Analyses of epithelial Na+ channel variants reveal that an extracellular β-ball domain critically regulates ENaC gating

Epithelial Na+ channel (ENaC)-mediated Na+ transport has a key role in the regulation of extracellular fluid volume, blood pressure, and extracellular [K+]. Among the thousands of human ENaC variants, only a few exist whose functional consequences have been experimentally tested. Here, we used the Xenopus oocyte expression system to investigate the functional roles of four nonsynonymous human ENaC variants located within the β7-strand and its adjacent loop of the α-subunit extracellular β-ball domain. αR350Wβγ and αG355Rβγ channels exhibited 2.5- and 1.8-fold greater amiloride-sensitive currents than WT αβγ human ENaCs, respectively, whereas αV351Aβγ channels conducted significantly less current than WT. Currents in αH354Rβγ-expressing oocytes were similar to those expressing WT. Surface expression levels of three mutants (αR350Wβγ, αV351Aβγ, and αG355Rβγ) were similar to that of WT. However, three mutant channels (αR350Wβγ, αH354Rβγ, and αG355Rβγ) exhibited a reduced Na+ self-inhibition response. Open probability of αR350Wβγ was significantly greater than that of WT. Moreover, other Arg-350 variants, including αR350G, αR350L, and αR350Q, also had significantly increased channel activity. A direct comparison of αR350W and two previously reported gain–of–function variants revealed that αR350W increases ENaC activity similarly to αW493R, but to a much greater degree than does αC479R. Our results indicate that αR350W along with αR350G, αR350L, and αR350Q, and αG355R are novel gain–of–function variants that function as gating modifiers. The location of these multiple functional variants suggests that the αENaC β-ball domain portion that interfaces with the palm domain of βENaC critically regulates ENaC gating.

The epithelial Na ϩ channel (ENaC) 2 is a member of the ENaC/degenerin family of nonvoltage-gated ion channels.
ENaCs are expressed in the apical plasma membranes of specific epithelia and, in parallel with the basolateral Na ϩ ,K ϩ -AT-Pase, mediate the absorption of Na ϩ from the lumen of the aldosterone-sensitive distal nephron (ASDN), the distal colon, and the airway and alveolae. ENaC-mediated Na ϩ absorption plays significant roles in the regulation of extracellular fluid volume and blood pressure and fluid volume in airways and alveolae (1)(2)(3)(4). ENaC-mediated Na ϩ absorption is also tightly linked to K ϩ secretion in the ASDN, and changes in extracellular [K ϩ ] influence activity of the Na ϩ -Cl Ϫ cotransporter in the distal convoluted tubule and blood pressure (5,6). Inherited forms of human hypertension or hypotension are largely associated mutations in specific genes that encode either renal tubular Na ϩ transporters or their regulators (7,8). Other than well-defined Liddle syndrome mutations that disrupt or result in a loss of a Pro-Tyr (PY) motif in the C terminus of the ␤or ␥-subunit, correlating nonsynonymous ENaC variants that alter channel activity with predicted changes in blood pressure in humans has been challenging. This may reflect that fact that only two of the common ENaC nonsynonymous variants alter ENaC function in heterologous expression systems, and these have not been clearly linked to changes in blood pressure in humans (9 -14). Other functional ENaC nonsynonymous variants that we and others have described are rare or of low-frequency (15)(16)(17)(18)(19). It is difficult to show that these rare functional human ENaC variants affect blood pressure in epidemiological studies, and the effects of specific ENaC variants on blood pressure in animal models have not yet been addressed. Another barrier is that the vast majority of ENaC variants, including synonymous and nonsynonymous ones, have no defined functional roles.
The resolved crystal structure of an acid-sensing ion channel 1 (ASIC1), a member of the ENaC/degenerin family, has provided important insights regarding the highly-organized structure of the extracellular domains of ENaC-subunits and has recently been confirmed by a resolved structure of ␣␤␥ENaC (20,21). A central core is formed by multiple ␤-strands that form the ␤-ball (␤2, ␤4, ␤5, ␤7, and ␤8) and palm domains. The ␤-ball domain contributes to an acidic pocket in ASIC1 that has a role in fine-tuning acid activation of the channel (20). Its func-tional role in ENaC has not been clearly defined. A recently resolved cryo-EM structure of the extracellular domain of human ENaC revealed that it is strikingly similar to ASIC1, with a few notable differences. The ␣and ␥-subunits have an embedded inhibitory track in their finger domains, and the region encompassing this track is formed by ␤-strands and is not present in ASICs (21). We identified several nonsynonymous variants in the ␤7-strand and its following loop of the human ␣-subunit that is part of the ␤-ball (␣R350W, rs181065138; ␣V351A, rs139861603; ␣H354R, rs753035419; and ␣G355R, rs189376498). We found that all these variants except ␣H354R alter human ENaC activity when expressed in Xenopus oocytes. Among these variants, ␣R350W exhibited the most robust effect on enhancing channel activity and open probability (P o ).

