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Acta Histochemica
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acta histochemica a Journal of Structural Biochemistry. Cell and Tinaun Imaging
Expression of p11 and heteromeric TASK channels in mouse adrenal cortical cells and H295R cells
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Hidetada Matsuoka ª, Keita Harada ª, Akira Sugawara , Donghee Kim , Masumi Inoue a, *, 1
a Department of Cell and Systems Physiology, University of Occupational and Environmental Health School of Medicine, Kitakyushu 807-8555, Japan
b Department of Molecular Endocrinology, Tohoku University Graduate School of Medical Science, Sendai 980-8575, Japan
” Department of Physiology and Biophysics, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064-3095, USA
ARTICLE INFO
Keywords:
TASK1
TASK3 Adrenal cortical cell P11
Angiotensin II H295R
ABSTRACT
TWIK-related acid-sensitive K+ (TASK) channels are thought to contribute to the resting membrane potential in adrenal cortical (AC) cells. However, the molecular identity of TASK channels in AC cells have not yet been elucidated. Thus, immunocytochemical and molecular biological approaches were employed to investigate the expression and intracellular distribution of TASK1 and TASK3 in mouse AC cells and H295R cells derived from human adrenocortical carcinoma. Immunocytochemical study revealed that immunoreactive materials were mainly located in the cytoplasm for TASK1 and at the cell periphery for TASK3 in mouse AC cells. A similar pattern of localization was observed when GFP-TASK1 and GFP-TASK3 were exogenously expressed in H295R cells. In addition, p11 that is known to suppress the endoplasmic reticulum exit of TASK1 was localized in the cytoplasm in mouse AC and H295R cells, but not in adrenal medullary cells. Proximity ligation assay (PLA) suggested formation of heteromeric TASK1-3 channels that were found predominantly in the cytoplasm and weakly at the cell periphery. A similar distribution was observed following exogenous expression of tandem TASK1-3 channels in H295R cells. When stimulated by angiotensin II, however, tandem TASK1-3 channels were present mainly in the cytoplasm in all H295R cells. In contrast to that in H295R cells, tandem channels were exclusively located at the cell periphery in all non-stimulated and exclusively in the cytoplasm in stimulated PC12 cells, respectively. From these results, we conclude that TASK1 proteins are present mainly in the cyto- plasm and minimally at the cell periphery as a heteromeric channel with TASK3, whereas the majority of TASK3 is at the cell periphery as homomeric and heteromeric channels.
1. Introduction
The mammalian adrenal cortex comprises the zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR), whose main ste- roids are aldosterone, glucocorticoids, and adrenal androgens, respec- tively (Vinson, 2016). These adrenal steroid hormones are produced in response to G protein-coupled receptor (GPCR) stimulation and/or an increase in external K+ concentration (Spät and Hunyady, 2004). One of the most well studied signaling pathways for GPCR-regulated secretion is angiotensin II (AngII)-induced aldosterone release (Ota et al., 2014). AngII activates the angiotensin receptor type 1 (AT1R) with the subse- quent inhibition of K+ channels (Czirják and Enyedi, 2002a; Penton
et al., 2012). K+ channel inhibition then produces depolarization in the resting membrane potential (Cohen et al., 1988; Lotshaw, 2001), resulting in an increase in intracellular Ca2+ concentration with the subsequent facilitation of transcription of enzymes involved in steroid synthesis (Szekeres et al., 2009; Spät et al., 2016). Interestingly, a similar mechanism has been proposed for ACTH-mediated increase in cortisol synthesis in human ZF cells (Enyeart and Enyeart, 2013). Thus, the identification of K+ channels contributing to the resting membrane potential is important to elucidate the signal transduction mechanism for GPCR-mediated steroid synthesis. Indeed, efforts have been made to identify the K+ channels in adrenal cortical (AC) cells (Gomez-Sanchez and Oki, 2014; Bandulik et al., 2015). Molecular biological and
Abbreviations: TASK, TWIK-related acid-sensitive K+; ZG, zona glomerulosa; GPCR, G protein-coupled receptor; AngII, angiotensin II; AT1R, angiotensin receptor type 1; PLA, proximal ligation assay; IR, immunoreactive.
