Abstract
The gene kcnma1 encodes the α-subunit of high-conductance calcium- and
voltage-dependent K^+ (BK) potassium channel. With the development of
generation gene sequencing technology, many KCNMA1 mutants have been
identified and are more closely related to generalized epilepsy and
paroxysmal dyskinesia. Here, we performed a genetic screen of 26
patients with febrile seizures and identified a novel mutation of
KCNMA1 (E155Q). Electrophysiological characterization of different
KCNMA1 mutants in HEK 293T cells, the previously-reported R458T and
E884K variants (not yet determined), as well as the newly-found E155Q
variant, revealed that the current density amplitude of all the above
variants was significantly smaller than that of the wild-type (WT)
channel. All the above variants caused a positive shift of the I-V
curve and played a role through the loss-of-function (LOF) mechanism.
Moreover, the β4 subunit slowed down the activation of the E155Q
mutant. Then, we used kcnma1 knockout (BK KO) mice as the overall
animal model of LOF mutants. It was found that BK KO mice had
spontaneous epilepsy, motor impairment, autophagic dysfunction,
abnormal electroencephalogram (EEG) signals, as well as possible
anxiety and cognitive impairment. In addition, we performed
transcriptomic analysis on the hippocampus and cortex of BK KO and WT
mice. We identified many differentially expressed genes (DEGs). Eight
dysregulated genes [i.e., (Gfap and Grm3 associated with astrocyte
activation) (Alpl and Nlrp10 associated with neuroinflammation) (Efna5
and Reln associated with epilepsy) (Cdkn1a and Nr4a1 associated with
autophagy)] were validated by RT-PCR, which showed a high concordance
with transcriptomic analysis. Calcium imaging results suggested that BK
might regulate the autophagy pathway from TRPML1. In conclusion, our
study indicated that newly-found point E155Q resulted in a novel
loss-of-function variant and the dysregulation of gene expression,
especially astrocyte activation, neuroinflammation and autophagy, might
be the molecular mechanism of BK-LOF meditated epilepsy.
Keywords: BK channel, KCNMA1, loss-of-function variants, epilepsy,
neuroinflammation, autophagy
Introduction
Ion channels are expressed throughout the body and perform important
physiological functions, such as neuronal excitability and the tone of
smooth muscle. Ion channel disease, also known as ion channelopathy, is
unusually considered to be caused by the gene mutation and abnormal
function of ion channel subunits ([52]Zheng and Trudeau, 2015;
[53]Bailey et al., 2019). BK channel is widely expressed in neurons and
muscles ([54]Fagerberg et al., 2014), and is also related to functions
such as membrane potential repolarization, neuronal excitability
control, neurotransmitter release, innate immunity, and cochlear hair
cell regulation ([55]Petersen and Maruyama, 1984; [56]Murrow and Fuchs,
1990; [57]Brayden and Nelson, 1992; [58]Robitaille and Charlton, 1992).
Human kcnma1 encodes the α-subunit of high-conductance calcium- and
voltage-dependent K^+ (BK) potassium channel. The α subunit of BK
channel contains seven transmembrane fragments (S0-S6) and a large
intracellular COOH terminal, consisting of two RCK domains (responsible
for calcium sensing through the high-affinity Ca^2+ binding sites), and
S1-S4 acts as the voltage sensor, S5 and S6 fragments as well as P-loop
form the pore region of the channel, and the (TVGYG) sequence of S6 is
considered as a selective filter for potassium ions ([59]Latorre et
al., 2017). BK channel is allosterically activated by the changes of
not only intracellular calcium concentration, but also membrane
potential. The main sources of BK channel dysfunction are de novo and
genetic nucleotide changes, which are roughly divided into
gain-of-function (GOF) and loss-of-function (LOF) ([60]Bailey et al.,
2019). LOF mutation changes the channel activity by reducing the
current amplitude or duration, while GOF mutation activates faster,
increases Ca^2+ sensitivity and current amplitude ([61]Du et al., 2005;
[62]Moldenhauer et al., 2020).
In 2005, the abnormality of the BK channel was associated with human
diseases for the first time. The substitution of aspartic acid at
position 434 of the alpha subunit of BK channel by glycine would induce
repolarization of action potentials, accelerate the firing rate, and
increase the overall excitability of neurons, leading to systemic
epilepsy ([63]Du et al., 2005). Interestingly, the GOF phenotype of
D434G mutant is due to increased BK channel Ca^2+ sensitivity ([64]Du
et al., 2005), but in N995S (also called N999S and N1053S) mutants, the
mechanism for BK GOF is the left shift of conductance-voltage (G-V)
curve ([65]Moldenhauer et al., 2020). However, different from the
phenotype of the mutants above, BK channel C413Y, P805L, D984N
([66]Bailey et al., 2019) and G354S-LOF ([67]Du et al., 2020) mutations
have been found to reduce the channel currents, with varying presence
of seizures, dyskinesia, and dystonia ([68]Bailey et al., 2019). In
addition to the genetic mutation and abnormal expression of the channel
itself, the physiological characteristics of the BK channel are also
affected by the interaction of its α subunit and auxiliary subunits
(e.g., β1-4 subunits or γ1-4 subunits). The β4 subunit is an auxiliary
subunit specifically expressed by neurons, dominantly expressed in
brain. The BK channel composed of it and the α subunit activates
relatively slowly than the channel composed of only the α subunit. The
mice lacking the β4 subunit show significant symptoms of temporal lobe
epilepsy ([69]Brenner et al., 2005). In addition, mutations in the β3
subunit also cause epilepsy ([70]Lorenz et al., 2007).
The unique physiological behavior of the BK channel enables it to both
enhance and reduce the excitability of neurons. Therefore, the role of
BK channel in the pathogenesis of epilepsy is still controversial and
is an increasingly intense research field ([71]Zhu et al., 2018).
Here, we report a novel de novo KCNMA1 mutant (E155Q) in a patient.
Electrophysiological results show that E155Q, R458T (not yet
determined), and E884K (not yet determined) mutant all present a LOF
phenotype. A series of behavioral experiments show that BK KO mice (as
the overall animal model of LOF) have spontaneous epilepsy, motor
impairment, abnormal electroencephalogram (EEG) signals, autophagic
dysfunction, as well as possible anxiety and cognitive impairment. This
study expands the mutation spectrum of KCNMA1-epilepsy, explores the
possible mechanism of epilepsy by transcriptome, reveals the
relationship between KCNMA1-LOF and epilepsy, and provides a possible
molecular template for individualized treatment of epilepsy.
Methods
Mutation Screening
Genomic DNA was extracted from peripheral blood with kit (DP348,
Tiangen, Beijing, China). The samples were assessed by Shanghai
Biotechnology Corp, China. KCNMA1 variants were examined in children
with febrile seizures by whole-exome sequencing. This study was
approved by the Institutional Review Board at Children’s Hospital of
Fudan University, Shanghai (National Children’s Medical Center, Fudan
University). And informed consent was given by the parent or guardian.
Site-Directed Mutagenesis of KCNMA1 (hBKα) Plasmids
The plasmids containing hSloα ([72]U23767) and β4 (KCNMB4; AF 160967.1)
were gifts from J.D. Lippiat (Leeds university) ([73]Lippiat et al.,
2003). Three individual point-mutations (E155Q, R458T, and E884K) were
constructed. Each amino-acid substitution was introduced into the hSloα
plasmid using a Hieff Mut™ Site-Directed Mutagenesis Kit (11004ES10,
Yeasen, Shanghai, China) according to the manufacturer’s protocol.