Functional variants do not alter levels of ENaC surface expression
We examined whether the differences in whole-cell currents we observed with the gain-of-function variants (␣R350W and ␣G355R) or loss-of-function variant (␣V351A) in oocytes reflected changes in numbers of channels at the plasma membrane. Levels of surface expression of WT and mutant human ENaCs in oocytes were determined using a chemiluminescence assay, using a human ␤-subunit construct with an extracellular epitope FLAG tag (17). As shown in Fig. 3, oocytes expressing either WT or mutant ENaCs had similar levels of surface expression.
ENaC trafficking and expression may vary in different cells, and the three subunits may traffic to the cell surface in a noncoordinate fashion (24). To confirm our observations in oocytes, we examined ENaC surface expression in a mammalian cell line (Fisher rat thyroid, FRT cells) by co-transfection of nontagged human ␤and ␥ENaCs, and WT or mutant human ␣ENaC with N-terminal HA tag and C-terminal V5 tag (25). Biotin-labeled surface proteins were purified and immunoblotted with anti-HA antibody. ENaC-subunit maturation in the biosynthetic pathway involves furin cleavages of both ␣and ␥-subunit (26 -28). As shown in Fig. 4, all three mutants (␣R350W, ␣V351A, and ␣G355R) and WT showed similar surface levels of both full-length (90 kDa) and cleaved (22 kDa) All four residues are shown as sticks with carbons in cyan, oxygen in red, and nitrogen in blue. C, sequence alignments of ENaC/degenerin members. Alignments were performed using Vector NTI 11 (Thermo Fisher Scientific). Only sequences of the ␤7-strand and its following residues are shown. Amino acid numbers of the first residue in all sequences are shown in parentheses. Four residues where variants of this study reside are shown in red letters.
ENaC ␤-ball domain forms of ␣ENaC (Fig. 4, A, C, E, and G). All four groups also had similar levels of expression of the full-length and cleaved forms of ␣ENaC in whole-cell lysates (Fig. 4, B, D, F, and G).
These results suggest that the increases in whole-cell currents seen with ␣R350W␤␥ and ␣G355R␤␥, as well as the reduction in current seen with ␣V351A␤␥, compared with WT, likely reflected a change in channel open probability and/or single channel conductance.

Gain-of-function variants ␣R350W and ␣G355R suppress the Na ؉ self-inhibition response
In addition to transporting Na ϩ , ENaC open probability is suppressed by extracellular Na ϩ , a process referred to as Na ϩ self-inhibition (4,29,30). We examined whether the increase in current seen with the ␣R350W variant reflected a loss of the inhibitory effect of extracellular Na ϩ . A typical Na ϩ self-inhibition current trace recorded in oocytes expressing WT ENaC is shown in Fig. 5A. An increase in bath [Na ϩ ] from 1 to 100 mM was associated with a rapid increase in inward Na ϩ current reaching a peak value (I peak ), followed by a slower reduction in inward Na ϩ current that reflects Na ϩ self-inhibition, with the current reaching a steady state (I ss ). We used the ratio of I ss to I peak as measure of the magnitude of the Na ϩ self-inhibition response. Oocytes expressing ␣R350W␤␥ had a blunted Na ϩ self-inhibition response, with an I ss /I peak of 0.86 Ϯ 0.04 (n ϭ 19, p Ͻ 0.0001 versus 0.52 Ϯ 0.05, n ϭ 20 for WT, Fig. 5, A and B).

ENaC ␤-ball domain
Another gain-of-function ␣G355R variant also reduced Na ϩ self-inhibition response (Fig. 5, G and H). These results suggest that the increases in current seen with ␣R350W␤␥ and ␣G355R␤␥ reflect an increase in ENaC open probability. Unexpectedly, the loss-of-function ␣V351A variant did not show an enhanced Na ϩ self-inhibition (Fig. 5, C and D). The "silent" ␣H354R variant modestly, but significantly, reduced Na ϩ selfinhibition (Fig. 5, E and F).