* Correspondence to: University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan. E-mail address: minoue@med.uoeh-u.ac.jp (M. Inoue).
1 ORCID:0000-0003-4180-5238
electrophysiological approaches were used to show that TASK3 channels are mainly responsible for the resting membrane potential in rat ZG cells (Czirják and Enyedi, 2002a). AT1R stimulation is thought to suppress TASK3 channels probably via diacylglycerol (DAG) binding to its C-terminus (Wilke et al., 2014). Consistent with the findings in rats, aldosterone production in mouse ZG cells was constitutively enhanced by deletion of the Task3 gene (Bandulik et al., 2013). Furthermore, intracellular Ca2+ concentration was found to oscillate in AC cells of Task3 knockout (KO) mice and AngII-induced increase in intracellular Ca2+ concentration was significantly diminished or abolished in Task3-KO AC cells (Penton et al., 2012). These results indicate that TASK3 channels contribute to the resting membrane potential in mouse AC cells including ZG cells and AngII suppresses TASK3 channels. However, whether AngII-induced inhibition of TASK3 channels can be entirely ascribed to a change in gating remains an open question (Inoue et al., 2020).
In contrast to TASK3, the evidence for the contribution of TASK1 to the resting membrane potential is relatively weak. Depletion of the Task1 gene also results in an increase in aldosterone production (Heitzmann et al., 2008). However, this increase is ascribed to the ectopic expression of aldosterone synthase in ZF cells. The reason that aldosterone synthase is ectopically expressed in TASK1-KO mice re- mains unknown. Furthermore, deletion of the Task1 gene resulted in depolarization of ~5 mV in AC cells (Heitzmann et al., 2008), a value which was apparently smaller than that (~27 mV) in TASK3-depleted AC cells (Penton et al., 2012). In-situ hybridization study revealed that TASK1 is expressed at the mRNA level in the whole adrenal cortex of mice (Davies et al., 2008). However, the expression at the mRNA level may not correlate with that at the protein level (Barry et al., 1995; Inoue et al., 2008). Thus, the physiological role of TASK1 proteins in AC cells still remains to be elucidated. Exactly how knockout of the Task1 gene results in the extension of aldosterone synthase expression to the ZF is totally unknown. Furthermore, whether a low level of depolarization noted in AC cells deleted of TASK1 proteins is due to the abolition of homomeric TASK1 channels in the plasma membrane remains an open question. We have elucidated that muscarinic receptor stimulation re- sults in internalization of TASK1 channels in PC12 cells and exogenous expression of p11 facilitates heteromeric channel formation of TASK1 and TASK3. Thus, if p11 is expressed in AC cells, heteromeric TASK1-3 channels are expected to be formed and subjected to regulation by GPCRs. To explore the molecular identify of TASK channels and regu- lation of channel activity by AngII, immunocytochemical and molecular biological approaches were employed to study the distribution of TASK proteins in mouse AC cells and/or H295R cells derived from human adrenocortical carcinoma cells (Gazdar et al., 1990) that were and were not subjected to AngII stimulation.
2. Materials and methods
2.1. Animals
Male wild-type C57BL/6, Task1-KO, and Task3-KO mice (2-6-month old) were used for the experiments. Task1-KO and Task3-KO mice were kindly provided by Dr. W. Wisden (Imperial College London, London, UK) and described elsewhere (Aller et al., 2005; Brickley et al., 2007). Briefly, the first coding exons in both Task1 and Task3 genes were dis- rupted, and no mutant allele was transcribed. Mice were housed in standard cages with free access to food and water, and kept under a light/dark cycle of 12/12 h. All procedures for the care and treatment of animals were carried out according to the Japanese Act on the Welfare and Management of Animals and the Guidelines for the Proper Conduct of Animal Experiments issued by the Science Council of Japan. The experiments were approved by the Institutional Animal Care and Use Committee of the University of Occupational and Environmental Health in Kitakyushu (permit AE10-010). Procedures complied with the Ani- mal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.
All efforts were made to minimize the potential animal distress.
