The absence of NIPA2 enhances neural excitability through BK (big potassium) channels

Summary Aim To reveal the pathogenesis and find the precision treatment for the childhood absence epilepsy (CAE) patients with NIPA2 mutations. Methods We performed whole‐cell patch‐clamp recordings to measure the electrophysiological properties of layer V neocortical somatosensory pyramidal neurons in wild‐type (WT) and NIPA2‐knockout mice. Results We identified that layer V neocortical somatosensory pyramidal neurons isolated from the NIPA2‐knockout mice displayed higher frequency of spontaneous and evoked action potential, broader half‐width of evoked action potential, and smaller currents of BK channels than those from the WT mice. NS11021, a specific BK channel opener, reduced neuronal excitability in the NIPA2‐knockout mice. Paxilline, a selective BK channel blocker, treated WT neurons and could simulate the situation of NIPA2‐knockout group, thereby suggesting that the absence of NIPA2 enhanced the excitability of neocortical somatosensory pyramidal neurons by decreasing the currents of BK channels. Zonisamide, an anti‐epilepsy drug, reduced action potential firing in NIPA2‐knockout mice through increasing BK channel currents. Conclusion The results indicate that the absence of NIPA2 enhances neural excitability through BK channels. Zonisamide is probably a potential treatment for NIPA2 mutation‐induced epilepsy, which may provide a basis for the development of new treatment strategies for epilepsy.

channel gene, are primarily associated with ion channels. 5 CAE is also related to nonion channel genes, such as NIPA2, a highly selective magnesium transporter gene. 6 NIPA2 encodes nonimprinted Prader-Willi/Angelman syndrome region protein 2. 7 It belongs to NIPA family that includes four family members, namely, NIPA1, NIPA2, NIPA3, and NIPA4.
NIPA family members are nonselective magnesium transporters except NIPA2, 6 which is highly selective to the extracellular-to-intracellular transfer of magnesium. 8,9 NIPA2 mutations associated with CAE have also been reported, at least in the Han Chinese population. 10,11 A previous study illustrated that loss-of-function NIPA2 mutations may cause the accumulation of NIPA2 proteins in the endoplasmic reticulum, block magnesium transport, and affect neuronal excitability. 12 Various receptors and ion channels regulate neuronal excitability. Among them, big-conductance potassium (BK) channels [13][14][15][16][17] are controlled by magnesium, 15 calcium, 18 and membrane voltage. 19 BK channels engage in action potential repolarization and play a key role in regulating action potential firing. 20 Moreover, changes in BK channel functions can alter neuronal excitability. Genes encoding BK channels have two mutation types, namely, gain of function [20][21][22] and loss of function. [23][24][25] Both types may increase neuronal excitability.
The electrophysiological mechanism of NIPA2 affecting neuronal excitability remains unknown. Here, we hypothesized that the dysfunction of NIPA2 may reduce BK channel currents, and the decreased currents of BK channels enhance neuronal excitability.

| Preparation of the transverse brain slices
All experimental processes involving the use of animals were approved by the Experimental Animal Sciences of Peking University Health Science Center Institutional Animal Care and Use Committee.
Animal pain was minimized, and the number of animals was reduced F I G U R E 1 Functional characterization of spontaneous APs in the neocortical somatosensory pyramidal neurons. A, A 40× view of representative photograph of the layer V neocortical somatosensory pyramidal neurons. B, Schematic diagram of action potential. C, Representative trace of spontaneous APs in the neurons isolated from the WT mice; scale bars represent 10 s (x-axis) and 25 mV (y-axis). D, Spontaneous APs in the neurons isolated from the NIPA2-knockout mice. Scale bars are identical to that in C. E, Number of spontaneous APs in the neurons isolated from the WT mice and the NIPA2-knockout mice within 5 min. Statistical significance was obtained by Wilcoxon signed rank test. *P < 0.05. F, Amplitude of spontaneous APs in the neurons isolated from the WT mice and NIPA2-knockout mice (by t test). APs, action potentials; n.s., no significance; sAP, spontaneous action potential; WT, wild-type They were stored at 22°C-24°C.

