Twik-1 KO mice produce an internal region-deleted TWIK-1 mutant protein
Twik-1 KO mice, first described in 2005, were generated by deleting exon 2 of the Twik-1 gene.8 Exon 2 of the Twik-1 gene encodes the pore domain, the transmembrane domain (TM)2, and TM3 of the channel. In Twik-1 KO mice, the deletion of exon 2 (396 nucleotides) of the Twik-1 gene was confirmed through PCR using their genomic DNA (Supplementary Fig. 1A). In addition, we confirmed the expression of exon 2-deleted Twik-1 (Twik-1 ΔEx2) mRNA using RT-PCR and sequencing analysis, in Twik-1 KO mice (Supplementary Fig. 1B). Twik-1 ΔEx2 mRNA results from the direct linkage of exon 1 to exon 3 without a frameshift mutation.8 Exon 2 of the Twik-1 gene corresponds to the amino acid residues, 119 − 250 of the TWIK-1 channel protein. Therefore, it is possible that the Twik-1 ΔEx2 mRNA does not produce a premature termination codon; this results in exon 2 being skipped while the reading frame is retained, potentially producing an abnormal protein (an internally deleted protein) rather than undergoing nonsense-mediated decay. However, it is not confirmed whether Twik-1 ΔEx2 mRNA can be used for protein expression in Twik-1 KO mice.
We firstly verified that the expression of the C-terminus of the TWIK-1 channel, which is encoded by exon 3, is not deleted in Twik-1 KO mice. We detected a TWIK-1 protein band in Twik-1 KO mice with a size approximately 14.87 kDa smaller than that in the wild-type (WT) mice through western blotting (Supplementary Fig. 1C). We also confirmed the exon 2-deleted Twik-1 KO mice showed no difference in astrocytic passive conductance in accordance with earlier reports (Supplementary Figs. 1D and E).13 These data raised the possibility that Twik-1 KO mice unintentionally expressed an internal region-deleted TWIK-1 mutant protein (TWIK-1 ΔEx2 protein), which might be act as a functional channel. To test this possibility, we developed a new C-terminus-specific antibody against the mouse TWIK-1 channel (Supplementary Figs. 1F and G). Immunohistochemistry using the antibody indicated strong immunohistochemical signals against TWIK-1 protein in the various brain regions of Twik-1 KO mice; these include the hippocampus, cerebellum, striatum, and neocortex (Figs. 1A and B). In addition, we observed immunohistochemical signals against the TWIK-1 protein in the hippocampal neurons and astrocytes of Twik-1 KO mice (Fig. 1C). Taken together, these data strongly suggest that the TWIK-1 ΔEx2 protein in Twik-1 KO mice, like the WT TWIK-1 channel protein, is highly expressed in the various previously reported brain regions and cell types.
TWIK-1 ΔEx2 protein mediates potassium current in the primary cultured astrocytes from Twik-1 KO mice
Most potassium channels are composed of tetramers because four pore domains are required to make a functional channel. However, in the case of K2P channels, one protein with two pore domains can constitute a functional channel as a dimer.14, 15 Twik-1 KO mice expressed the protein in a form lacking the second and third transmembrane domains and one pore domain of the TWIK-1 channel, which is encoded by exon 2 (schematic diagram of the channel shown in Fig. 1A). Therefore, it is possible that the TWIK-1 ΔEx2 protein could function as one subunit if it has one remaining pore domain. In particular, the 119th glycine and 120th tyrosine residues, which are part of the GYG motif also known as a potassium selectivity filter, are still present in the TWIK-1 ΔEx2 protein,16 suggesting that it could mediate K+ current.
To confirm this possibility, electrophysiological experiments were performed using specific shRNAs against each exon of Twik-1 gene. Exon-specific shRNAs targeting exon 2 (sh-Ex2) and exon 3 (sh-Ex3) of the Twik-1 gene were constructed and validated (Figs. 2A and B). The expression of TWIK-1 proteins was reduced in both sh-Ex2 and sh-Ex3 treatments in primary cultured astrocytes from WT mice, while that was not affected in the control scrambled shRNA (Sc shRNA). However, the expression of TWIK-1 proteins was only reduced in the sh-Ex3 treatment in the primary cultured astrocytes from Twik-1 KO mice. We also measured K+ currents in the presence of Twik-1 shRNAs through electrophysiological experiments. Under Twik-1 knockdown conditions, K+ currents, both inward and outward, were significantly reduced by sh-Ex2 and sh-Ex3, in the primary cultured astrocytes from WT mice (Figs. 2C and D). In the primary cultured astrocytes from Twik-1 KO mice, there was no effect on K+ currents in the sh-Ex2 treatment; however, sh-Ex3 effectively reduced both inward and outward K+ currents (Figs. 2E and F).