2.2. Immunocytochemistry
Acutely dissociated adrenal cells were obtained, as described else- where (Inoue et al., 2019). Briefly, six animals for each type of mouse were killed by cervical dislocation, and the adrenal glands were excised, and immediately immersed in ice-cold Ca2+-deficient balanced salt so- lution, in which 1.8 mM CaCl2 was omitted from standard salt solution. The standard balanced salt solution contained 137 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, 0.53 mM NaH2PO4, 5 mM D-glucose, and 5 mM Hepes, and pH of the solution was adjusted to 7.4 with 4 mM NaOH. The adrenal cortex was removed from the adrenal medulla with microscissors and forceps under stereoscopic observations, and the ad- renal cortex was minced into small pieces of preparations. The adrenal cortical and medullary preparations were individually incubated in a 0.5% collagenase-containing Ca2+-free solution at 36 ℃ for 30 min. Then, one or two pieces of each preparation was placed in a dish with non-fluorescent glass (P35GC-0-14-C: MatTek, Ashland, MA, USA), and cells were dissociated using fine needles under a microscope. Dissoci- ated cells were allowed to attach to the glass for 30 min, fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.2) for 1 h, and pre-incubated in PBS with 5% fetal bovine serum (FBS) (172012: Sigma-Aldrich, Tokyo, Japan) and 0.3% Triton-X 100 for 30 min. For indirect immunofluorescence studies, cells were treated overnight with rabbit anti-TASK1 antibody (Ab) (sc-28635: Sant Cruz Biotechnology; Dallas, TX, USA) (RRID: AB_661017), goat anti-TASK3 Ab (sc-11317) (RRID: AB_2131233), or goat anti-p11 Ab (AF2377; R&D systems, Minneapolis, MN, USA) (RRID: AB_2183469). Specific immunoreactiv- ities of anti-p11, anti-TASK1, and anti-TASK3 antibodies used have been immunocytochemically confirmed in our previous experiments where the proteins tagged with green fluorescent protein (GFP) or myc were exogenously expressed in PC12 cells (Inoue et al., 2008; Matsuoka et al., 2013), and the Task1 or Task3 gene was deleted in the mouse germ line (Inoue et al., 2019). After incubation, cells were washed three times with PBS and treated with a respective secondary antibody conjugated with Alexa Fluor 546 (Molecular Probes, Eugene, OR, USA). Fluorescence was observed under a laser confocal microscope (LSM5 Pascal: Carl Zeiss, Tokyo, Japan). The objective lens was an oil-immersion with a magni- fication of 63x and a numerical aperture of 1.4. For Alexa Fluor 546, a 543 nm laser was used and the emission above 560 nm was observed (rhodamine-like fluorescence), whereas for GFP, a 514 nm laser was used and 530-600 nm emission was observed. The cell periphery was defined as an area of 1 um width at the cell boundary, which was determined in a differential interference contrast (DIC) image. Immu- nofluorescence of the whole cell and at the cell periphery was measured with ImageJ software (NIH, Bethesda, MD, USA), and the coincidence of the two fluorescence was assessed with Metamorph software (Universal imaging, Downingtown, PA, USA).
2.3. Cell culture and transfection
PC12 cells (RRID: CVCL_0481) originating from male rat adrenal medullary cells (Greene and Tischler, 1976) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco: Life Technologies, Tokyo, Japan) supplemented with 10% FBS, as was previously described (Matsuoka et al., 2013). H295R (RRID: CVCL_0458) originating from human adrenal cortical cartinoma were cultured in Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (DMEM/F-12) (Gibco) supple- mented with 2.5% FBS, 5.35 µg ml-1 linoleic acid (Sigma-Aldrich) and Insulin-Transferrin-Selenium (ITS-G) (414000045: ThermoFisher Sci- entific, Tokyo, Japan) (containing 6.25 µg ml-1 insulin, 6.25 µg ml-1 transferrin, and 6.25 µg ml-1 selenium). The Lipofectamine 2000 re- agent (Invitrogen: Life Technologies) was used to transfect cells with expression vectors, according to the manufacturer’s instructions. The transfected cells were placed onto glass coverslips coated with collagen
Type I (BD Biosciences, San Jose, CA, USA) and then cultured for 24 h. The cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After washing three times with PBS, cells were incubated in PBS containing 0.1% Triton X-100 for 30 min and then with PBS containing 1% FBS for 1 h at room temperature. The cells were treated with primary and secondary antibodies. Coverslips were mounted in 50% glycerol containing 1 mg ml-1 4-diaminobenzene.