␣R350W increases ENaC open probability
If a mutation is considered as a gating modifier, it should cause a change in channel open probability in a predicted manner. We used a cell-attached path-clamp technique to determine the open probability of WT ␣␤␥, ␣R350W␤␥, and ␣V351A␤␥ human ENaCs in oocytes. The open probability of ␣R350W␤␥ channels was 0.35 Ϯ 0.12 (n ϭ 11), significantly greater than that of WT (0.23 Ϯ 0.09, p Ͻ 0.05, n ϭ 10; Fig. 6, A and E). In contrast, the open probability of ␣V351A␤␥ channels was 0.19 Ϯ 0.13, similar to that of WT (p Ͼ 0.05, n ϭ 7; Fig. 6, A and E). Both NP o and N (number of channels within patches) in ␣R350W␤␥-expressing oocytes were moderately greater than in WT-expressing cells (Fig. 6, C and D). Single channel conductances measured with 110 mM LiCl in patch pipettes were similar between WT, ␣R350W␤␥, and ␣V351A␤␥ (Fig. 6B).

Comparison of gain-of-function variants located in the ␣-subunit
Besides the well-known gain-of-function mutations in ␤and ␥ENaCs that cause Liddle syndrome, emerging evidence indicates that gain-of-function mutations/variants in the ␣-subunit present novel causes of salt-related clinical disorders (15,16,31). The point mutation ␣C479R was reported in a family with Liddle syndrome (31). In addition, ␣W493R variant was found in a group of patients with a cystic fibrosis phenotype and with either a single mutant cystic fibrosis transmembrane conductance regulator (CFTR) allele or no CFTR mutation (15,16). We compared ␣R350W, ␣C479R, and ␣W493R for functional changes in ENaC activity, normalized to WT. Relative amiloride-sensitive currents of ␣R350W␤␥ (3.0 Ϯ 1.5, n ϭ 49) were similar to that of ␣W493R␤␥ (3.2 Ϯ 1.9, n ϭ 44, p Ͼ 0.05), and both were significantly greater than that of both ␣C479R␤␥ (1.4 Ϯ 0.7, n ϭ 46, p Ͻ 0.0001) and WT (1.0 Ϯ 0.4, n ϭ 43, p Ͻ 0.0001, Fig. 8A). As shown in Fig. 8B, Na ϩ self-inhibition response was dramatically reduced in all three variants (n ϭ 17-19, p Ͻ 0.0001 versus WT). The increases in I ss /I peak seen with ␣W493R were modestly greater than that seen with ␣R350W, and both were significantly greater than that observed with ␣C479R.

DISCUSSION
The increasing number of sequenced human genomes has been accompanied with the identification of an increasing number of human ENaC missense variants. We and others have found human ENaC variants located in the extracellular domains of ENaC subunits that affect channel activity when expressed in oocytes, primarily though changes in open probability that are associated with changes in Na ϩ self-inhibition (16 -19). Although functional human ENaC variants have been described in the thumb, finger, knuckle, and palm domains, the only functional variant in the ␤-ball previously described was a  ENaC ␤-ball domain loss-of-function variant associated with pseudohypoaldosteronism type 1 (␣C133Y (7, 32)). We found that the human ENaC variants ␣R350W, ␣V351A, ␣G355R, located in ␤7 and the succeeding loop of the ␤-ball domain, significantly altered ENaC activity when expressed in oocytes. ␣R350W and ␣G355R are novel gain-of-function variants, and for ␣R350W, this gain-of-function was associated with a reduction in Na ϩ self-inhibition along with an increase in channel open probability. The changes in ENaC activity seen with ␣R350W, ␣V351A, and ␣G355R were not accompanied by a change in channel surface expression as assessed by a chemiluminescence-based assay in oocytes and a surface biotinylation assay in FRT cells, although patch-clamp analysis suggested that the channel number in patches of ␣R350W was modestly greater than WT. These differing results may reflect differences in the sensitivities of these assays to detect changes in ENaC surface expression. Although we did not perform single channel recordings of ␣G355R␤␥ channels, the increase in current and the accompanying decrease in Na ϩ self-inhibition suggest that channels with the ␣G355R variant have an increase in open probability when compared with WT (30). Although the ␣V351A variant significantly reduced ENaC currents, it did not affect surface expression level, Na ϩ self-inhibition response, nor open probability. At present, it is unclear what caused the current reduction. Figure 5. ␣R350W, ␣H354R, and ␣G355R reduce the Na ؉ self-inhibition response. A, C, E, and G, representative recordings in oocytes expressing WT or mutant ENaCs to show the Na ϩ self-inhibition response. Oocytes were clamped at Ϫ100 mV, whereas bath Na ϩ concentration was increased from 1 mM (NaCl-1, gray bar) to 110 mM (NaCl-110, black bar). Traces were superimposed with the same time and current scales. B, D, F, and H, I ss /I peak represents the magnitude of Na ϩ self-inhibition. Values were obtained from amiloridesensitive I ss and I peak . Horizontal bars are mean Ϯ S.D. Data were collected from three batches of oocytes. The p values were from Student's t tests, NS, not significant.

