KCNG2 and KCNS3 are unlikely to fully explain the strongest GWAS associations identified for these loci. While there are a multitude of reasons why bulk tissue eQTL may fail to detect mechanistic convergence including alternative splicing, post translational modifications (PTMs), or cell type specific regulatory effects not visible to bulk tissue use, it is clear that the dissociation between strong GWAS signals and eQTL effects across both KCNG2 and KCNS3 warrant more targeted approaches. Other limitations of the eQTL study were that the GWAS data used was limited to those of European ancestry, the assignment of variants to nearest genes may not reflect the true causal gene, and the relatively small brain tissue sample sizes available in GTEx. Future work should prioritize cell type specific eQTL analysis in disease relevant populations, formal colocalization methods, and direct electrophysiological characterization of disease associated variants to better study their biophysical consequences.

As we have made the case for in this paper, KvS subunits play critical roles in effectively changing the biophysical properties (voltage dependence and gating kinetics) of Kv2 mediated currents, altering the interactions of PTM’s, as well as mediating crucial cellular functions at the ER-PM junction. While there is yet plenty study to be done on Kv2/KvS channels in terms of their actual structure and function.

Final thoughts

For decades, electrically silent Kv subunits have been defined by their flaw: their inability to conduct potassium ions on their own. We have argued in this paper that this framing obscures their real significance. Far from being passive accessories to ion channels, KvS subunits are active, tissue-specific regulators of excitability that remodel the biophysical identity of Kv2 channels. In fact, KvS subunits are one or two of the alpha Kv subunits that form a functional channel. Therefore, they are integral determinants of heteromeric Kv2/KvS channel properties rather than conventional auxiliary subunits.

Heteromeric Kv channels containing KvS subunits are biophysically unique molecular machines. Each of these heteromeric channels shape the membrane potential dynamics with a direct effect on action potentials duration and firing frequency to finally modulate the synaptic output in case of a neuron.

The physiologically significant stoichiometries of these heteromeric assemblies containing KvS subunits represent a current bottle neck in the field. Without that properly defined, the rational development of new drugs to potentially act as therapeutics in the future is halted. Structural biology must help with that soon, but the massive expression required from those studies must blur any definitive conclusions drawn by these approaches.

Also important is the KvS subunits associations with diseases by GWAS. Despite being disavowed by the technicalities of hairy statistics-based calculations, one cannot deny these subunits are involved in physiological and pathological processes. As an example, regular genetic panels normally come with variants in genes encoding hepatic enzymes, and this data is clinically discarded as traits or simply as not relevant to probands’ phenotypes.

KvS subunits, in our view, represent the next frontier for treating diseases involving excitable cells. KvS subunits are currently unexplored as drug targets, and the knowledge acquired by current studies aiming at unveiling their detailed structural aspects, tissue-specific expressions, and not less importantly their gating mechanisms will open large avenues for drug development.

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Footnotes

Edited by: Isabel D. Alves, Centre National de la Recherche Scientifique (CNRS), France

Reviewed by: Yang Yaxiong, Sun Yat-sen University, China

Noor Zulfiqar, University of Agriculture, Pakistan