Abstract:Kv1.2 channels, encoded by the KCNA2 gene, are localized in the central and peripheral nervous system, where they regulate neuronal excitability. Recently, heterozygous mutations in KCNA2 have been associated with a spectrum of symptoms extending from epileptic encephalopathy, intellectual disability, and cerebellar ataxia. Patients are treated with a combination of antiepileptic drugs and 4-aminopyridine (4-AP) has been recently trialed in specific cases. We identified a novel variant in KCNA2, E236K, in a Se… Show more
“…Neuronal firing behavior can be further modulated by K V 1.2-subunit interaction with other K V 1 subunits: e.g., an A-type conductance is formed by K V 1.2 and K V 1.4 heteromeric channels ( 3 , 5 , 6 ). A growing number of both gain- and loss-of-function KCNA2 variants are implicated in epileptic encephalopathy ( 7 – 10 ).…”
Significance
A child with epilepsy has a previously unreported, heterozygous mutation in
KCNA2
, the gene encoding K
V
1.2 proteins. Four K
V
1.2 assemble into a potassium-selective channel, a protein complex at the neuronal cell surface regulating electrical signaling. K
V
1.2 subunits assemble with other K
V
1-family members to form heterotetrameric channels, contributing to neuronal potassium-channel diversity. The most striking consequence of this mutation is preventing K
V
1.2-subunit trafficking, i.e., their ability to reach the cell surface. Moreover, the mutation is dominant negative, as mutant subunits can assemble with wild-type K
V
1.2 and K
V
1.4, trapping them into nontrafficking heterotetramers and decreasing their functional expression. Thus, K
V
1-family genes’ ability to form heterotetrameric channels is a double-edged sword, rendering K
V
1-family members vulnerable to dominant-negative mutations in a single member gene.
“…Neuronal firing behavior can be further modulated by K V 1.2-subunit interaction with other K V 1 subunits: e.g., an A-type conductance is formed by K V 1.2 and K V 1.4 heteromeric channels ( 3 , 5 , 6 ). A growing number of both gain- and loss-of-function KCNA2 variants are implicated in epileptic encephalopathy ( 7 – 10 ).…”
Significance
A child with epilepsy has a previously unreported, heterozygous mutation in
KCNA2
, the gene encoding K
V
1.2 proteins. Four K
V
1.2 assemble into a potassium-selective channel, a protein complex at the neuronal cell surface regulating electrical signaling. K
V
1.2 subunits assemble with other K
V
1-family members to form heterotetrameric channels, contributing to neuronal potassium-channel diversity. The most striking consequence of this mutation is preventing K
V
1.2-subunit trafficking, i.e., their ability to reach the cell surface. Moreover, the mutation is dominant negative, as mutant subunits can assemble with wild-type K
V
1.2 and K
V
1.4, trapping them into nontrafficking heterotetramers and decreasing their functional expression. Thus, K
V
1-family genes’ ability to form heterotetrameric channels is a double-edged sword, rendering K
V
1-family members vulnerable to dominant-negative mutations in a single member gene.
“…described a heterozygous de novo variant in KCNA2 , E236K. 35 This variant caused a hyperpolarizing shift of the voltage-dependence of activation and slowed kinetics of activation and deactivation, resulting in an overall mixed GOF/LOF. Variants without a clear net overall functional effect were excluded from our model's training set.…”
Section: Resultsmentioning
confidence: 99%
“…For both use cases presented here ( KCNA1, KCNA2 ), possible precision therapies based on variant function have been proposed. 15 , 34 , 35 Figure 3 Radar plots of class probability distributions for each variant, with class probabilities shown in the legend. a: KCNA1 p.P403S was correctly predicted as LOF (P405L and P405S are not shown).…”
“…Second, it may result in attenuated action potentials: these may well propagate in excitatory neurons, which tend to have simple and myelinated axons; in contrast, inhibitory neurons tend to have more branched axons, with less myelination, where attenuated action potentials may fail to propagate (Huguenard, 2000; Swadlow et al., 1980). The latter is supported by the adoption of potassium‐channel blocker 4‐aminopyridine (4‐AP, fampridine), currently used to increase neuronal excitability in multiple sclerosis, as a potential therapeutic strategy to alleviate the symptoms of KCNA2 gain‐of‐function encephalopathy (Hedrich et al., 2021; Imbrici et al., 2021). Loss of K V β1‐dependent inactivation of K V 1.2(H310Y) channels would augment the aforementioned mechanisms.…”
Two KCNA2 variants (p.H310Y and p.H310R) were discovered in paediatric patients with epilepsy and developmental delay. KCNA2 encodes KV1.2‐channel subunits, which regulate neuronal excitability. Both gain and loss of KV1.2 function cause epilepsy, precluding the prediction of variant effects; and while H310 is conserved throughout the KV‐channel superfamily, it is largely understudied. We investigated both variants in heterologously expressed, human KV1.2 channels by immunocytochemistry, electrophysiology and voltage‐clamp fluorometry. Despite affecting the same channel, at the same position, and being associated with severe neurological disease, the two variants had diametrically opposite effects on KV1.2 functional expression. The p.H310Y variant produced ‘dual gain of function’, increasing both cell‐surface trafficking and activity, delaying channel closure. We found that the latter is due to the formation of a hydrogen bond that stabilizes the active state of the voltage‐sensor domain. Additionally, H310Y abolished ‘ball and chain’ inactivation of KV1.2 by KVβ1 subunits, enhancing gain of function. In contrast, p.H310R caused ‘dual loss of function’, diminishing surface levels by multiple impediments to trafficking and inhibiting voltage‐dependent channel opening. We discuss the implications for KV‐channel biogenesis and function, an emergent hotspot for disease‐associated variants, and mechanisms of epileptogenesis.
imageKey points
KCNA2 encodes the subunits of KV1.2 voltage‐activated, K+‐selective ion channels, which regulate electrical signalling in neurons. We characterize two KCNA2 variants from patients with developmental delay and epilepsy. Both variants affect position H310, highly conserved in KV channels.
The p.H310Y variant caused ‘dual gain of function’, increasing both KV1.2‐channel activity and the number of KV1.2 subunits on the cell surface.
H310Y abolished ‘ball and chain’ (N‐type) inactivation of KV1.2 by KVβ1 subunits, enhancing the gain‐of‐function phenotype.
The p.H310R variant caused ‘dual loss of function’, diminishing the presence of KV1.2 subunits on the cell surface and inhibiting voltage‐dependent channel opening.
As H310Y stabilizes the voltage‐sensor active conformation and abolishes N‐type inactivation, it can serve as an investigative tool for functional and pharmacological studies.
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