2.4. Plasmids
The following mammalian expression vectors were used: GFP-tagged TASK1 (GFP-TASK1) and GFP-tagged TASK3 (GFP-TASK3) where rat TASK1 and TASK3 cDNAs were subcloned into pEGFP (Berg et al., 2004); myc-DDK-tagged p11 (myc-p11) where mouse p11 cDNA was into pCMV6-Entry; hemagglutinin (HA)-tagged tandem TASK1-3 where tandem TASK1-3 cDNA was made by insertion of a three-residue (gly- cine-serine-alanine) between the C-terminal amino acid of TASK1 and the start methionine of TASK3 and the resulting tandem TASK1-3 cDNA was subcloned into pcDNA3.
2.5. Proximal ligation assay (PLA)
To detect heteromeric TASK1-TASK3 channel formation in H295R cells more specifically, an in situ PLA, which visualizes the close appo- sition of two proteins within 40 nm, was performed using the Duolink in situ PLA kit (Olink Bioscience, Uppsala, Sweden), according to the
manufacturer’s instructions. Fixed cells were treated overnight with a combination of rabbit anti-TASK1 and goat anti-TASK3 Abs or mouse anti-GFP (sc-9996) (RRID: AB_627695) and rabbit anti-myc Abs (sc-789) (RRID: AB_631274). After treatment with primary Abs, cells were incubated with the secondary ones conjugated with the oligonucleotides (PLA probe MINUS and PLA probe PLUS). Oligonucleotides of two PLA probes were hybridized by ligase if they were in close proximity. A rolling-circle amplification was carried out with polymerase and fluo- rescently labeled oligonucleotides, resulting in amplification of the fluorescent signal. The fluorescence from PLA products was observed with the LSM 5 Pascal Microscope System.
2.6. Sources of reagents
The plasmid for myc-DDK-tagged p11 (MR226807) was purchased from OriGene Technologies (Rockville, MD, USA); mouse anti-HA antibody (sc-7392) (RRID: AB 1537399) was from Santa Cruz Biotech- nology; mouse anti-Flag antibody (200-301-383) (RRID: AB_627809) was from Rockland Immunochemicals (Limerick, PA, USA).
2.7. Statistics
Data were expressed as means ± SEM. Significance of differences between experimental and control results was assessed with unpaired Student’s t-test for data with a normal distribution (Shapiro-Wilk test). Otherwise, Mann-Whitney rank sum test was used. A p value < 0.05
A
WT
TASK1KO
WT
TASK3KO
B
100
P < 0.001
Cell periphery / whole cell (%)
80
TASK1
TASK3
60
40
20
Merge
Merge
0
王
TASK1 TASK3
5 um
D
100
P < 0.001
Cell periphery / whole cell (%)
80
C
GFP
Merge
GFP
Merge
60
40
GFP- TASK1
GFP- TASK3
20
王
0
GFP- GFP- TASK1 TASK3
5 um
defined a statistically significant difference. Statistical analysis was performed with Sigma Plot v13.0 software (Systat Software, San Jose, CA, USA).
3. Results
3.1. Distribution in the cells
Confocal laser microscopy was used to locate immunocytochemically TASK proteins in dissociated mouse AC cells. TASK1-like immunoreac- tive (IR) material was mainly present in the cytoplasm, whereas the majority of TASK3-like IR material was located at the cell periphery (Fig. 1, A and B). As was previously noted (Inoue et al., 2019), the IR fluorescence was abolished by deletion of each gene, indicating that IR material represents each TASK isoform. The cytoplasmic distribution of TASK1 in AC cells is contrasted to the plasma membrane presence in adrenal medullary (AM) and PC12 cells (Matsuoka et al., 2013). Thus, to further explore the distribution of TASK proteins in AC cells, GFP-TASKs were exogenously expressed in H295R cells originating from human adrenocortical carcinoma cells (Gazdar et al., 1990). The different dis- tributions of TASK1 and TASK3 were reproduced by the exogenous expression of each GFP fusion protein in H295R cells: GFP-TASK1 and
GFP-TASK3 were mainly located in the cytoplasm and at the cell pe- riphery, respectively (Fig. 1, C and D). These results clearly show that in contrast to AM cells and PC12 cells (Matsuoka et al., 2013), TASK1 is mainly located in the cytoplasm, whereas TASK3 is present at the cell periphery in AC and H295R cells.