To exclude the possibility of off-target effects by shRNA,17 we conducted a similar experiment using the CRISPR/Cas9 system to target each exon of the Twik-1 gene. The efficiency of the Twik-1 targeting guide RNA was confirmed through immunocytochemistry in primary cultured astrocytes from both WT and Twik-1 KO mice (Supplementary Fig. 2). Using electrophysiological experiments, we confirmed that the K+ currents were significantly reduced by all CRISPR/Cas9 vectors targeting each exon of the Twik-1 gene in the astrocytes cultured from WT mice, while the currents was not affected by control the control guide RNA (Figs. 2G and H). However, targeting only exon 2 did not result in any changes in the K+ current in the astrocytes cultured from Twik-1 KO mice (Figs. 2I and J). Therefore, our data strongly suggested that the TWIK-1 ΔEx2 protein can act as a functional channel for mediating astrocytic K+ currents in Twik-1 KO mice.
TWIK-1 ΔEx2 protein mediates astrocytic passive conductance in hippocampal slices of Twik-1 KO mice
Next, we assessed whether knockdown of TWIK-1 using AAV-expressing TWIK-1 Ex3 shRNA affected the astrocytic passive conductance of Twik-1 KO mice in vivo. We stereotaxically injected AAVs carrying either scrambled or Ex3 shRNA into the hippocampal stratum radial region of WT and Twik-1 KO mice, respectively. After 2 weeks, brains were harvested and stained with antibodies against TWIK-1, mCherry, and the astrocytic marker GFAP (Fig. 3A and Supplementary Fig. 3A). Astrocytic TWIK-1 immunoreactivity was significantly reduced when the mice were injected with AAV-mChe-TWIK-1 Ex3 shRNA compared to that in those injected with AAV-mChe-Sc shRNA, in both WT and Twik-1 KO groups (Fig. 3B and Supplementary Fig. 3B). In addition, densitometric analysis of the TWIK-1 staining intensity in AAV-mChe Sc or TWIK-1 Ex3 shRNA treated astrocytes (mChe + and GFAP + cells) showed no difference between WT and Twik-1 KO mice (Supplementary Fig. 3B). We also confirmed the effect of TWIK-1 Ex3 shRNA on astrocytic passive conductance in the hippocampal slices of WT and Twik-1 KO mice. TWIK-1 Ex3 shRNA significantly reduced astrocytic passive conductance in both WT and Twik-1 KO mice (Fig. 3C). The outward currents at + 20 mV and inward currents at −140 mV decreased by about 50% in both WT and Twik-1 KO mice (Figs. 3D and E). Input resistance at −140 mV was significantly increased in AAV-mChe-TWIK-1 Ex3 shRNA-injected mice compared to that in control Sc shRNA-injected mice, in both WT and Twik-1 KO groups (Fig. 3F). Taken together, the TWIK-1 ΔEx2 protein mediates an astrocytic passive conductance, similar to the full-length TWIK-1 protein, even in vivo.
New Twik-1 KO mice with exon 1 targeting exhibit deficiency of TWIK-1 protein in vivo
Since the exon2 deleted Twik-1 KO mice express TWIK-1 ΔEx2 protein, which can perform functions similar to that of full-length TWIK-1, we concluded that the exon2 deleted Twik-1 KO mice would not be suitable for the functional studies of TWIK-1 channel. This led us to generate a new strain of Twik-1 KO (nKO) mouse targeting the protein coding sequence (CDS) region of exon 1 using the CRISPR/Cas-9 system. Targeting with dual single-guided RNAs (sgRNA) results in a higher frequency of indels than using single sgRNAs;18 therefore, we designed two sgRNAs targeting the CDS region of exon 1 and the contiguous intron region (Fig. 4A). We obtained a heterozygous KO founder (F1) and confirmed that the target site was effectively deleted in the genomic DNA obtained from the tail of Twik-1 nKO mice. The homozygous KO mice (F2) were generated from an intercross between F1 heterozygous KO mice and confirmed through PCR genotyping. Primers were designed to amplify a 788 bp or a 442 bp fragment from WT or Twik-1 nKO mice, respectively (Fig. 4B).
Next, we confirmed that in situ hybridization analysis using the deleted part of exon 1 as a probe did not detect any signals in the brains of Twik-1 nKO mice (Fig. 4C). Twik-1 mRNA band was not detected in the RT-PCR analysis using primers targeting the deleted region of exon 1 and exon 2 (Fig. 4D). Consequently, sequence analysis of the mRNA showed that the deletion of the rear part of exon 1 and partial insertion of intron in CDS region and frame shift occurs in the Twik-1 mRNA of Twik-1 nKO mice, leading to early termination (Supplementary Figs. 4A and B). We also verified the expression of TWIK-1 in Twik-1 nKO mice using the specific antibody against the C-terminus of TWIK-1 (Supplementary Figs. 1F and G). Immunohistochemistry analysis showed no TWIK-1 signal in the whole brain, including the hippocampus and cerebellum in the brain slice of Twik-1 nKO mice compared to that in the brain slice from the littermate controls (Fig. 4E). This tendency was also observed in hippocampal neurons and astrocytes (Fig. 4F). These data show that Twik-1 nKO mice express a TWIK-1 protein prematurely terminated in the N-terminal region, which does not contain the pore domain required for forming the potassium ion channels.