Site-directed mutagenesis was performed with the following primers [P1:
5′-CCAGCCGACCTGGGCGGCCACTG-3′, P2:
5′-CAGTGGCCGCCCAGGTCGGCTGG-3′ (E155Q); P1:
5′-CCACCTGAGTAAAATGTGTTTTGAACAGAGCTTCAAGCTCCAG-3′, P2:
5′-CTGGAGCTTGAAGCTCTGTTCAAAACACATTTTACTCAGGTGG-3′
(R458T); P1: 5′-GACGCCAAGATGCATTTCTTGTCCTGCAGCGAA-3′, P2:
5′-TTCGCTGCAGGACAAGAAATGCATCTTGGCGTC-3′ (E884K)]. All mutant
constructs were verified by sequencing (GENEWIZ, Jiangsu, China).
Cell Culture and Transfection
All experiments were performed on HEK 293T cell lines. HEK 293T cells
were obtained from Shanghai cell bank of Chinese Academy of Science.
The cells were both cultured in Dulbecco’s modified Eagle medium (DMEM;
Life Technologies, Grand Island, NY) supplemented with 10%
heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY).
Culture dishes were incubated at 37°C in a humidified atmosphere
containing 5% CO[2], and subcultured approximately every 2–3 days. One
day before transfection, HEK 293T cells were transferred to 24 well
plates. At 90% confluence, cells were transiently transfected using
Lipofectamine-3000 (Invitrogen, United States) at a ratio of 2 µl
reagent with 1 µg total plasmid per well. Electrophysiological
recordings from fluorescent cells were made 48 h after transfection.
Electrophysiological Recordings
Whole-cell voltage-clamp experiments were performed following the
procedures described previously ([74]Hamill et al., 1981), using an
EPC-9 amplifier (HEKA Eletronik, Germany) at room temperature
(21–25°C). Patch pipettes were fabricated from glass capillary tubes by
PC-10 Puller (Narishige, Japan) with the resistance of 2–3 MΩ. Data
acquisition and stimulation protocols were controlled by a Pentium III
computer (Legend, Beijing, China) equipped with Pulse/PulseFit 8.3
software (HEKA Eletronik, Germany). Capacitance transients were
cancelled. Cells with a seal resistance (Rseal) below 1 GΩ were
omitted. Series resistance (Rs) was compensated (80–85%) to minimize
voltage errors, and cells with an uncompensated Rs above 10 MΩ were
omitted. Leak subtraction was performed using P/6 protocol. Data were
low-passed at 10 kHz. Unless stated specially, for HEK 293T cells, the
holding potential was −80 mV. BK channel currents were elicited by the
step pulses ranging from −100 to +150 mV for 200 ms with the increments
of 10 mV. The holding potentials were held at −80 mV for BK channel.
Current density calculation formula (pA/pF), where pA represents the
current of BK channel and pF represents the membrane area of measured
cell.
Solutions
In the patch-clamp recordings, the standard bath solution for HEK 293T
cells was consisted of the following components (in mM): NaCl 135, KCl
5, MgCl2·6H2O 1, CaCl2 1.8, HEPES 10, glucose 10 (pH 7.4 with NaOH).
Pipette solutions for HEK 293T cells were composed of the following
components (in mM): NaCl 10, KCl 117, MgSO4 4, HEPES 10, EGTA 1 (pH 7.2
with KOH). The total Ca^2+ to be added to give the desired free
concentration was calculated using the program WEBMAXC STANDARD
([75]https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/web
maxc/webmaxcS.htm).
Animals and Genotyping
The BK knockout (BK KO, kcnma1 ^−/−) mice were established by breeding
BK^+/− males and females. These breeding pairs provided wild-type
(kcnma1 ^+/+), heterozygous (kcnma1 ^+/−) and BK KO (kcnma1 ^−/−)
littermates ([76]Wang et al., 2019), and three-month-old (10–12 weeks)
male BK KO mice were subjected to subsequent experiments, such as gait
analysis, morris water maze, field potential recordings, etc. The
kcnma1 knockout was generated by a frameshift mutation (− 16bp) in exon
4. Genotyping was performed with the following primers: P1:
5′-CTTCCTGCTTGTCCTTCCTC-3′, P2: 5′-CATTGCTTCAAACCCTTCCT-3′,
and the PCR products were directly sequenced. In this study, all
animals were randomly fed and watered in SPF standard animal facilities
and were fed and watered in 21°C, 50% humidity, 12 h light: under the
schedule of 12 h dark, five animals were raised in each cage, and all
the animal work was carried out under the moral permission of the
ethics review group of Shanghai University of traditional Chinese
Medicine.
FP Recordings
Four BK KO and WT male mice were implanted with electrodes for the
field potential (FP) recordings. At the age of 3 months (10–12 weeks),
the animals were first anaesthetized by pentobarbital sodium [in a dose
of 40 mg/kg through intraperitoneal injection (i.p.)]. Then, they were
put in a stereotaxic frame (Narishige, Tokyo, Japan). Their heads were
shaved and sterilized with povidone-iodine. In order to expose the
sagittal, coronal, and lambdoid sutures, cut the scalp along the
center. Subsequently, the recording electrode was also inserted into
the lateral dorsal hippocampus (AP −2.0 mm posterior to bregma, V
1.5 mm ventral to the dura surface, and L 1.5 mm lateral to the skull
midline). The reference wire is located in the electrode bundle, and
the grounding electrode is placed at the front of the skull. The wound
surface was sealed with dental cement. The electrode was fixed
simultaneously. Data were collected only for animals with accurately
positioned electrode. The FP recorded after the mice were awake. The FP
signals together with synchronized video could be recorded with the
OmniPlex (Plexon, United States). The mount of the head was linked to a
preamplifier which is tied with the analog-digital converter box.
According to Nesquet’s sampling theory, take 1 Hz as the sampling
frequency of local FP recording and set 50 Hz high-pass filter and
300 Hz low-pass filter to record for more than 30 min continuously. The
results of local FP recording were exported as the *.pl2 file format,
and offline sorter v4 software was used for visualization preview.
Local FP analysis selected the same channel through MATLAB (MathWorks,
United States) program to export data. The wavelet transform is used to
decompose the signal of different frequencies of local FP and get the
physiological rhythm of different frequencies (δ: from 0 to 4 Hz, θ:
from 4 to 8 Hz, α: from 8 to 13 Hz: β: from 13 to 30 Hz, and γ: from 30
to 100 Hz). The Welch method, hamming window, and fast Fourier
transform method were used to calculate the frequency domain
information of the local FP in power spectrum analysis. The time domain
of energy change is calculated by weighted operation. The PSD
calculations follow the formula given below.
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P=lim
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mi>t)dt=12
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Behavioral Observation of Epilepsy
In order to eliminate the possibility of the human error, behavioral
observations are double-blind during experiments. The Racine’s
five-point scale ([77]Racine, 1972), improved by [78]Fathollahi et al.
(1997), was employed to classify the seizure-like behavior at different
stages. Stage 0 is termed no response; stage 1 is called facial and ear
twitching; stage 2 is myoclonic jerks without an upright position;
stage 3 represents myoclonic jerks and upright position with bilateral
forelimb clonus; stage 4 stands for clonic-tonic seizure; stage 5 is
named generalized clonic-tonic seizures and loss of postural control.