3.2. Expression of p11
p11 (Rescher and Gerke, 2008) has been shown to suppress the exit of TASK1 from the endoplasmic reticulum (ER) via binding to its C-terminus (Renigunta et al., 2006). Thus, whether the cytoplasmic presence of TASK1 in AC cells is due to retention by p11 or not was explored. As expected, p11 was immunocytochemically found to be expressed in all mouse AC cells (n = 11), whereas the protein was not in any of AM cells (n = 21) where TASK1 was located at the cell periphery (Fig. 2A). Similarly, p11-like IR material was detected in the cytoplasm in 13 of 14 (93%) H295R cells examined (Fig. 2B), where exogenous GFP-TASK1 was mainly present in the cytoplasm (Fig. 3A).
3.3. Heteromeric TASK channel formation
We have reported that the exogenous expression of p11 in PC12 cells
A
TASK1
p11
IR
Merge
IR
Merge
AM
AM
AC
AC
5 um
B
p11
IR
Merge
H295R
*
-
5 um
A
GFP-TASK1
anti-TASK3
Merge
B
60
50
P < 0.001
Coincidence (%)
40
30
GFP-TASK3
anti-TASK1
Merge
20
10
0
TASK3/
GFP-TASK1
TASK1/
GFP-TASK3
5 pm
C
PLA (GFP/TASK3)
GFP-TASK1
Merge
D
100
P < 0.001
Cell periphery / whole cell (%)
80
60
PLA (GFP/TASK1)
40
GFP-TASK3
Merge
P = 0.076
20
0
PLA
GFP
PLA
GFP
TASK1
TASK3
5 pm
results in the heteromeric channel formation of TASK1 and TASK3 (Inoue et al., 2019). Therefore, the fact that TASK1, TASK3, and p11 are endogenously expressed in AC cells and H295R cells raises the possi- bility that TASK1 and TASK3 are present as a heteromeric channel in these cells. This possibility was first investigated with the immunocy- tochemical method. When GFP-TASK1 was expressed in H295R cells, GFP-TASK1 associated with TASK3-like IR material constituted 37% of the total GFP-TASK1. On the other hand, the fraction of GFP-TASK3 associated with TASK1-like IR material in GFP-TASK3-expressing H295R cells was at most 11% of the total GFP-TASK3 (Fig. 3, A and B). These results suggest that the significant fraction of TASK1 proteins formed heteromeric channels with TASK3, whereas that of TASK3 did not. Thus, whether heteromeric channels were formed or not was directly investigated by using PLA. As is evident in Fig. 3, C and D, PLA products developed between GFP-TASK1 and endogenous TASK3 or between GFP-TASK3 and endogenous TASK1 in H295R cells expressing GFP fusion proteins. In addition, the majority of both PLA products were present in the cytoplasm, and the fraction of PLA products present at the
cell periphery did not differ between in GFP-TASK1- and GFP-TASK3-expressing H295R cells. The results suggest that the small fraction of heteromeric TASK1-3 channels are trafficked to the cell membrane, irrespective of whether GFP-TASK1 or GFP-TASK3 was exogenously expressed in H295R cells.