ENaC ␤-ball domain
We used a ␤-subunit with an extracellular epitope tag to determine surface expression of ␣␤␥ENaCs. As ␤-subunits alone do not transit to the plasma membrane at a measurable level in Xenopus oocytes (33), ␤-subunits at the surface level largely reflect ␣␤␥ channels (34 -36). We observed similar levels of surface expression of WT and mutant ENaCs in Xenopus oocytes and FRT cells, using either an epitope-tagged ␤-subunit or an epitope-tagged ␣-subunit.
The identification of multiple functional variants in the ␤-ball domain of ␣ENaC strongly suggests that this domain plays an important role in ENaC-gating regulation. The ␤-ball is a structure formed by five ␤-strands and is surrounded by the helical finger, thumb, and knuckle domains and the ␤-sheet palm domains (Fig. 1) (20, 21). In ASICs, Arg-191 in the ␤-ball interacts with protonable residues at the acidic cavity, potentially contributing to acid sensing (20). Extracellular protons in the physiological range selectively activate human ENaCs by presumably interacting with multiple ␤and ␥ENaC residues (37,38). It would be interesting to examine whether ␣Arg-350 plays any role in the pH regulation of ENaC. In MEC-4, touchdisrupting mutations were identified in the ␤-ball domain (39). However, there have been few studies examining the specific roles of residues in ENaC ␤-ball domains (32,(40)(41)(42). Mutations of a pair of Cys residues within the rat ENaC ␤-ball (first and sixth extracellular Cys forming a sulfide bridge) greatly hampered channel delivery to plasma membrane (32), suggesting that the structural integrity of the ␤-ball domains in all three-subunits is essential for efficient functional expression of ENaCs. We previously observed that mutations of the same pair of Cys residues of mouse ␣ENaC suppressed the Na ϩ self-inhibition response (40), which could reflect some degree of misfolding. Edelheit et al. (41) reported that ␣R350A significantly reduced whole-cell current that was associated with a moderately reduced surface expression. Although the magnitude of the Na ϩ self-inhibition response was not altered, the rate of the decrease in Na ϩ current in response to a rapid increase in [Na ϩ ] was enhanced (41). The homologous mutation in ␥-subunit (␥K328A) led to a reduced channel current, associated with reduced channel surface expression (42). The authors also noted a reduced Na ϩ self-inhibition response, which should increase ENaC activity. We found that four substitutions (Trp, Gly, Gln, and Leu) at ␣Arg-350 led to increased channel currents, associated with a suppressed Na ϩ self-inhibition response. Taken together, our results and previous studies suggest that the ␤-ball domains have important roles in the regulation of ENaC gating.
The increases in current seen with channels expressing the ␣R350W and ␣G355R variants were similar in magnitude to that seen with the ␣W493R variant and significantly greater that that seen with the ␣C479R variant (Figs. 2 and 8) (16, 31). The observation that the ␣C479R variant was present on one allele of a sibling pair with a Liddle syndrome phenotype (31) suggests that individuals with an ␣R350W, ␣G355R, or ␣W493R variant are at risk for hypertension presenting as Liddle syndrome.