The severity of seizures in BK KO mice was evaluated according to the
grade and time of seizures. The least interval between two countable
seizures was set as 5 s during the quantification of all seizure
numbers.
Immunofluorescence Staining
Frozen sections were permeabilized with 0.5% Triton X-100, and blocked
for 1 h at room temperature (RT) with 5% bovine serum albumin. Without
washing, sections were incubated overnight at 4°C with primary
antibodies. These include against LC3B (1:200 dilution; ab48394;
Abcam), LAMP1(1:500 dilution; ab25630; Abcam), and Iba-1 (1:500
dilution; ab178846; Abcam). washing four times in PBS (4 × 5 min),
sections were incubated with goat anti-rabbit antibody conjugated with
Alexa Fluor 594 (for LC3B; 1:200 dilution; ab150080; Abcam); goat
polyclonal secondary antibody to mouse conjugated with Alexa Fluor 488
(for LAMP1; 1:200 dilution; ab150113; Abcam) and Alexa Fluor
350-labeled goat anti-rabbit IgG (H + L) (for Iba-1; 1:500 dilution;
A0408; Beyotime) for 1 h at RT, washing four times in PBS (4 × 5 min).
Slices were cover-slipped with 50% glycerin. Fluorescence images were
captured using a Virtual/Digital Slice Microscope (Olympus, Tokyo,
Japan). Quantification was performed using ImageJ software.
Calcium Imaging
HEK293T cells, co-transfected with pcDNA3-TRPML1-GCaMP3, gifted from
Prof. Xu HX (University of Michigan, United States), were kept in HBSS
(Hank’s balanced salt solution) at RT for 30 min. Confocal imaging was
performed by using a Zeiss confocal LSM 880, a laser scanning
microscope system (Zeiss, Germany). GCaMP3 was evoked with a laser
wavelength at 488 nm, and the fluorescence images were collected with
the resolution of 512 × 512 pixels. The ROI (regions of interest; 3 × 3
pixels) were selected in individual HEK293T cells by Zeiss LSM Image
Browser (Zeiss, Germany) to track the changes in the ratio of
fluorescence intensity. The ratio (F/F0) of fluorescence intensity was
calculated by dividing fluorescence intensity at time t (F) with the
beginning fluorescence intensity (F0) of the experiment.
Y-Maze Test
Y‐maze test was performed as previously described ([79]Watanabe et al.,
2011). Exploratory activity was measured using a Y‐maze apparatus (arm
length: 40 cm, arm bottom width: 10 cm, arm upper width: 10 cm, height
of wall: 12 cm). The floor and the wall of the maze is made of black
PVC plastic. Each subject was placed in the arm Ⅰ of the Y‐maze field.
The alteration (%) and total distance (m) was recorded using a modified
version of the image software. Data were collected for a period of
10 min.
Gait Analysis
We analyzed gait of the mice during walk/trot locomotion by ventral
plane videography as described ([80]Hampton et al., 2004; [81]Koshimizu
et al., 2014), using digigait imaging system (Mouse Specifics Inc.).
This system enables mice to walk on a motorized transparent treadmill
belt, and the software automatically identifies the stance and swing
components of stride and calculates stride length, print area, mean
intensity, and swing speed. Briefly, we placed the mice on a treadmill
belt that moves at constant speed. We collected digital video images of
mice.
Morris Water Maze Test
A dark blue pool (diameter: 120 cm, depth: 50 cm) was placed in a quiet
room and filled with white-colored water. The water was equilibrated to
room temperature (between 22 and 23°C) before the MWM test. Colored
papers with a variety of different shapes were posted around the pool
as visual cues. A platform of 10 cm in diameter was used. For hidden
platform trials, the platform was positioned 1.5 cm below water
surface. The MWM test was performed in the period of 11AM—3PM to
minimize circadian effects. The BK KO and control mice were tested in
same days, and testing sequences for individual mice were altered in
each test day. A protocol with 5 days of visible platform trials and
1 day of hidden platform trials were employed. In the trials,
individual mice underwent four trials per day, and the maximal time for
each trial was 60 s. If mice did not find the platform within 60 s,
they were guided to the platform by the experimenter’s hand and allowed
to stay on the platform for 60 s. For the probe tests in which the
platform was removed from the pool, individual mice underwent a trial
of 60 s in each quadrant. If mice exhibited convulsions shortly before
or during a trial, they were allowed to recover for 20–30 min before
next trial. Any trial interfered with convulsions were excluded from
analysis. Frequency and the time in aim-quadrant during the probe
trials were analyzed. Group data for the BK KO and WT mice were
compared.
Open Filed Test
The open field behavior experiment device is a very effective device
for measuring spontaneous and exploratory behaviors to measure the
degree of anxiety in mice. Place the animal in an unknown environment
with walls around it, and the rodents will spontaneously tend to move
on the edges rather than the open center of the area. In this
experiment, the device is composed of a black polystyrene box (50 × 50
× 50 cm), which is divided into two areas: the outer square (periphery)
and the inner square (center). Each mouse was placed in the center of
the box and explored freely for 10 min. Observe their behavior through
the animal video monitoring with the behavior software, and measure the
stay time (s), the total distance of movement in the central area (m),
the average speed (mm/s) through the computer tracking system, evaluate
spontaneous and exploratory behavior.
Tissue Sample Collections
Three different BK KO and WT adult mice were randomly sampled,
sacrificed, and their cortices and hippocampi extracted under 2-min,
frozen, and stored at − 80°C.
RNA Extraction
Total RNA was extracted from the mouse cortex/hippocampus using TRIzol
Reagent (15596018, Invitrogen) according to the manufacturer’s
protocol. Using the a nanodrop (Thermo Scientific NanoDrop 2000c
Spectrophotometer), the RNA concentration of each sample was determined
by measuring the absorbance at 260 nm (A260), and its purity was
determined by the ratio of the absorbance measured at 260 nm (A260) and
280 nm (A280). The ratios of A260/A280 between 1.9 and 2.1 were
considered acceptable.
Library Preparation for Transcriptome Sequencing
A total amount of 2 μg RNA per sample was used as input material for
the RNA sample preparations. Sequence libraries were generated using
NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, United States)
following manufacturer’s recommendations and index codes were added to
attribute sequences in each sample. Briefly, mRNA was purified from
total RNA using poly-T oligo-attached magnetic beads. Fragmentation was
carried out using divalent cations under elevated temperature in
NEBNext First Strand Synthesis Reaction Buffer (5×). First strand cDNA
was synthesized using random hexamer primer and M-MuLV Reverse
transcriptase (RNase H). Second strand cDNA synthesis was subsequently
performed using DNA Polymerase I and RNase H. Remaining overhangs was
converted into blunt ends to exonuclease/polymerase activities. After
adenylate of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin
loop structure was ligated to prepare for hybridization. In order to
select cDNA fragments of preferentially 200–250 bp in length, the
library fragments were purified with AMPure XP system (Beckman Coulter,
Beverly, United States). Then 3 μl USER Enzyme (NEB, United States) was
used with size-selected, adaptor-ligated cDNA at 37°C for 15 min
followed by 5 min at 95°C before PCR. Then PCR was performed with
Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index
(X) Primer. At last, PCR products were purified (AMPure XP system) and
library quality was assessed in the Agilent Bioanalyzer 2,100 system.