3.4. Endocytosis of TASK1 channels by AngII
Muscarinic M1 receptor stimulation has been reported to induce endocytosis of homomeric TASK1 and heteromeric TASK1-3 channels, but not homomeric TASK3 channels, in a clathrin-dependent manner in PC12 and AM cells (Matsuoka and Inoue, 2017; Inoue et al., 2020). Thus, we investigated the effects of AT1R activation on the distribution of TASK channels in H295R cells where either GFP-TASK1 or GFP-TASK3 was exogenously expressed. Activation of AT1R, which is coupled with the same pertussis-toxin-insensitive G protein as the M1 receptor, is expected to phosphorylate a tyrosine residue in the C-terminus of TASK1 with a consequent change in its conformation (Matsuoka and Inoue,
2017; Inoue et al., 2020), resulting in AP-2 binding to a dileucine motif (Traub, 2003; Matsuoka et al., 2013). Thus, if exogenously expressed GFP-TASK3 proteins form heteromeric channels with endogenous TASK1 in H295R cells, GFP-TASK3 in the plasma membrane will be translocated to the cytoplasm in response to AT1R activation. As is evident in Fig. 4, A and B, exogenously expressed GFP-TASK3 proteins were located at the cell periphery in all the non-stimulated H295R cells, whereas AngII-stimulated cells were divided into two groups: GFP-TASK3 proteins were located at the cell periphery in one group of cells (55%) and in the cytoplasm in the other (45%). On the other hand, GFP-TASK1 proteins expressed in H295R cells were predominantly located in the cytoplasm, irrespective of whether the cells were stimu- lated by AngII or not (Fig. 4, A and B). In contrast to H295R cells, GFP-TASK3 proteins exogenously expressed in PC12 cells (where TASK3 does not form a heteromeric channel with TASK1 because of the lack of p11 expression) were not internalized in response to AngII stimulation. On the other hand, GFP-TASK1 proteins in PC12 cells were translocated from the plasma membrane to the cytoplasm in response to AngII (Fig. 4, C and D).
3.5. Tandem TASK1-3 channels
The results hitherto mentioned suggested that some of GFP-TASK3 proteins form heteromeric channels with TASK1 and are trafficked to the plasma membrane and that such heteromeric channels of GFP-
TASK3 and TASK1 are internalized in response to AngII stimulation. Thus, this notion was further explored with expression of tandem TASK1-3 channels in H295R cells (Fig. 5, A and B). H295R cells expressing tandem TASK1-3 channels were divided into two groups: in one group of cells (60%) tandem channels were predominantly located at the cell periphery; in the other of cells (40%) they were present in the cytoplasm. On the other hand, in all the H295R cells stimulated by AngII the tandem channels were present in the cytoplasm. In contrast to H295R cells, exogenously expressed tandem channels were predomi- nantly located at the cell periphery and in the cytoplasm in all the non- stimulated and stimulated PC12 cells, respectively (Fig. 5, A and B).
4. Discussion
4.1. Molecular identity of TASK channels
Genetic manipulations of the Task genes have advanced our under- standing of the physiological role of TASK channels in AC cells and unveiled new questions, such as the role for the regulation of aldoste- rone synthase expression (Heitzmann et al., 2008). Deletion of TASK3 resulted in ~18 mV depolarization in mouse AC cells (Penton et al., 2012), whereas that of TASK1 produced just 5 mV depolarization, but had a profound effect on aldosterone production. Aldosterone synthase becomes expressed in ZF cells in female mice lacking the TASK1 (Heitzmann et al., 2008). These results indicate that TASK3 proteins, but
A
GFP-TASK1
GFP-TASK3
B
GFP- TASK1
GFP-
GFP-
TASK3
TASK3
(-)
(-)
100
Cell periphery / whole cell (%)
80
60
40
P = 0.004
+AngII
+AngII
20
@
0
0
0.5
0
0.5
0
0.5
5 um
+AngII (min)
C
D
GFP-
GFP-
GFP-TASK1
GFP-TASK3
TASK1
TASK3
P < 0.001
100
(-)
(-)
Cell periphery / whole cell (%)
80
60
40
20
+AngII
+AngII
0
0
0.5
0
0.5
5 um
+AngII (min)
A
B
HA-TASK1-3
HA-TASK1-3
100
H295R
PC12
+Angll
0
0.5 (min)
0
0.5 (min)
8
80
Cell periphery / whole cell (%)
60
H295R
PC12
40
O
20
0
-
5 um
0
0.5
0
0.5 (min)
+Angll
not TASK1, predominantly contribute to the resting membrane potential in mouse AC cells, whereas TASK1 plays a yet unknown role for the regulation of aldosterone synthase expression in AC cells. The findings in the present immunocytochemical experiment supported the conclusion derived from the functional analysis of the effects of deletion of each of TASK1 and TASK3 on the resting membrane potential. The majority of TASK3 proteins were located at the cell periphery in mouse AC cells, whereas TASK1 proteins were mainly present in the cytoplasm. This different distribution of TASK1 and TASK3 was reproduced by exoge- nous expression of GFP-fusion proteins in H295R cells, which were used as a model cell for ZG (Matsuda et al., 2014; Yarimizu et al., 2015) and ZF cells (Nishi et al., 2013). In addition, p11 was immunocytochemically detected in the cytoplasm in mouse AC and H295R cells. Thus, p11, which is known to bind to a variety of membrane proteins, such as the serotonin receptor type 1B (5-HT1BR) (Svenningsson et al., 2006) and the tetrodotoxin-insensitive Na+ channel Nay1.8 (Okuse et al., 2002), may suppress the exit of TASK1 in the ER through binding to its C-ter- minus (Renigunta et al., 2006; Inoue et al., 2019).