| Electrophysiological recordings
After the brain slices were prepared, the electrophysiological properties of the whole-cell patch-clamp recording procedures were determined. An incubated slice was transferred to the recording chamber, placed under an upright microscope (BX51WI; Olympus), and continuously perfused with oxygenated ACSF Germany). Recording was rejected when series resistance was more than 50 MΩ, when series resistance was changed by more than 20%, or when the resting membrane potential (RMP) was more positive than −55 mV. Only one neocortical somatosensory pyramidal neuron was recorded per slice, and 4-5 slices were examined per mouse.

| Experimental protocols
In the current-clamp mode, the spontaneous action potential (AP) of layer V neocortical somatosensory pyramidal neurons was recorded at RMP, which was measured in the current-clamp mode with a 0 pA holding current.
In the current-clamp mode, at the resting membrane potential, a series of positive current injections (0 pA to +140 pA with a step of +20 pA, 1 second duration) was applied to evoke a set of AP. The evoked AP and the firing pattern were recorded at each evoking potential (0 pA to +140 pA). The evoked AP frequency was determined as the spike number in 1 second. Figure 1B shows details about how to measure the amplitude and the half-width of action potential. The AP threshold was obtained by a special voltage stimulus from −50 to −30 mV with a step of 1 mV and a duration of 100 ms, and the voltage evoking the initial current was the AP threshold. 26 The currents of the BK channels were evoked as described previously. 27 In the voltage-clamp mode, under the holding potential of −80 mV, a series of depolarizing step voltage pulses (+40 mV to +180 mV with a step of +20 mV, 75 ms duration) was applied to evoke BK channel currents. At a potential of +40 mV to +180 mV, the curve of BK channel currents was the difference between that of the total currents before and after paxilline (blocking BK channels) and tetrodotoxin (TTX; blocking sodium channels) were used. The current density was calculated by dividing the corresponding amplitude of BK channel currents by the cell capacitance at each evoking potential (+40 mV to +180 mV). The current density-voltage curve was drawn.

| Chemical application
All chemicals were bath applied through a peristaltic pump system at a steady perfusion and suction rate. The time required for the solution to flow to the recording slice through stopcocks was approximately 1 minute. All drugs, including paxilline (a specific BK channel blocker 28,29 ), were purchased from Sigma-Aldrich (St. Louis, MO, USA), except NS11021 (a specific BK channel opener 27,30,31 ) and TTX (a sodium channel blocker), which were bought from Tocris Bioscience (Bristol, UK). All chemicals were dissolved in dimethyl sulfoxide (except TTX that was dissolved in distilled water) at 1000 times final concentration, stored at −20°C, and diluted to the final concentration in an oxygenated ASCF solution immediately before use.

| Statistical analysis
All numerical data were expressed as mean ± SEM, except abnormal distribution data, which were presented as the median ± quar- Magnesium is a vital regulatory factor of many ion channels, such as BK channels, associated with neuronal excitability. To investigate the effect of NIPA2 mutations on neuronal excitability, we performed whole-cell current-clamp recordings between the two independent groups, namely, the wild-type (WT) mouse group and the NIPA2-knockout mouse group. In the current-clamp mode, we measured spontaneous and evoked APs of the layer V neocortical somatosensory pyramidal neurons isolated from WT or NIPA2-knockout mice. The neurons isolated from the WT mice generated minor or minimal spontaneous APs within 5 minutes. Correspondingly, the frequency of the spontaneous APs of the neurons isolated from the NIPA2-knockout mice was significantly higher than that from the WT mice ( Figure 1B-E, WT: 1.5 ± 1.0, n = 10 vs NIPA2 knockout: 4.0 ± 10, n = 10, P = 0.038).
We also measured the evoked AP firing activities in the layer V neocortical somatosensory pyramidal neurons and found that the frequency of the evoked APs of the neurons isolated from the NIPA2-knockout mice was significantly higher than that of the neurons from the WT mice through a train of current injection ( Figure 2).
The neurons from the NIPA2-knockout mice were more excitable at current injections of 80, 100, 120, and 140 pA and showed a sustained activity at high current injections, such as 80 pA (