Clustering and Sequencing
The clustering of the index-coded samples was performed on a cBot
Cluster Generation System using TruSeq PE Cluster Kit v4-cBot-HS
(Illumia) according to the manufacturer’s instructions. After cluster
generation, the library preparations were sequenced on an Illumina
Hiseq 4,000 platform and paired-end 150 bp reads were generated.
Differential Expression Genes Analysis
Differential expression analysis of two conditions/groups was performed
using the DESeq R package (1.10.1). DESeq2 provides statistical
routines for determining differential expression of digital gene
expression data using a model based on the negative binomial
distribution. The resulting p values were adjusted using the Benjamini
and Hochberg’s approach to controlling the false discovery rate. Genes
with pvalue <0.05 and |log2FC| > 0.58 found by DESeq were assigned as
differentially expressed.
GO Enrichment Analysis
Gene Ontology (GO) enrichment analysis of the DEGs was implemented by
the GOseq R packages based Wallenius non-central hyper-geometric
distribution, which can adjust for gene length bias in DEGs.
KEGG Pathway Enrichment Analysis
KEGG is a database resource for understanding high-level functions and
utilities of the biological system, such as the cell, the organism and
the ecosystem, from molecular level information, especially large-scale
molecular datasets generated by genome sequence and other
high-throughput experimental technologies
([82]http//:www.genome.Jp/kegg/) ([83]Jiang et al., 2018). We used
KOBAS ([84]Mao et al., 2005) software to test the statistical
enrichment of differential expression genes in KEGG pathways.
PCR Primer Design and Testing
We first looked through NCBI’s Primer-Bank
([85]https://pga.mgh.harvard.edu/primerbank/) to find PCR primers for
selected genes. Primers were checked for specificity using NCBI’s
Primer-BLAST program
([86]https://www.ncbi.nlm.nih.gov/tools/primer-blast/) against RefSeq
RNA to ensure no non-specific matches. When designing primers that
could match multiple transcript variants, their sequences were aligned
in ClustalW ([87]http://www.genome.jp/tools/clustalw/), and only the
primers that amplified a consensus region were used. All PCR primers
were purchased from GENEWIZ (Jiangsu, China) and listed in
[88]Supplementary Table S3 with sizes of the resulting PCR products in
base pairs (bp).
Real-Time RT-PCR Validation of Selected DEGs
To validate the transcriptome gene expression data, six DEGs identified
by transcriptome were validated by real-time RT-PCR using QuantityNova
SYBR Green PCR Kit (208052, Qiagen, Valencia, CA). RT- PCR was
performed in a one-step RT-PCR process according to the protocol on
Roche480II using 30 ng RNA. Housekeeping gene β-actin was used as
endogenous control. RNA was first reverse transcribed into cDNA at 65°C
for 5 min. After enzyme activation at 95°C for 2 min, PCR was carried
out at 95°C for 5 s and 60°C for 10 s for 45 cycles. For RT-PCR
analysis, each sample was run in triplicates. Comparative Ct method
(delta Ct method) was used to calculate the fold differences between BK
KO and WT groups.
Statistical Analysis
Data were analyzed with Origin 8.5 (OriginLab, United States), Excel
2016 (Microsoft, WA) and Prism 6 (GraphPad software, San Diego, CA).
Data are presented as the mean ± standard error of the mean (SEM).
Student’s t-test or one-way ANOVA was used to assess the statistical
significance of differences. When p < 0.05, differences were accepted
as significant.
Results
Identification of a de novo Variant in KCNMA1 (hbkα) From a Patient With
Febrile Seizures
In this study, among the 26 clinical patients, two patients had a
history of head trauma, two patients had a history of asphyxia and
oxygen inhalation, two patients had premature delivery and one patient
had severe pneumonia, which might be the inducement of epilepsy. In
addition, eleven patients had abnormal EEG, five patients had abnormal
MRI, and two patients had a family history of epilepsy ([89]Table 1).
TABLE 1.
Clinical data of 26 patients with one LOF variants in BK channels.
Patient ID genomic variant 2 p. E155Q 13 NA 4 p. E471G 20 c. 344A > G
Gene KCNMA1 SCN1A UPF3B Panel ASXL3
De novo variant Yes NA NA NA
Gender M M F F
Age 6 6 17 3
Course (years) 5 5 8 2
Age of seizure onset (years) 1 1 9 1
Epilepsy details Tonic-clonic limbs, unconsciousness, eyes rolled up
and staring, no answer Tonic-clonic limbs, unconsciousness, foaming at
the mouth, lasting Often onset in sleep and early morning, accompanied
by disturbances in consciousness and urinary incontinence Series of
spasmodic seizures
2–10min
Anticonvulsant treatment VPA VPA, LEV, CLB, Ketogenic diet OXC, VPA VPA
EEG features Sharp wave, sharp slow wave and spike slow wave. Rhythmic
energy intensity of δ and θ increased Sharp wave and sharp slow wave A
few sharp waves, sharp slow waves and spike slow waves Peak rhythm
disorder with intermittent phenomenon
MRI NA Normal Normal Brain dysplasia, left brain atrophy
Family history of epilepsy No No No Yes
Other details Premature delivery 2/26
History of head trauma 2/26
History of severe pneumonia 1/26
History of suffocation and oxygen 2/26
Developmental delay 3/26
EEG abnormality 11/26
Abnormal MRI 5/26
Family history of epilepsy 2/26
[90]Open in a new tab
F, female; M, male; NA, data not available; VPA, valproate acid; LEV,
levetiracetam; CLB, clobazam; OXC, oxcarbazepine.
A novel variant c.463G > C [p.(E155Q)] in a patient with febrile
seizures was identified. The patient, male, full-term born on August
26, 2015, bw3500 g, had a history of oligohydramnios and intrauterine
distress, and no family history of epilepsy. He had several histories
of febrile convulsions, which was characterized by tetanic clonus of
limbs, unconsciousness, upturned eyes, unable to call, hyperactivity,
repeated daze, poor language development and developmental delay. There
was no abnormality in blood tandem mass spectrometry, and the energy of
urine tandem mass spectrometry was disordered. EEG showed that there
were some sharp waves, and spike waves on both sides of the brain. We
predicted that the location of amino-acid E155 was in the domain S1/S2
linker of BK α ([91]Figure 1A). A previous study already reported that
R458T and E884K mutants were located in the domain S6/RCK1 cytoplasmic
linker and the RCK2 domain of BKα ([92]Figure 1A). Therefore, we
constructed three plasmids: KCNMA1-E155Q, KCNMA1-R458T, and
KCNMA1-E884K ([93]Figure 1B).
FIGURE 1.
FIGURE 1
[94]Open in a new tab
Location and sequencing of the KCNMA1 variants. (A) Predicted
transmembrane topology of KCNMA1 depicting the location of the
variants. (B) DNA sequencing identified the mutations in the
constructed hBKα plasmid. The mutation sites are marked by a red
square.
Electrophysiological Characteristics of BK Channel LOF Variant E155Q
E155Q variant is highly conserved among different species during
evolution ([95]Figure 2A). To determine whether the c.463 G > C [p.
(E155Q)] variant had an effect on BK channel function, we expressed
mutant E155Q channel and control wild-type (WT) channel, recorded
potassium currents, and analyzed the current voltage relationship. The
results showed that in 1 and 10 μM free Ca^2+ concentration, the macro
currents amplitude of E155Q mutant was always smaller than that of WT
([96]Figure 2B). Moreover, we found that the I-V curve of the mutant
E155Q moved in the direction of positive potential ([97]Figures 2C,D).