We reported earlier that p11 facilitates heteromeric channel forma- tion of TASK1 and TASK3 (Inoue et al., 2019, 2020). In fact, in H295R cells where GFP-TASK1 was exogenously expressed, 37% of the exoge- nous GFP-TASK1 coincided with endogenous TASK3 in the cytoplasm, whereas in H295R cells exogenously expressing GFP-TASK3, the ma- jority of GFP-TASK3 proteins were located at the cell periphery, and their small fraction (11%) coincided with the endogenous TASK1. These results suggest that a large fraction of TASK1 proteins exist as hetero- meric TASK1-3 channels in the cytoplasm, whereas that of TASK3 does as homomeric TASK3. The detailed analysis of PLA products between exogenous GFP fusion proteins and endogenous TASK revealed that PLA products were mainly present in the cytoplasm, but their small fraction was definitely located at the cell periphery, irrespective of whether GFP-TASK1 or GFP-TASK3 proteins were transiently expressed in H295R cells. The results, taken together with the immunocytochemical findings, suggest that the majority of TASK3 proteins at the cell pe- riphery is present as homomeric TASK3, and a small fraction is present as heteromeric TASK1-3 channels. This notion is supported by trans- location of GFP-TASK3 in H295R cells in response to AngII. GFP-TASK3 proteins expressed in H295R cells were predominantly located at the cell periphery. On the other hand, H295R cells stimulated by AngII were divided into two groups based on GFP-TASK3 localization: the cells in
one group (55%) had GFP-TASK3 at the cell periphery, whereas those in the other (45%) had GFP-TASK3 predominantly in the cytoplasm. The GFP-TASK3 proteins located in the cytoplasm in the stimulated cells may include internalized heteromeric TASK1-3 channels in response to AngII. First, in PC12 cells where p11 is not endogenously expressed and thus heteromeric TASK1-3 channels are not formed, endogenous or exogenous TASK3 proteins are not internalized in response to muscarine (Matsuoka and Inoue, 2017) and AngII (present results). Secondly, tandem TASK1-3 channels in H295R and PC12 cells were internalized in response to AngII.
Our results suggest that GFP-TASK3 predominantly present at the cell periphery in the stimulated cells represents homomeric TASK3 channels. Additionally, homomeric TASK3 and heteromeric TASK1-3 channels represent 55% and 45% of TASK3 proteins at the cell periph- ery, respectively. These findings are consistent with the earlier report that 75% of TASK channel activity in rat AC cells were suppressed by ruthenium red (Czirják and Enyedi, 2002a), which selectively inhibits homomeric TASK3, but not heteromeric TASK1-3 channels (Czirják and Enyedi, 2002b; Kim et al., 2009). TASK channel inhibition by GPCR activation have been shown to be partly mediated by DAG binding to a six-amino-acid (VLRFLT) motif at the most proximal intracellular C-terminus of TASK1 and TASK3 (Wilke et al., 2014). Thus, AngII may suppress homomeric TASK3 channels by reducing its activity through DAG binding to the VLRFLT motif. Collectively, our data suggest that at the plasma membrane, the majority of TASK3 proteins is homomeric TASK3 and a small fraction is heteromeric TASK1-3.