| Absence of NIPA2 reduced the currents of BK channels
The difference in the evoked AP half-width between the WT group and the NIPA2-knockout group suggested that potassium channels might be involved in NIPA2-regulated neural excitability. BK channels were activated by intracellular magnesium and played a vital role in AP repolarization. NIPA2 dysfunction could decrease intracellular magnesium concentrations. Thus, we inferred that the absence of NIPA2 might enhance neuronal excitability by decreasing BK channel currents. In our study, after 0.5 μmol/L TTX were F I G U R E 4 Evoked AP changes in NIPA2-knockout neurons after the use of BK channel opener. A, Exemplar traces of evoked APs in NIPA2-knockout neurons in response to test currents ranging from 0 pA to 140 pA in 20 pA increments for 1 s in the absence of NS11021, a selective BK channel opener. Scale bars are identical to that in B. B, Recorded evoked APs under the same test current injections with A in NIPA2-knockout neurons in the presence of NS11021. Scale bars represent 200 ms (x-axis) and 20 mV (y-axis). C, Frequency of evoked APs in response to the current injections in the neurons without NS11021 (brown, n = 10) or with NS11021 (black, n = 10). The neurons without NS11021 exhibited higher evoked APs at current injections of 40, 100, 120, and 140 pA. Statistical significance was obtained by t test separately. *P < 0.05. D, Amplitude of evoked first AP responded to 80 pA current stimulus without or with NS11021. E, Rheobase of evoked initial APs in the neurons without or with NS11021. APs, action potentials used at least 5 minutes to block sodium channels, 10 μmol/L paxilline was provided for 10 minutes in 0.5 μmol/L TTX-containing bath to completely inhibit the BK channel currents, and the maximum inhibitory effects were measured 1-2 minutes after the end of paxilline and TTX bath perfusion. The amplitude of the BK currents was measured as the difference between the total currents after a +180 mV voltage step and at an initial holding potential of +40 mV. We found that the BK channel currents of the neurons isolated from the NIPA2-knockout mice were significantly smaller than those of the neurons from the WT mice at voltage injections of 40-160 mV ( Figure 3A-C), although no significance was observed at +180 mV stimulus (WT: 2.9 ± 0.63 nA, n = 8 vs NIPA2 knockout: 1.7 ± 0.24 nA, n = 13, P = 0.062). The current density was obtained by dividing the amplitude of the BK channel currents at each voltage injection by the cell capacitance. A significant decrease was observed in the NIPA2-knockout group in terms of current density compared with that in the WT group ( Figure 3D, WT: 159 ± 35 pA/ pF, n = 8 vs NIPA2 knockout: 57 ± 10 pA/pF, n = 13, P = 0.0031).
These data revealed that the absence of NIPA2 decreased the BK channel currents, possibly hindering AP repolarization and

| Increased BK channel currents reduced action potential firing in NIPA2-knockout mice
To demonstrate whether the increased BK channel currents in the NIPA2-knockout mice could decrease neuronal excitability, we com-

| Decreased BK channel currents enhanced action potential firing in wild-type mice
To further observe the effect of BK channels on AP, we added The rheobase of the evoked AP declined significantly after paxilline was added ( Figure 5E, without paxilline: 80 ± 7.9 pA vs with paxilline: 54 ± 6.0 pA, P = 0.00075). However, the evoked AP amplitude was not significantly changed by paxilline ( Figure 5D, without paxilline: 95 ± 2.3 mV vs with paxilline: 92 ± 1.9 mV, P = 0.11). These results illustrated that decreased BK channel currents enhanced AP firing in WT mice.