Regardless of whether the free calcium concentration was 1 μM (p < 0.05
at 70 mv, p < 0.01 at 80–90 mv, p < 0.001 at 100–150 mv) or 10 μM (p <
0.05 at 70 mv, p < 0.01 at 80 mv −100 mv, p < 0.001 at 110 mv −150 mv),
the E155Q mutant significantly reduced the current density of BK when
the stimulation voltage reached 70–150 mv ([98]Figure 2E, n =
6–14/group). Thus, the E155Q mutant is identified as a LOF variant.
FIGURE 2.
[99]FIGURE 2
[100]Open in a new tab
Electrophysiological characterization of variant E155Q. (A) The E155Q
variant occur at an evolutionarily conserved amino acid residues. (B)
Representative macroscopic currents of WT and mutant BK channels with
variant E155Q from whole-cell patch experiments in the presence of 1
and 10 μm Ca^2+. (C) The I-V curves of WT and E155Q mutant BK channels
are shown at 1 and 10 μm Ca^2+. The I-V curves are fitted by Boltzmann
function (solid lines) with V1/2 and slope factor at nominal at 1 μm
Ca^2+ [97.5 ± 4.1 mV, 21.4 ± 2.1 WT, and 113.4 ± 6.4 mV, 33.2 ± 3.3
p.(E155Q)] and at 10 μm Ca^2+ [30.4 ± 2.4 mV, 15.9 ± 0.8 WT, and 34.2 ±
2.9 mV, 14.6 ± 1.6 p.(E155Q)]. (D) Scatter plots of voltage at
half-maximal activation (V1/2) for WT and variants. (E) The current
density of WT and E155Q mutant BK channels are shown at 1 and 10 μm
Ca^2+. The data are presented as mean ± SEM. (Compared with WT-1um, *p
< 0.05, **p < 0.01, ***p < 0.001. Compared with WT-10um, #p < 0.05, ##p
< 0.01, ###p < 0.001. n = 6–14/group).
Effect of the β4 Subunit on the BK Channel Variant E155Q
The β4 subunit is an auxiliary subunit specifically expressed by
neurons, dominantly expressed in brain. To determine whether the β4
subunit and the E155Q mutation interact, we co-transformed the β4
subunit and the E155Q mutant in HEK 293T cells. Compared with E155Q
mutant, the β4 subunit had no effect on the macro current density
amplitude, I-V curve, or V1/2 ([101]Supplementary Figures S1A–D).
Further analysis of the activation time constant (τ) revealed that τ of
the E155Q+ β4 mutant was significantly greater than that of the E155Q
mutant ([102]Supplementary Figure S1E, n = 6–9/group), and thus the β4
subunit slowed down the activation of the E155Q mutant.
Electrophysiological Characteristics of BK Channel LOF Variant R458T
R458T variant is highly conserved among different species during
evolution ([103]Figure 3A). We used the HEK 293T cell system to express
and compare WT and mutant R458T channels. The results showed that R458T
mutant significantly reduced the macro currents and current density
amplitude of BK channel ([104]Figures 3B,E). The I-V curve of R458T
mutant shifted to the positive voltage direction ([105]Figures 3C,D).
Noticed, the smaller Ca^2+-induced leftward shift of the I–V in the
mutant suggests that its apparent Ca^2+ sensitivity is less than that
of the WT, further exacerbating the LOF phenotype.
FIGURE 3.
[106]FIGURE 3
[107]Open in a new tab
Electrophysiological characterization of variant R458T. (A) The R458T
variant occur at an evolutionarily conserved amino acid residues. (B)
Representative macroscopic currents of WT and mutant BK channels with
variant R458T from whole-cell patch experiments in the presence of 1
and 10 μm Ca^2+. (C) The I-V curves of WT and R458T mutant BK channels
are shown at 1 and 10 μm Ca^2+. The I-V curves are fitted by Boltzmann
function (solid lines) with V1/2 and slope factor at nominal at 1 μm
Ca^2+ [97.5 ± 4.1 mV, 21.4 ± 2.1 WT, and 111.9 ± 6.3 mV, 27.5 ± 2.4
p.(R458T)] and at 10 μm Ca^2+ [30.4 ± 2.4 mV, 15.9 ± 0.8 WT, and 48.2 ±
2.6 mV, 13.7 ± 1.1 p.(R458T)]. (D) Scatter plots of voltage at
half-maximal activation (V1/2) for WT and variants. (E) The current
density of WT and R458T mutant BK channels are shown at 1 and 10 μm
Ca^2+. The data are presented as mean ± SEM. (Compared with WT-1um, *p
< 0.05, **p < 0.01, ***p < 0.001. Compared with WT-10um, #p < 0.05, ##p
< 0.01, ###p < 0.001. n = 6–14/group).
E884K Variant in the RCK2 Domain Markedly Reduced the Amplitude of the BK
Currents
E884K variant is highly conserved among different species during
evolution ([108]Figure 4A). The E884K variant significantly reduced the
amplitude of the BK currents ([109]Figure 4B). Moreover, the I-V curve
of E884K variant shifted to the positive voltage direction similar to
E155Q and R458T ([110]Figures 4C,D). Regardless of whether the free
calcium concentration was 1 μM (p < 0.01 at 50 mv, p < 0.001 at
60–150 mv) or 10 μM (p < 0.05 at 50–60 mv, p < 0.01 at 70–130 mv, p <
0.001 at 140–150 mv) the E884K mutant markedly reduced the current
density of BK when the stimulation voltage reached 50–150 mv
([111]Figure 4E). Therefore, the E884K variant is also considered as a
LOF variant.
FIGURE 4.
[112]FIGURE 4
[113]Open in a new tab
Electrophysiological characterization of variant E884K. (A) The E884K
variant occur at an evolutionarily conserved amino acid residues. (B)
Representative macroscopic currents of WT and mutant BK channels with
variant E884K from whole-cell patch experiments in the presence of 1
and 10 μm Ca^2+. (C) The I-V curves of WT and E884K mutant BK channels
are shown at 1 and 10 μm Ca^2+. The I-V curves are fitted by Boltzmann
function (solid lines) with V1/2 and slope factor at nominal at 1 μm
Ca^2+ [97.5 ± 4.1 mV, 21.4 ± 2.1 WT, and 112.0 ± 6.7 mV, 34.8 ± 4.2
p.(E884K)] and at 10 μm Ca^2+ [30.4 ± 2.4 mV, 15.9 ± 0.8 WT, and 39.3 ±
2.2 mV, 19.3 ± 1.4 p.(E884K)]. (D) Scatter plots of voltage at
half-maximal activation (V1/2) for WT and variants. (E) The current
density of WT and E884K mutant BK channels are shown at 1 and 10 μm
Ca^2+. The data are presented as mean ± SEM. (Compared with WT-1um, *p
< 0.05, **p < 0.01, ***p < 0.001. Compared with WT-10um, #p < 0.05, ##p
< 0.01, ###p < 0.001. n = 6–14/group).