4.2. Roles of TASK channels in the ER
The expression of p11 results in hindrance of ER exit of TASK1. Thus, ER TASK channels may be homomeric TASK1 and heteromeric TASK1-3 in p11-expressing cells, such as AC cells. Therefore, knockout of the Task1 gene results in deletion of TASK channels in the ER. Heteromeric TASK1-3 channels will be substituted for by homomeric TASK3 chan- nels in TASK1-KO mice (Turner and Buckler, 2013), channels which are expected to be trafficked to the cell membrane because of the lack of retention through TASK1 by p11. Therefore, knockout of the Kask1 gene results in lack of TASK channels in the ER.
A decrease in K+ channel activity in the ER impairs the operation of K+,H+-exchangers with the consequent decrease in Ca2+ uptake
capacity (Kuum et al., 2012; Kuum et al., 2015; Richter et al., 2016), as explained below. The Ca2+ pump in the ER, SERCA, takes up Ca2+ into the ER in a manner coupled with H+ efflux, and the consequent decrease in H+ concentration in the ER is compensated by the H+, K+-exchanger-mediated H+ influx. The K+ loss from the ER is then compensated by K+ channel activity. Several kinds of K+ channels, such as small conductance Ca2+-dependent K+ (SK) channels (Richter et al., 2016), have been reported to be located in the ER membrane. In addi- tion, TALK-1 channels, one of K2p channel family of K+ channels (Enyedi and Czirják, 2010; Feliciangeli et al., 2015), have recently been shown to participate in ER Ca2+ homeostasis in pancreatic ß cells (Vierra et al., 2017). A decrease in ER Ca2+ uptake is expected to lead to an increase in an intracellular Ca2+ concentration (Kuum et al., 2012). The mechanism for an increase in aldosterone synthase expression in ZF cells in TASK-KO female mice has not been elucidated yet. The present finding raises the possibility that an increase in intracellular Ca2+ con- centration in ZF cells due to a decrease in ER Ca2+ uptake is responsible for an increase in aldosterone synthase expression in TASK1-KO mice (Kobuke et al., 2018). It is worth to note that TASK1-KO-induced aldo- steronism does develop in male mice before puberty, but not in adult male. In addition, administration of androgens in female mice with TASK1-KO ameliorates aldosteronism (Heitzmann et al., 2008). Why androgens are inhibitory on TASK1-KO-induced aldosteronism remain an open question. One possibility is that androgens might enhance K+ channel activity in the ER. In fact, androgens have been shown to in- crease expression of several K+ channels (Brouillette et al., 2003; Zhao et al., 2018; Khatun et al., 2018). However, whether androgens enhance K+ channel activity in the ER membrane or not requires a further experiment.
In conclusion, TASK1 was mainly present in the cytoplasm and TASK3 was mainly localized at the cell periphery in both mouse AC and H295R cells. The majority of the TASK1 proteins in AC cells formed heteromeric channels with TASK3 due to the expression of p11, whereas TASK3 proteins were present mostly as homomeric TASK3 channels. AngII stimulation in AC cells resulted in translocation of TASK1 and TASK1-3 channels from the cell periphery to the cytoplasm through AP- 2 binding to the C-terminus of TASK1. These results support the view that AngII-induced depolarization in AC cells is in part due to endocy- tosis of TASK channels. The limitation of the present experiment is qualitative, but not quantitative, analysis. How much TASK channel endocytosis contributes to AngII-induced depolarization in AC cells will be a next issue.
Author contributions
H. M., K. H., A. S., and M. I. contributed to the conception and design of the work; M. I. and A. S. made the preparation; D. K. made the DNA constructs; H. M. acquired the data; H. M. and K. H. analyzed the data; M. I and H. M. wrote the manuscript. All authors have read and approved the final manuscript.
Conflict of Interests
The authors have no conflicts of interest to declare.
Data Availability
Data will be made available on request.
Acknowledgments
This study was in part supported by grants of JSPS KAKENHI, Japan (17K08555 to M. I. and 18K06865 to H. M.).
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