ENaC ␤-ball domain
Low-frequency and rare variants likely influence the heritability of complex disorders (43-45). Furthermore, normal physiological processes may be modified by rare variants (45). All functional variants in this study are rare. The ␣Arg-350 variants (Trp, Gly, Gln, and Leu) have reported allele frequencies of less than 0.001 (dbSNP Build 152). ␣R350Q was reported as a de novo mutation in an individual with nonfamilial Brugada syndrome together with a KCNB2 mutation (46), and SCNN1A has been included in the current list of genes associated with Brugada syndrome (47). The ␣R350Q variant was reported in an individual with Dent disease (48), although its contribution to the disease is unclear. As sequencing efforts increase, we expect to see reports of additional associations of ENaC variants with human diseases.
The variants ␣R350W, ␣W493R, and ␣C479R suppress the Na ϩ self-inhibition response (Fig. 7) (16). All three residues are located at an intersubunit interface (Fig. 8C), highlighting the important role of intersubunit interfaces in ENaC gating (17, 21, 31, 41, 42, 49 -52). The ␣Arg-350 side chain in the resolved ENaC structure projects toward the ␤-subunit palm domain (21). Examination of the structure in the vicinity of ␣Arg-350 indicates that most ␤ENaC residues near ␣Arg-350 are polar (Fig. 8D). We speculate that ␣Arg-350 and nearby ␣-subunit polar residues form a hydrophilic patch interacting with their counterparts in the ␤-subunit palm domain, facilitating the Na ϩ self-inhibition response. Replacing ␣Arg-350 with Trp or Leu would interfere with these hydrophilic interactions between the ␣and ␤-subunits and with the Na ϩ self-inhibition response.
Based on structural information from ASIC1 (53), we previously suggested ␣Trp-520 in mouse ENaC (equivalent to human ␣Trp-493) and nearby residues in the ␣-subunit form a hydrophobic patch that facilitates interactions with neighboring structures within ENaC (54). The orientation of ␣Trp-493 in the resolved human ENaC structure (21) is consistent with this notion. Shobair et al. (55) suggested a different mechanism of ENaC activation by ␣W493R based on a heterotetrameric ␣␤␣␥ model, where W493R on one ␣-subunit interfaces with ␥Glu-348, and W493R on the other ␣-subunit interfaces with residues on the ␤-subunit. The resolved structure of ENaC does not support their model, as ENaC is an ␣␤␥ trimer (21), where ␣Trp-493 is in proximity to hydrophobic and aromatic residues, including residues in loops connecting the ␤6 and ␤7 and the ␤8 and ␤9 in the ␥-subunit. Multiple substitutions at ␣Trp-493 (Ala, Cys, or Glu) result in an ϳ2.5-fold increase in amiloride-sensitive current (16). This observation suggests that it is the loss of ␣Trp-493 interactions with neighboring residues when other amino acids are placed at this site that leads to the loss of Na ϩ self-inhibition, rather than interactions between ␣W493R and ␥Glu-348 (55).
In summary, ␣R350W, ␣G350G, ␣R350Q, ␣R350L, and ␣G355R are novel gain-of-function human ENaC variants, whereas ␣V531A is a loss-of-function variant. When expressed in Xenopus oocytes, ␣R350W shares similar features with the ␣W493R and ␥L511Q variants (16, 17) as well as ␣C479R mutation implicated in Liddle syndrome (31). These variants increase whole-cell Na ϩ currents and open probability and suppress the Na ϩ self-inhibition response. When examined, little or no effect on the number of channels expressed at the plasma membrane has been observed. These variants form a new class of ENaC extracellular gain-of-function variants with properties that are distinct from classic Liddle mutations targeting the PY motif. Further studies are needed to determine the contributions of these variants to human disorders.

Materials
All reagents were purchased from Sigma unless otherwise noted.

Site-directed mutagenesis
Point mutations in human ␣ENaC cDNA were generated using QuikChange II XL site-directed mutagenesis kit (Agilent, Santa Clara, CA). Target mutations were verified by direct DNA sequencing. Mutant and WT human ENaC cRNAs were synthesized with either SP6 or T7 RNA polymerase (Thermo Fisher Scientific, Waltham, MA), using linearized plasmids as templates. Synthesized cRNAs were purified with an RNA purification kit (Qiagen, Germantown, MD), and concentrations were quantified by spectrophotometry.