BK Channel Knockout Mediates Epilepsy
BK channel knockout (BK KO, kcnma1 ^−/−) mice were generated by the
deletion of exon four of kcnma1 (gene encoding the α subunit of BK,
BKα) using the CRISPR/Cas9 strategy ([114]Figure 5A) ([115]Wang et al.,
2019). The BK KO mice, which carried a 16 bp fragment deletion in exon
4, was identified by PCR ([116]Figure 5B) and confirmed by sequencing
([117]Figure 5C). By observing movie ([118]Supplementary Movies S1,
S2), it is more intuitive to show that BK KO mice have an epileptic
phenotype. Through continuous recording for 2 h, it was found that BK
KO mice showed nearly 25% of grade 4–5 convulsive seizures ([119]Figure
5D, n = 3). In addition, the seizure time and grade of BK KO mice were
significantly greater than those of WT mice ([120]Figure 5D, p < 0.001,
n = 3). Thus, we found that BK KO mice have spontaneous epileptic
symptoms, mainly manifested as generalized tonic clonic seizures and
absence seizures ([121]Figure 5D), which corresponds to the epileptic
phenotype of human BK channel functional inactivation gene mutation (BK
channel frameshift mutation) ([122]Tabarki et al., 2016).
FIGURE 5.
[123]FIGURE 5
[124]Open in a new tab
Construction of BK KO mice with spontaneous epilepsy. (A) Schematic
outlining the generation of BK knockout mice using the CRISPR/Cas9
system. The targeting sites of KCNMA1 (gene encoding the α subunit of
BK, BKα) are shown. (B,C) BK KO mice were established by breeding
BK^+/− males and females. The targeted fragment of KCNMA1 was amplified
by PCR using genomic DNA templates, and the BK channel deletion was
confirmed by sequencing. Genome sequencing of BK KO mice showed a
frameshift mutation (- 16 bp) in exon 4. (D) Spontaneous epileptic
behavior of BK KO mice. The proportion of different seizure stages in
BK KO/WT mice was observed for 2 h (***p < 0.001, n = 3).
FP Characteristics of BK KO Mice
Then, to determine differences of BK KO and control mice in EEG levels,
the power spectral density (PSD) of BK KO and control mice on was
directly measured. We compared the field potential (FP) signals of BK
KO and control mice, and differences in FP activity were visualized via
using heat maps of spectral density generated by OmniPlex software
(Plexon, United States). Compared with the control mice, BK KO mice had
no significant differences on the FP activity ([125]Figure 6A). The
results showed that the EEG of WT mice presented basic waves with low
frequency and amplitude, and there were no abnormal epileptic waves.
The EEG of BK KO mice showed the basic wave with lower frequency and
amplitude, accompanied by a small number of spike-waves ([126]Figure
6B). The energy intensity values of five common rhythms collected by
EEG were counted, δ Wave (0.5–4 Hz) belongs to a slow wave, which is
the main rhythm in the sleep state of mice. Compared with control
group, the δ rhythmic energy intensity of BK KO mice decreased
significantly ([127]Figure 6C, p < 0.001, n = 4). θ wave (4–7 Hz) and δ
wave is similar to the rhythm that appears during sleep. θ rhythmic
energy intensity in BK KO mice was lower than that in control group
([128]Figure 6C, p < 0.001, n = 4). α wave (8–13 Hz) is the normal
brain wave of mice. Compared with control group, α rhythmic energy
intensity of BK KO mice increased significantly ([129]Figure 6C, p <
0.001, n = 4). β wave (15–30 Hz) is the main rhythm when the brain is
excited. Compared with control group, β rhythmic energy intensity in BK
KO mice increased significantly ([130]Figure 6C, p < 0.001, n = 4). γ
wave (>30 Hz) belongs to a fast wave that occurs during rapid eye
movement sleep and γ rhythmic energy intensity in BK KO mice decreased
significantly ([131]Figure 6C, p < 0.01, n = 4). Moreover, the PSD of
total frequency waves of BK KO mice was obviously smaller than that of
the control group ([132]Figure 6D). Thus, the slow-wave power was
reduced, normal and excited wave power was enhanced in BK KO mice.
FIGURE 6.
[133]FIGURE 6
[134]Open in a new tab
In vivo multichannel EEG recording of mice. (A,B) FP signals and
spectral heat maps from a representative WT (black) and BK KO (red)
mice are shown, respectively. (C) Spectral analysis of PSD values on
different frequency δ, θ, α, β, and γ waves in each group. (D) The PSD
of mice in each group (Compared with control group, *p < 0.05, **p <
0.01, ***p < 0.001, n = 4).
Motor Impairment in BK KO Mice
Using the catwalk gait analysis system, a number of gait abnormalities
were identified. By observing movie ([135]Supplementary Movies S3, S4),
it can be intuitively found that BK KO mice have abnormal gait and
shorter stride phenotype ([136]Figures 7A–C). The print area (RF, RH,
LF, LH) of BK KO mice was narrower than that of control mice
([137]Figure 7D, p < 0.001, n = 5). Moreover, the mean intensity (RF,
LF) of BK KO mice less than that of control mice ([138]Figure 7D, p <
0.01, n = 5). Swing speed and stride length were smaller, with an
unsteady gait pattern in the BK KO mice, compared to the gait of
control mice ([139]Figure 7D, n = 5). Results revealed BK KO mice
presented motor impairment.
FIGURE 7.
[140]FIGURE 7
[141]Open in a new tab
Gait analysis was performed by DigiGait imaging system. (A–C) Schematic
diagram of WT and BK KO mouse footprints. (D) Print area (cm^2), mean
intensity and Swing speed (cm/s) of the right front (RF), the right
hind (RH), the left front (LF), the left hind (LH) limb were chosen as
the observation index. The Data are presented as means ± SEM. (Compared
with control group, *p < 0.05, **p < 0.01, ***p < 0.001, n = 5).
In addition, the total distance ([142]Supplementary Figure S2A, p <
0.001, n = 8), average speed ([143]Supplementary Figure S2C, p < 0.001,
n = 8) of BK KO mice significantly reduced in the open field, and the
total distance ([144]Supplementary Figure S2G, p < 0.001, n = 8) of BK
KO mice markedly decreased in the Y-maze test. Thus, BK KO mice also
showed motor impairment in the Y-maze and open field test. What’ more,
BK KO mice showed smaller time in the central area ([145]Supplementary
Figure S2B, p < 0.001, n = 8), frequency and aim-quadrant stay time
([146]Supplementary Figures S2D,E, p < 0.001, p < 0.05, n = 6), and the
percentage of spontaneous alternation ([147]Supplementary Figure S2F, p
< 0.001, n = 8) as compared with the WT mice. Although motor impairment
suggested that it might have an impact on anxiety and cognitive
impairment of BK KO mice, there were also literatures supporting that
BK KO have the cognitive impairment phenotype without the interference
factor of locomotion ([148]Sausbier et al., 2004). This strongly
suggested that BK KO mice might be accompanied by anxiety and cognitive
impairment in addition to the phenotype of motor impairment.
Transcriptome Sequencing and Analysis of Hippocampus and Cortex of BK KO Mice
In this study, we used RNA-Seq to analyze the transcriptome of BK KO/WT
mice hippocampus and cortex, and performed transcriptome profiling to
characterize the differentially expressed genes. A total of 652 genes
were screened with the threshold of significance at p < 0.05 and
|log2foldchange| > 0.58, among which 382 genes were down-regulated and
270 genes were up-regulated in hippocampus tissues. In cortex tissues,
we detected a total of 561 differentially expressed genes with the
threshold of significance at p < 0.05 and |log2foldchange| > 0.58,
including 162 up-regulated genes and 399 down-regulated genes
([149]Figure 8A).
FIGURE 8.
[150]FIGURE 8
[151]Open in a new tab
Transcriptome profiling in the hippocampus and cortex tissues of mice.
