KCNA1 (K_v1.1) and KCNA2 (K_v1.2) have been associated with epilepsy (Cooper, 2012; Robbins and Tempel, 2012; Boutry-Kryza et al., 2015). The Kcna family codes for the pore-forming “K_v1” subunits, which may compose either A-type or delayed rectifier channels (Raab-graham and Niere, 2017). A-type currents are rapidly activating and fast inactivating, while delayed rectifiers open slowly and remain open (Raab-Graham and Niere, 2017). Depending on the brain region and cellular composition, the most abundantly expressed α subunits of the K_v1 subfamily are K_v1.1, K_v1.2, and K_v1.4 (Robbins and Tempel, 2012:1). Interestingly, K_v1.1 codes for a delayed rectifier, while K_v1.4 codes for the A type family. However, K_v1.1 in conjunction with a K_vβ1 or K_v1.4 subunit, can have properties of the A type family (D’Adamo et al., 2020). K_v1 channels are responsible for resetting the resting membrane potential and titrating synaptic release in neurons (Foust et al., 2011; Robbins and Tempel, 2012). Interestingly, reduced expression of either K_v1.1 and K_v1.2 channels have been associated with epilepsy, as each knockout mouse presents with seizures that resemble the development of human epilepsy (Robbins and Tempel, 2012). Additionally, mutations in K_v channels that disrupt coassembly with other α or β subunits reduces channel functional and/or expression (D’Adamo et al., 2020). Together, Kv1 channels prevent “runaway” depolarization, increased firing rates, and excessive neurotransmitter release, all of which can lead to seizures.

It should be noted, that cross referencing the transcriptome of human TSC cortical tubers and those genes with associated epilepsies, transcripts coding for K_v auxiliary subunits K_vβ1 and K_vβ2 were detected (Tables 1, 2; Boer et al., 2010). Interestingly, other K_v subunits such as K_v1.1, K_v1.2, K_v1.4, and K_v4.2 in have been implicated in mTOR-related epilepsy models (Table 2; Brewster et al., 2013; Niere and Raab-Graham, 2017). Together, these data suggest that the K_v1 class should be further investigated in TSC.

Among the K_v4 subunits, KCND2 (K_v4.2) and KCND3 (K_v4.3) also belong to A-type voltage-gated potassium channel class. These channels are abundantly found in the nervous system within somatodendritic compartment of neurons (Zemel et al., 2018). Interestingly, several studies have shown that down regulation of the A-current leads to increased excitability (Bernard et al., 2004; Liu et al., 2014). For example, a mutation in K_v4.2 (V404M) leads to impairments in inactivation after channel opening. This mutation has been associated with infant-onset epilepsy and autism (Lin et al., 2018). Thus, examining the K_v4 subunit class maybe be a potential interest to TSC and associated seizure types. Altogether, we predict that these channels to be dysfunctional in TSC, specifically the potassium genes listed in Tables 1–3.

Since the discovery of on demand local protein synthesis occurring at the synapse, dysregulation of protein synthesis can lead to misexpression of ion channel subunits and alter the membrane potential and consequently lead to seizure-like conditions (Switon et al., 2017). Pathways involving mTOR, are known to regulate local synthesis at the synapse. Under conditions where mTOR is active, local synthesis of K_v1.1 is repressed on dendrites without altering axonal expression (Raab-Graham et al., 2006; Niere and Raab-Graham, 2017). Additionally, others have shown that both K_v1.2, and K_vβ2 at the synapse are reduced when mTOR is active (Niere and Raab-Graham, 2017). Together, these findings suggest that mTOR activity toggles expression of potassium channels as a local feedback mechanism that ensures optimized synaptic function (Raab-Graham and Niere, 2017).

If mTOR activity is left unregulated, as seen in TSC, repression of K_v channel expression may lead to an increase in neuronal excitability and to eventual epileptogenesis (Cho, 2011; Meng et al., 2013; Jeong and Wong, 2016). This is suggested by Brewster and colleagues who utilized a model of pilocarpine-induced status epilepticus (SE) model and examined ion channel expression in presence and absence of rapamycin, an mTOR inhibitor. With the development of epileptogenesis, reduced expression of K_v1.4 and K_v4.2 in the hippocampus was observed. With the addition of rapamycin, which has been shown to reduce seizure frequency (Zeng et al., 2009) in SE rodents, protein levels of K_v1.4 and K_v4.2 increases to similar levels as seen in the vehicle treated rodents (Brewster et al., 2013). Altogether, these independent studies indicate that alterations in K_v expression could result in altered neuronal excitability and further studies are needed to implicate K_v channels to seizures such as those experienced in TSC.