| Zonisamide reduced action potential firing in NIPA2-knockout mice
Previous study showed that an antiepileptic drug of zonisamide had an anticonvulsant effect by activating BK channels. 32 In our study, brain slices of NIPA2-knockout mice were treated by zonisamide to observe neural excitability after the treatment. Specifically, we

| Absence of NIPA2 induces neural hyperexcitability
We demonstrated that the layer V neocortical somatosensory pyramidal neurons isolated from the NIPA2-knockout mice showed significantly excessive neuronal excitability. The spontaneous and evoked AP frequency of the neocortical somatosensory pyramidal neurons of the NIPA2-knockout mice was higher than the WT mice.
In WAG/Rij rats of absence epilepsy model, the somatosensory pyramidal neurons of the cortex with low hyperpolarization-activated cation currents exhibit high frequency in response to current injections. 33 For CAE, the cortico-thalamo-cortical network oscillation plays an important role in the occurrence and development of absence epilepsy. 34 Abnormal discharges of CAE originate from the thalamocortical network and are finally projected into the neocortical somatosensory pyramidal neurons. Furthermore, neocortical somatosensory pyramidal neurons are a key part of the neurological circuit of absence epilepsy, 33 and abnormal discharges of neocortical somatosensory pyramidal neurons are the feature of absence epilepsy. 35 In the present study, the neocortical somatosensory pyramidal neurons isolated from the NIPA2-knockout mice showed significantly excessive neuronal excitability. Thus, the absence of NIPA2 induced the hyperexcitability of neocortical somatosensory pyramidal neurons, thereby affecting the thalamocortical network to trigger absence epilepsy.

| Absence of NIPA2 enhances neuronal excitability by decreasing BK channel currents
Neuronal excitability is regulated by various receptors and ion channels. Among them, BK channels and N-methyl-D-aspartic acid receptor (NMDAR) channels 36 are the two common magnesium-regulated ones.
In this study, we found that the absence of NIPA2 enhanced

| Zonisamide is probably a potential treatment for epilepsy patients with NIPA2 loss-offunction mutation
Precision medicine has been extensively used to treat patients with epilepsy. For example, quinidine can be applied to treat KCNT1positive epilepsy, 41,42 and retigabine is utilized for KCNQ2-positive epilepsy. 43,44 We suggested that zonisamide might be a precision treatment for epilepsy patients with NIPA2 loss-of-function mutation. We identified three NIPA2 mutations (c.532A > T, c.731A > G, c.1002_1003insGAT) from children with CAE. 10,11 However, we did not find NIPA2 variations in children with epilepsy and intellectual/ developmental disabilities through targeted next-generation sequencing or Sanger sequencing. 45 A subsequent functional study has verified that these three mutations are loss of function. 12 In the present study, the dysfunction of NIPA2 enhanced the excitability of neocortical somatosensory pyramidal neurons by decreasing BK channel currents. NIPA2 loss-of-function mutations might serve as pathogenic mutations in children with CAE. If a drug could rescue NIPA2-induced pathogenic process, then this drug would save patients with epilepsy. Zonisamide, as a commercially available antiepileptic drug, elicits an anticonvulsant effect by activating BK channels, and it reduces neuronal hyperexcitability by increasing BK channel currents. 32 Therefore, this drug might be used to treat the epilepsy patients with NIPA2 loss-of-function mutation. Previous preclinical studies on zonisamide emphasized that its biological metabolism is prototype metabolism without drug attenuation. In this study, brain slices of NIPA2-knockout mice were treated by zonisamide, and we have found that zonisamide reduce action potential firing in NIPA2knockout mice. It suggests that zonisamide may be an effective treatment for NIPA2-knockout mice. We believe that zonisamide is probably a potential treatment for NIPA2 mutation-induced epilepsy.
In summary, the dysfunction of NIPA2 enhances neuronal excitability by decreasing BK channel currents. NIPA2 loss-of-function mutations are pathogenic in patients with CAE. Zonisamide may be used to treat the epilepsy patients with NIPA2 loss-of-function mutation.

| CON CLUS ION
In this study, we found that the absence of NIPA2 enhanced the excitability of neocortical somatosensory pyramidal neurons by decreasing the currents of BK channels. Zonisamide, reducing action potential firing in NIPA2-knockout mice, is probably a potential treatment for NIPA2 mutation-induced epilepsy, which may provide a basis for the development of new treatment strategies for epilepsy.

ACK N OWLED G M ENTS
We would like to express our gratitude to the research team of Pro.
Zhiqing Xu for providing experimental platform of whole-cell patchclamp and to the research group headed by Pro. Zhi-Qing Xu for the helpful discussion and pertinent opinions.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.