(A) Heatmap of the DEGs. (B) Enriched Biological Process pathway,
cellular component and molecular function in GO analysis (p < 0.05).
(C) KEGG enrichment analysis of DEGs. The intensity of the color
depends on the p value. The size of plot depends on the gene count. (n
= 3).
In order to better understand the potential functions of differentially
expressed genes, Gene Ontology (GO) enrichment analysis was carried out
to assess the involved pathways. Biological process pathway in GO
analysis results showed that development process and biological
adhesion were enriched ([152]Figure 8B). The results of kyoto
encyclopedia of genes and genomes (KEGG) showed that insulin secretion,
axon guidance, p53 signaling pathway, HIF-1 signaling pathway and
calcium signaling pathway were enriched in hippocampus ([153]Figure
8C). On the other hand, cortical KEGG results showed that cell adhesion
molecules (CAMs), ECM-receptor interaction, phagosome and calcium
signaling pathway were enriched ([154]Figure 8C).
Genes Were Verified
The transcriptomic results were compared with NCBI genebank
([155]https://www.ncbi.nlm.nih.gov/gene/?term=), and it was found that
DEGs in [156]Supplementary Tables S1, S2 were closely related to
epilepsy, astrocyte activation, neuroinflammation and microglia
autophagy. Through RT-PCR, we used three groups of the cortex and
hippocampus between WT and BK KO mice to verify the bold eight genes in
the [157]Supplementary Tables S1, S2. (Foldchange was larger in the
same group of genes). Gfap and Cdkn1a gene highly expressed in BK KO
mice ([158]Figure 9, p < 0.01, n = 3). There were three genes that were
lowly expressed in BK KO mice, Grm3, Alpl and Nr4a1 ([159]Figure 9, p <
0.05, n = 3). In all, the results of RT-PCR and transcriptomics were
highly consistent. We continued to explore the possible mechanisms of
epilepsy through transcriptome.
FIGURE 9.
FIGURE 9
[160]Open in a new tab
Differential expression of mRNAs between the cortex and hippocampus of
WT (Black) and BK KO (Red) mice validated by RT-PCR. Gfap and Grm3
associated with astrocyte activation, Alpl, and Nlrp10 associated with
neuroinflammation, Efna5 and Reln associated with epilepsy, Cdkn1a and
Nr4a1 associated with autophagy. Gfap, Grm3, Nlrp10, Alpl in cortex,
and Efna5, Reln, Cdkn1a, Nr4a1 in hippocampus. Ns (no significant
difference, p > 0.05), ∗p < 0.05, ∗∗p < 0.01.
Abnormal Autophagy in BK KO Mice
Autophagy is a normal catabolic process in cells. Various types of
biological macromolecules undergo degradation and circulation through
lysosomal digestion to maintain cell homeostasis. Improving the
neuroinflammation response in the pathogenesis of multiple sclerosis
(MS) can be achieved by enhancing autophagy ([161]Liang and Le, 2015;
[162]Feng et al., 2017). This suggests that neuroinflammation and
autophagy can influence each other, which in turn affects the
progression of CNS-related diseases.
Transcriptomics results showed that there were 11 differentially
expressed genes (DEGs) related to microglia autophagy in hippocampus
and 5 DEGs related to microglia autophagy in cortical tissues
([163]Supplementary Table S2). In order to further explore the role of
autophagy in epilepsy, immunofluorescence experiments were performed on
the hippocampus of WT and BK KO mice. In the hippocampal CA3 region of
Ctrl group mice, autophagosome marker LC3B, lysosome marker LAMP1 and
microglia marker IBA-1 were co-labeled, indicating that the interaction
and fusion of autophagosome and lysosome in hippocampal microglia was
normal ([164]Figure 10A), but in the hippocampus CA3 region of BK KO
mice, LC3B, LAMP1 and IBA-1 were partially not co-labeled, indicating
abnormal interaction and fusion of autophagosomes and lysosomes in
microglia of BK KO mice ([165]Figure 10B). During epilepsy, there may
be abnormal interaction and fusion between autophagosomes and lysosomes
in hippocampal microglia. TRPML1 (key calcium channel of autophagy)
promotes the fusion of autophagosomes and lysosomes, and the lysosomal
calcium release of TRPML1 is closely related to autophagy ([166]Scotto
Rosato et al., 2019; [167]Zhang et al., 2019). BK channel and
TRPML1-GCaMP3 were co-expressed in HEK293T. The opener NS1619 of BK
channel could induce the calcium outflow of lysosomes. Paxilline (PAX),
a specific inhibitor of BK channel, can significantly inhibit the
lysosomal calcium outflow ([168]Figure 10C). In general, the activation
of BK channel could activate lysosomal TRPML1. It suggested that BK
might regulate the autophagy pathway from TRPML1.
FIGURE 10.
[169]FIGURE 10
[170]Open in a new tab
Autophagy in BK KO mice was abnormal. Calcium imaging found that the
activation of BK channel could activate lysosomal trpml1 (autophagy key
calcium channel). (A) Co labeling of microglia, LC3B, IBA-1, and LAMP1
in control mice. (B) Co labeling of microglia, LC3B, IBA-1, and LAMP1
in BK KO mice. (C) NS1619 was applied to HEK293T transfected with BK
channel and TRPML1-GCaMP3 in order to detect its regulation of
lysosomal calcium outflow (^∗∗ p < 0.01, n = 6).
Discussion
In this study, we identified three KCNMA1-LOF mutants (E155Q, R458T,
E884K), of which E155Q was a de novo mutant. All three variants showed
profound effects on BK channel function, and played its role through
LOF mechanism (BK channel current decreased and I-V curve shifted to
the positive voltage direction). Faster BK current activation directly
increases neuronal firing rate by causing faster repolarization of
action potentials ([171]Jaffe et al., 2011; [172]Contet et al., 2016),
which might be the cause of BK-GOF mediated epilepsy. The causes of
BK-LOF mediated epilepsy include the inhibition of repolarization of
action potential, resulting the increase of neuronal excitability, and
the role of neuroimmune inflammation. What’s more, hβ4 had no effect on
the I-V curve and current amplitude density of E155Q mutant, but
activation time constant (τ) of E155Q+β4 channel was greater than that
of E155Q mutant ([173]Supplementary Figure S1).
Compared with wild-type littermates, kcnma1 ^−/− mice lost weight,
interestingly, so did the kcnma1 ^+/− mice. Susan T Halm et al.
speculated that the lack of kcnma1 allele leads to insufficient grip in
mice, which may limited the cubs’ access to nutrition ([174]Halm et
al., 2017). It was worth noting that the fertility of adult BK KO mice
decreased and failed without exception in the process of 15 mating
(Transcriptomics results suggested that Adam18, Cabyr and other genes
related to sperm function were abnormal, and Eqtn regulated the
abnormality of sperm and egg plasma membrane fusion). In addition, the
results of KEGG ([175]Figure 8C) showed that insulin secretion was
enriched, which provided possible evidence for the imbalance of body
weight and fat in BK KO mice ([176]Halm et al., 2017). Disturbance of
insulin release may damage health and cause signals to convert energy
for growth into fat storage. Of note, the malnutrition and
developmental delay caused by the weak grip of BK KO mice are
reminiscent of the developmental delay found in the patient with the
KCNMA1-LOF (E155Q) variant.