Of interest are Na_v1.1 and Na_v1.2, channels expressed in neurons, but more specifically the gene mutations affecting the α subunits of these channels. These mutations lead to inherited forms of epilepsy that differ based on type of α subunit defect (Mantegazza and Catterall, 2012). The SCN1A gene, which encodes the Na_v1.1 channel, has been associated with Dravet syndrome, which displays afebrile intractable seizures (Craig et al., 2012; Schmunk and Gargus, 2013). Missense mutations in the SCN1A gene (D322N), commonly display gain of function (GOF), that lead to enhanced sodium currents as a result of lack of inhibition on excitatory neurons (Menezes et al., 2020). Likewise, mutations in the SCN2A gene (A467T), that encodes the voltage-gated sodium channel Na_v1.2, have been shown to elicit seizure behavior such as in generalized epilepsy with febrile seizure plus (GEFS+) syndrome by also enhancing sodium currents (Schmunk and Gargus, 2013; Wolff et al., 2017). Additionally, LOF mutations in SCN2A have been linked to ASD and intellectual disability, all of which are commonly seen in patients with TSC. Thus, further research is needed to understand the mechanisms by which mutations in these genes leads to TSC.

Other voltage-gated sodium channels, such as Na_v1.6 and Na_v1.7 are associated with infantile spasms and febrile seizures, respectively, while Na_v1.3 has been associated with patients with epilepsy (Menezes et al., 2020). Notably, mutations in SCN8A gene, coding for Na_v1.6, affects the action potential threshold which increases spontaneous and repetitive firing leading to an increase in excitability (Menezes et al., 2020). Additionally, GOF and LOF mutations in Na_v1.3 and Na_v1.7, have been reported to alter the biophysical properties of neurons as these genes modify other sodium channels such as Na_v1.1, which can contribute to pathogenesis of epilepsy, whoever, more studies are needed to ascertain their direct involvement. Altogether, these channels are associated with types of seizures experienced within TSC, however, these channels remain uninvestigated, making these channels possible candidates to examine in TSC.

Voltage-gated calcium channels are required for different functions in the neuron, such as controlling neuronal excitability and regulating calcium-sensitive intracellular pathways (Cain and Snutch, 2012; Zamponi et al., 2015). There are three classes of voltage-gated calcium channels, Ca_v1, Ca_v2, and Ca_v3 (Table 3). Each class has subclasses of ion channel expression that vary based function, and kinetics. The channels are either high voltage (HVA) or low voltage activated (LVA), meaning the channel opens or activates at −40 and −60 mV, respectively (Cain and Snutch, 2012). The calcium channel, like the sodium and potassium channel, contain an α subunit that stands as the pore-forming unit that is selective for calcium (Cain and Snutch, 2012; Zamponi et al., 2015). They also have auxiliary subunits β, γ and α2δ that regulate the properties of the channel (Campiglio and Flucher, 2015). Because of the genetic diversity among the calcium channels, only a select few of the channels expressed in the brain will be discussed, specifically the HVA and LVA channel subunits listed in Tables 1–3.

The β auxiliary subunits play an important role in enhancing the biophysical properties of the α subunit, such as channel folding, channel trafficking, and alters gating kinetics and voltage-dependence (Dolphin, 2003, 2016). Interestingly, our comparison of the human TSC cortical tuber transcriptome cross referenced with genes associated with epilepsies, yielded elevated mRNA coding for Ca_vβ2 and Ca_vβ4 (Table 1; Boer et al., 2010). Interestingly, one study demonstrated that ablation of Ca_vβ1, Ca_vβ2, and Ca_vβ3 have no major impact on neuronal function (Ball et al., 2002; Vergnol et al., 2022). On the other hand, one study demonstrated Ca_vβ4 is associated with the lethargic mouse model of epilepsy (Burgess et al., 1997; Vergnol et al., 2022), while another study showed that disruption in the Ca_vβ2 gene leads to diminished L-type channel currents (Weissgerber et al., 2006). Although the biophysical properties of these two subunits have yet to be determined in TSC, this finding shows possible insights into voltage-gated calcium channels and whether they are disrupted.