The newly developed Twik-1 KO mice show reduction in astrocytic passive conductance and K+ buffering activity
Astrocytes contribute to K+ buffering against extracellular K+ increases in the brain.19 The passive conductance of astrocytes is important in the K+ buffering.5, 20, 21 Therefore, we investigated the passive conductance of hippocampal astrocytes in response to changes in external K+ concentration (from 3.5 to 7 mM and 15 mM) in the brain slices from WT and Twik-1 nKO mice. K+ currents were increased when the [K+]o changed from 3.5 to 7 mM and 15 mM in the hippocampal astrocytes of WT mice. However, the K+ currents in hippocampal astrocytes of Twik-1 nKO were not altered with the change in [K+]o (Fig. 5A). The I-V relationship of astrocytic passive conductance was always linear regardless of the changes in the extracellular K+ concentration in both WT and Twik-1 nKO mice (Fig. 5B). Comparing the I-V curves to K+ currents in WT mice, both outward and inward currents in Twik-1 nKO were dramatically reduced (Fig. 5C and D). These data clearly showed that the hippocampal astrocytes from Twik-1 nKO mice displayed significant reduction in passive conductance and K+ buffering.
The dysregulation in astrocytic potassium buffering in response to extracellular K+ concentration is involved in epileptic seizure.22 The potassium ion channels in neuron are also important in determining susceptibility to epileptic seizures.23, 24, 25 In addition, we recently reported that Twik-1 mRNA expression is increased in astrocytes and neurons in the hippocampus by kainic acid (KA) injection 26. Therefore, it is possible that TWIK-1 may act in the epileptic seizures. To test this possibility, we observed their seizure behavior following the intraperitoneal injection of 35 mg/kg KA in both the WT and Twik-1 nKO mice (Fig. 5E). We scored the behavioral seizure responses using the modified Racine scale for 90 min following KA injection. Twik-1 nKO mice showed a higher seizure score throughout the observation period compared to WT mice (Fig. 5F). In addition, the latency to score 3 was significantly shorter, and the cumulative score of seizure events was higher in Twik-1 nKO mice compared to that in WT mice (Figs. 5G and H). These data clearly showed that Twik-1 nKO mice are more susceptible to KA-induced seizures.
The deficiency of astrocytic TWIK-1 and the resulting dysfunction of passive conductance accelerate the onset of seizures
To elucidate the role of TWIK-1 mediated astrocyte passive conductance, we examine the sensitivity to seizures could be regulated by astrocytic TWIK-1 alone. To specifically deplete TWIK-1 in the astrocytes, we constructed AAV-R-CREon TWIK-1 shRNA27 and stereotaxically injected this virus into the stratum radiatum of the hippocampal CA1 region with the astrocyte-specific GFAP promoter-driven CRE virus27 (Fig. 6A). After 2 weeks, immunohistochemistry confirmed TWIK-1 depletion in astrocytes (Figs. 6B and C). We confirmed the AAV-R-CREon system selectively expressed TWIK-1 shRNA and fluorescent proteins in astrocytes. Next, we measured the passive conductance in these astrocytes and found that it was significantly reduced (Fig. 6D). The graph of mean I-V relationship showed that the hippocampal astrocytes of mice infected with AAV-R-CREon TWIK-1 shRNA/AAV-GFAPp-BFP-Cre showed approximately 45% less passive conductance in both the outward and inward currents (Fig. 6E and F). In addition, their input resistance was considerably increased compared to that in the hippocampal astrocytes of control AAV-R-CREon TWIK-1 shRNA/AAV-GFAPp-BFP-infected mice (Fig. 6G).
Next, we used this AAV system to selectively knockdown TWIK-1 in the hippocampal astrocytes and monitor KA-induced seizure behavior (Fig. 6H). The behavioral seizure responses during the 90 min observation indicated that AAV-R-CREon TWIK-1 shRNA/AAV-GFAPp-BFP-Cre-infected mice rapidly processed seizure onset compared to control AAV-R-CREon TWIK-1 shRNA/AAV-GFAPp-BFP-treated mice (Fig. 6I). The time to reach score 3 also decreased in AAV-R-CREon TWIK-1 shRNA/AAV-GFAPp-BFP-Cre-infected mice (Fig. 6J). However, there was no change in the cumulative score (Fig. 6K). We also evaluated the neuronal damage in the hippocampus using Fluoro-jade B (FJB) staining.28 The positive signals for FJB were significantly increased in the CA1 of the hippocampus after KA injection in AAV-R-CREon TWIK-1 shRNA/AAV-GFAPp-BFP mice (Figs. 6L and M). Taken together, deficiency of astrocytic TWIK-1 in hippocampus significantly reduces astrocytic passive conductance and accelerates the onset of KA-induced seizures.