Although the epileptic phenotypes of BK KO mice were similar with those
of clinical patients, such as developmental delay and interictal
epileptiform discharge (IED), the FP characteristics of BK KO mice were
different from those of clinical patients. Specifically, the intensity
of δ and θ energy rhythms in BK KO mice reduced, while rhythmic energy
intensity of δ and θ increased in the patient carrying the E155Q
mutation site. We guess that it is mainly caused by the difference in
detection method and detection area. In clinical testing, non-invasive
EEG recording is mainly used to detect the membrane potential of
neurons in the cortex, and we use in vivo multichannel
electrophysiological recording to detect the local field potential of
the hippocampus in animals. In addition, abnormal background activity
amplitude may also affect IED. Christine M. Muheim et al. pointed out
that the delta slow wave power of BK KO mice was reduced in cortex
(<4 Hz) ([177]Muheim et al., 2019). In our experiment, in addition to
the decrease of delta slow wave power, the power of θ wave (4–7 Hz) and
γ wave (>30 Hz) also decreased. Of note, the power of α wave and β wave
increased, which may be the cause of spontaneous epilepsy in BK KO
mice. In the motor cortex, beta waves are mainly involved in grasping,
muscle contraction and maintaining attention ([178]Khanna and Carmena,
2015). The abnormal up regulation of β wave may lead to abnormal
excitation of neurons, excessive increase of motor control ability, and
then lead to motor dysfunction.
In addition, we found that BK KO mice have a dyskinetic phenotype
through a series of behavioral experiments. And BK KO mice showed
smaller time in the central area ([179]Supplementary Figure S2B, p <
0.001, n = 8), frequency and aim-quadrant stay time ([180]Supplementary
Figures S2D,E, p < 0.001, p < 0.05, n = 6), and the percentage of
spontaneous alternation ([181]Supplementary Figure S2F, p < 0.001, n =
8). The frequency of seizures and the duration of the disease have a
negative impact on cognitive impairment ([182]Jokeit and Ebner, 1999;
[183]Allone et al., 2017). Although, motor impairment might have an
impact on anxiety and cognitive impairment of BK KO mice, one study
cleverly excluded interference of motor, proving that BK KO mice have
the cognitive impairment phenotype ([184]Sausbier et al., 2004). This
strongly suggested that lacking kcnma1 genes (BK KO) may cause anxiety
and cognitive impairment in BK KO mice, which required further study.
We further explored the possible molecular mechanisms of
BK-LOF-mediated epilepsy through transcriptomics in the hippocampus and
cortex. Gene Ontology (GO) analysis showed that DEGs were related to
protein labeling, protein binding transcription factor activity,
development process and biological adhesion ([185]Figure 8B). The kyoto
encyclopedia of genes and genomes (KEGG) showed these DEGs were mainly
enriched in insulin secretion, axon guidance, p53 signaling pathway,
HIF-1 signaling pathway, calcium signaling pathway, cell adhesion
molecules (CAMs), ECM-receptor interaction, and phagosome ([186]Figure
8C). In the process of epilepsy, including the changes of gene
expression, neuroinflammation, protein production and connection, these
may be the targets of inhibiting epilepsy. The results of KEGG showed
that Cdkn1a is closely related to the HIF-1 signaling pathway, and
dysregulated HIF-1 signaling may play a role in the pathogenesis of
epilepsy in hippocampus ([187]Merelli et al., 2018). A large number of
glial cells activate and proliferate, glutamate and the secretion of
inflammatory factors increases, which reduces the convulsion threshold
and increases the excitability of brain neurons, and accelerates
spontaneous recurrent convulsions ([188]Friedman and Dingledine, 2011;
[189]Huberfeld et al., 2015). Epilepsy like activity in vitro and
prolonged seizures in vivo lead to increased p53 accumulation and
transcriptional activity ([190]Sakhi et al., 1994; [191]Liu et al.,
1999; [192]Tan et al., 2002; [193]Araki et al., 2004). Abnormal axon
guidance may induce mossy fiber germination, and the “wrong” guidance
of mossy fiber may be a necessary process of dentate nerve circuit
homeostasis under the condition of epilepsy ([194]Koyama and Ikegaya,
2018). Meanwhile, the mammalian target of rapamycin (mTOR) is inhibited
by rapamycin to prevent mossy fiber sprouting and reduce seizures in
rodent models of acquired epilepsy ([195]Zeng et al., 2009). Cell
adhesion molecules (CAMs) may form trans synaptic complexes that are
essential to correctly identify synaptic partners and further for
determine the establishment and dynamics of synapses ([196]Gorlewicz
and Kaczmarek, 2018). Dysfunction of transsynaptic adhesion is
associated with epilepsy ([197]Gorlewicz and Kaczmarek, 2018). This
strongly suggested that the DEGs find in transcriptomics, particularly
those related to astrocyte activation, neuroinflammation and autophagy,
may be the molecular mechanism of BK-LOF mediated epilepsy.
In summary, we identified and functionally characterized three
different LOF variants in the BK channel (E155Q, R458T, E884K), of
which E155Q variant was a de novo mutant and affected one patient. All
the above variants caused a positive shift of the I-V curve and played
a role through the loss-of-function (LOF) mechanism. Moreover, the β4
subunit slowed down the activation of the E155Q mutant. BK KO mice had
spontaneous epilepsy, motor impairment, autophagic dysfunction,
abnormal electroencephalogram (EEG) signals, as well as possible
anxiety and cognitive impairment. In addition, BK might regulate the
autophagy pathway from TRPML1. Dysregulation of gene expression,
especially astrocyte activation, neuroinflammation and autophagy, might
be the molecular mechanism of BK-LOF meditated epilepsy.
Data Availability Statement
The data presented in the study are deposited in the GEO repository,
accession number [198]GSE191038.
Ethics Statement
The studies involving human participants were reviewed and approved by
the Children’s Hospital of Fudan University, Shanghai (National
Children’s Medical Center, Fudan University). Written informed consent
to participate in this study was provided by the participants’ legal
guardian/next of kin. The animal study was reviewed and approved by the
Ethics Committee of Shanghai University of traditional Chinese
Medicine. Written informed consent was obtained from the individual(s),
and minor(s)’ legal guardian/next of kin, for the publication of any
potentially identifiable images or data included in this article.
Author Contributions
ZL, JT, YY, and YJ designed the research. YY and XJ performed the data
analysis work. YY, DQ, LZ, YZ, and XC conducted experiments. YY and QZ
wrote and revised the manuscript. YY, LZ, and XC drew the figures. JT
was responsible for the statistical analyses. YY, DQ, and XJ are
co-first author. All authors read and approved the final manuscript.
Funding
This work was supported by the grants from National Key Research and
Development Program (2020YFA0803800), National Natural Science
Foundation of China (Nos. 31771191, 82074162, 81903995, 81973385, and
81874325), Medical Guidance Projects (Traditional Chinese Medicine) of
Shanghai Municipal Science and Technology Commission (No. 21Y11921700),
Young Elite Scientists Sponsorship Program by CACM (No.
CACM-2019-QNRC2-C10), Shanghai Municipal Commission of Health and
Family Planning Fund (Nos. 20184Y0086 and 2018JQ003), Project for
Capacity Promotion of Putuo District Clinical Special Disease
(2019tszb02), Science and Technology Innovation Project of Putuo
District Health System (Nos. ptkwws201902, ptkwws201908, and
ptkwws202107).
Conflict of Interest
The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.
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Supplementary Material
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References