The Cav2 family encompasses Ca_v2.1, Ca_v2.2, and Ca_v2.3 isoforms. these channels are comprised of a pore-forming α subunit and auxiliary β subunits. Together, they are responsible for regulating Ca2+ entry in response to depolarization and release of neurotransmitters (Mochida, 2019). These channels can undergo alternative splicing, and thus, have a wide spectrum of biophysical properties. Of particular interest are Ca_v2.1 and Ca_v2.3, as shown in Table 3, will be discussed further as these channels have more direct implications to seizure. There are several Ca_v2.1 channel mutations that generate epileptic phenotypes commonly seen within TSC. For example, TSC patients can suffer from absence epilepsy, whose mouse models, “leaner”, “tottering”, and “rocker,” display epileptic phenotypes as a result of different Ca_v2.1 channel mutations (Imbrici et al., 2004; Mochida, 2019). These mutations affect Ca_v2.1 current density by slowing channel inactivation as well as imbalances on inhibitory to excitatory neurotransmission leading to increased firing (Rajakulendran and Hanna, 2016). Similarly, Ca_v2.3 has also been demonstrated to play a role in absence epilepsy role (Zaman et al., 2011). Nevertheless, the contribution of Ca_v2.1 or Ca_v2.3 to TSC absence epilepsy remains to be determined and, thus, this remains a possible avenue of exploration.

T-type calcium channels, do not require auxiliary subunits (Simms and Zamponi, 2014). Because the Ca_v3 subunits have been shown to undergo alternative splicing, resulting in channel function diversity, the T-type channels that will be discussed in the context of TSC will be Ca_v3.1 (CACNA1G) and Ca_v3.2 (CACNA1H). CACNA1G channels are highly expressed in thalamocortical (TC) neurons (Chen et al., 2014). CACNA1H has been shown to be primarily expressed in the dorsal root ganglion, dentate of the hippocampus, and thalamus (Graef et al., 2011; Simms and Zamponi, 2014; Bernal Sierra et al., 2017). Ca_v3.2 mutations have been shown to lead to seizures in murine models, specifically absence epilepsy. Because of this implication in seizure commonality, and because this limbic seizure can precede from subcortical structures such as the thalamus, it is possible that T-channels play a role in initiating the spread to higher structures in the TSC brain.

As previously mentioned above, the HVA class also encompasses L-type calcium channels as they are integral to cell’s membrane complex that mediate influx of Ca2+ after a depolarization response (Hofmann et al., 2014). The “L” in L-type represents the long-lasting inward currents during depolarization which have distinguished them from their “transient current” T-type cousins (Zamponi et al., 2015). One L-type channel, Ca_v1.2, is composed of three subunits α1, α2δ, and β axillary subunits (Hofmann et al., 2014; Zamponi et al., 2015), and may provide more insight into TSC epileptic phenotypes. There have been case studies indicating the presence of febrile seizures among TSC patients (Kubo et al., 2011; Siddaraju et al., 2016). However, these case studies primarily served as documentation for the patients’ condition, and no other studies have followed up on the possibilities of febrile seizures in TSC models. Interestingly, one independent study demonstrated that febrile seizures in rat pups may be prevented with the use of nimodipine (Radzicki et al., 2013). Additionally, mTOR hyperactivation has been shown to differentially regulate L channel expression in TSC. An independent study has demonstrated that somatic Ca_v1.2 and Ca_v1.3 gene and protein expression are augmented in TSC2-null neurons. Hisatsune et al., 2021 also demonstrates that Cav1.3 triggers enhanced neuronal activity of TSC2^–/– neurons and could be a potential novel target for epilepsy in TSC (Hisatsune et al., 2021). On the other hand, Ca_v1.2 de novo protein synthesis was found to be reduced in the dendrites of hippocampal CA-1 neurons in a mouse model of TSC1 (Figure 2). Furthermore, Niere et al. (2023) found that an RNA binding protein DJ-1 coordinates the expression of Ca_v channel complex, including Cav1.2 and α2δ2, resulting in attenuated calcium signaling in the dendrites (Figure 2 and Table 2). Like the β subunits mentioned above, the α2δ auxiliary subunits play an important role in trafficking and gating of the α subunits (Dolphin, 2003, 2016). Additionally, α2δ1 was found to be overexpressed in conditionally knockout TSC hippocampal dendrites (Niere et al., 2023); however its role in TSC has not been established. Together, these two studies on L-type calcium channels, suggest that subcellular localization of these channels differentially affects calcium influx across the cell. Considering the importance of calcium channel to seizures, these findings give credence to further investigation of calcium channel dysfunction in TSC.