Na ؉ self-inhibition
Na ϩ self-inhibition was performed as reported previously (17,57). Na ϩ self-inhibition responses were recorded following a rapid transition from 1 mM Na ϩ bath solution (NaCl-1: 1 mM NaCl, 109 mM N-methyl-D-glucamine, 2 mM KCl, 2 mM CaCl 2 , and 10 mM HEPES, pH 7.4) to 110 mM Na ϩ solution (NaCl-110: 110 mM NaCl, 2 mM KCl, 2 mM CaCl 2 , and 10 mM HEPES, pH 7.4). Oocytes were then perfused with 110 mM Na ϩ solution containing 10 M amiloride to determine the amiloride-insensitive component of the whole-cell current. The ratio of steadystate amiloride-sensitive current (I ss ) in 110 mM Na ϩ solution, obtained 40 s after transition to the 110 mM Na ϩ solution, to the peak amiloride-sensitive current (I peak ) observed following the transition to 110 mM Na ϩ , reflects the magnitude of Na ϩ self-inhibition.

ENaC ␤-ball domain Patch clamp
Cell-attached patch clamp was performed in oocytes expressing WT ␣␤␥, ␣R350W␤␥, or ␣V351A␤␥ human ENaC. Bath solution contained 110 mM NaCl, 2 mM KCl, 2 mM CaCl 2 , 10 mM HEPES, pH 7.4. Pipette solution contained 110 mM LiCl, 2 mM KCl, 2 mM CaCl 2 , 10 mM HEPES, pH 7.4. Patch clamp was carried out using a PC-One patch-clamp amplifier (Dagan Corp.) and a DigiData 1440A interface connected to a PC. Cellattached patches were clamped at Ϫ80 or Ϫ100 mV (negative value of pipette potential). pClamp 10 software (Molecular Devices) was used for data acquisition and analyses. Singlechannel recordings were acquired at 5 kHz, filtered at 1 kHz with a built-in Bessel filter. Channel open probability was estimated with the single-channel search function of pClamp 10 from recordings that were a minimum of 5 min in length. Unitary currents at clamping voltages (i.e. membrane potentials) of 20, Ϫ20, Ϫ40, Ϫ60, Ϫ80, Ϫ100, and Ϫ120 mV were determined by cursor measurements and used to generate a current-voltage plot yielding single channel slope conductance.

Surface expression in Xenopus oocytes
ENaC surface expression was determined using a chemiluminescence assay and a human ␤ENaC construct with an extracellular epitope FLAG tag (58), as described previously (17). Oocytes were injected with 2 ng/subunit cRNAs for WT or mutant human ␣-subunit, WT human ␥-subunit, and human ␤-subunit with an extracellular FLAG epitope tag (DYKD-DDDK) that was inserted between residues Thr-137 and Arg-138. Oocytes injected with a WT ␤-subunit cRNA without the FLAG tag and WT ␣and ␥-subunit cRNAs were used as a negative control group. Surface expression was assayed 48 h after cRNA injection. All steps were performed on ice, except for the last step that was performed at room temperature. Briefly, following a 30-min incubation with MBS (without antibiotics) supplemented with 1% BSA (MBS/BSA), oocytes were incubated with MBS/BSA supplemented with 1 g/ml of a human anti-FLAG M2 mAb (Sigma) for 1.5 h. Oocytes were then washed six times for 5 min in MBS/BSA and incubated in MBS/BSA supplemented with 1 g/ml secondary antibody (peroxidase-conjugated AffiniPure F(abЈ) 2 fragment goat antimouse IgG; Jackson ImmunoResearch, West Grove, PA) for 1 h. Cells were extensively washed six times for 5 min in MBS/BSA and finally washed six times for 5 min in MBS without BSA. Individual oocytes were transferred into a white U-bottom 96-well plate, and 100 l of SuperSignal ELISA Femto Maximum Sensitivity Substrates (Thermo Fisher Scientific, Rockford, IL) was added to each well. Following a 1-min incubation at room temperature, chemiluminescence was quantified with a GloMax-Multi ϩ detection system (Promega, Madison, WI). Results are presented in relative light units.

Statistical analyses
Data are presented as either mean Ϯ S.D. alone or together with dot plots from individual datum points. Statistical significance was examined by the Student's t test for two group data and one-way ANOVA followed by Dunnett's (for comparison between a mutant and WT) or Tukey's post hoc test for multiple group data, using Prism 8 (GraphPad Software, San Diego). A p value of Ͻ 0.05 was considered statistically significant.