Expression and function of mitochondrial inhibitor factor-1 and TASK channels in adrenal cells

Abstract

Adrenal medullary chromaffin (AMC) cells in the perinatal period and carotid body glomus cells after birth respond to hypoxia with catecholamine secretion. The hypoxia detection mechanism in such O₂-sensitive cells is still not well defined. One hypothesis is that a decrease in cellular ATP may be involved in the hypoxia detection. This idea is based on ATP dependence of TASK channel activity that regulates the resting membrane potential and is suppressed by hypoxia in glomus cells. Mitochondrial ATPase inhibitor factor-1 (IF₁), a physiological regulator of ATP synthase, helps prevent ATP hydrolysis under hypoxic conditions. In cells where IF₁ expression is very low, exposure to hypoxia is expected to have no effect on TASK channel activity. This possibility was electrophysiologically and immunocytochemically explored. Single channel recordings revealed that 36-pS TASK3-like channels contribute to the resting membrane potential in young rat adrenal cortical (AC) cells. TASK3-like channel activity in a cell-attached patch was not affected by bath application of mitochondrial inhibitors. Consistent with this finding, IF₁-like immunoreactive material was well expressed in rat AC cells. In further support of our hypothesis, IF₁-like immunoreactive material was well expressed in adult rat AMC cells that are known to be hypoxia-insensitive and minimally expressed in newborn AMC cells that are hypoxia-sensitive. These results provide evidence for the functional relevance of IF₁ expression in excitability in O₂-sensitive cells in response to mitochondrial inhibition.

Introduction

Adrenal medullary chromaffin (AMC) cells in perinatal mammalian embryos and newborns [1–2], and carotid body (CB) glomus cells days after birth [3–4] are excited by a decrease in O₂ concentration, which leads to augmented catecholamine secretion. K⁺ channels are an important regulator for hypoxia-induced secretion in such O₂-sensitive cells [5–6]. In particular, TWIK-related acid-sensitive K⁺ (TASK) channels, which are formed by TASK1 and/or TASK3 [7–8], are known to contribute to the resting membrane potential in various kinds of cells [6, 9] and to be suppressed by hypoxia in glomus cells [6, 10]. Single channel recording in the cell-attached mode revealed that TASK channel activity recorded from the patch decreases when the cells are exposed to hypoxia [10–11]. However, detailed mechanisms for hypoxia detection by TASK channels are largely unknown. Although several hypotheses have been proposed for the hypoxia detection mechanisms, none of them are currently widely accepted [12–13]. AMC and glomus cells are developmentally close, originating from the neural crest [14]. Thus, similar mechanisms are likely involved in hypoxia detection in both types of cells. Recently, the hypothesis that mitochondria play a primary role for hypoxia detection has revived [15–16], although how mitochondria are involved in hypoxia detection remains largely unsettled.

TASK channel activity in rat CB glomus cells was found to depend on ATP and exposure to hypoxia resulted in a rapid decrease in cellular ATP [17], suggesting that a decrease in cellular ATP may be involved in hypoxia-induced suppression of TASK channels in glomus cells. In addition, the rank of order of mitochondrial inhibitors in evoking catecholamine secretion in guinea-pig AMC cells were similar to that in producing a decrease in intracellular ATP concentration [18]. These findings raise the possibility that ATP decrease may be involved in hypoxia detection in O₂-sensitive cells.

Mitochondria are a site for energy production under normoxic conditions, but a major site for energy consumption under hypoxic conditions [19]. Under normoxic conditions, H⁺ is actively transported from the matrix to the intermembrane space through complexes I, III, and IV, thereby generating a large gradient of H⁺ across the inner membrane. The electron transport through the electron transport chain stops with the consequent diminution of the H⁺ concentration gradient in the absence of O₂. Thus, F₁F₀-ATPase or complex V is expected to operate in the reversed mode with the consumption of ATP [19], consequently facilitating H⁺ efflux. However, the reverse operation of F₁F₀-ATPase may deteriorate the cellular conditions, because of a decrease in ATP contents. To prevent this cell deterioration, inhibitor factor 1 (IF₁), which blocks the reversed operation of F₁F₀-ATPase through binding to the F₀ component [19], is produced in cells.

Rat AMC cells have a capability of sensing O₂ during the perinatal period, but lose it shortly after birth [1, 20–21]. If the reverse operation of F₁F₀-ATPase is involved in hypoxia detection by AMC cells [22], the expression level of IF₁ in newborn rat AMC cells is expected to be small, compared with that in adult rat AMC cells. In fact, our earlier study showed that the expression level of IF₁ in adult guinea-pig AMC cells, which secrete catecholamine in response to hypoxia or mitochondrial inhibitors, is smaller than that in adult rat AMC cells, which secrete little in response to them. The aim of the present experiment was to elucidate whether TASK channels contribute to the resting membrane potential in adult rat adrenal cortical (AC) cells and, if so, to investigate the relationship between the effects of mitochondrial inhibitors on TASK channel activity and the expression of mitochondrial IF₁ in adrenal cells.

Materials and Methods

Animals

Male adult (2–6 months old) and pregnant Wister rats and Sprague-Dawley rats (4–6 weeks old) were used for the experiments. Rats were housed in standard cages with free access to food and water, and kept under a light/dark cycle of 12/12 h. The experiments were approved by the Institutional Animal Care and Use Committee of the University of Occupational and Environmental Health in Kitakyushu (permit AE03–012) and the Animal Care and Use Committee of Rosalind Franklin University (permit #18–02). At Rosalind Franklin University, the animal care and use program is accredited by American Association for Accreditation of laboratory Animal Care (AAALAC) and approved by the Office of Laboratory Animal Welfare (A3279–01). All procedures complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Every effort was made to minimize the potential animal distress.

Immunocytochemistry

Acutely dissociated adrenal cells were obtained, as described elsewhere [23]. Briefly, eight newborn and four adult Wister rats were killed by cervical dislocation, and the adrenal glands were excised, and immediately immersed in ice-cold Ca²⁺-deficient balanced salt solution, 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 CaCl_2, 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 roughly removed from the adrenal medulla with microscissors and forceps under stereoscopic observations. The adrenal medullary preparations were incubated in a 0.5% collagenase-containing Ca²⁺-free solution at 36°C for 30 min. Then, one 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. Dissociated 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 mouse anti-ATPase IF₁ antibody conjugated with Alexa Fluor 488 (ab198075: Abcam Com., Tokyo, Japan) or a combination of mouse anti-ATPase IF₁ and goat anti-chromogranin A (sc-1488: Santa Cruz Biotechnology, San Antonio, TX, USA) [24] or goat anti-CYP11A antibodies (sc-18043: Santa Cruz Biotechnology) [25]. After incubation, cells were washed three times with PBS and treated with anti-goat IgG 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 magnification 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 Alexa Fluor 488, a 514 nm laser was used and 530 to 600 nm emission was observed. Immunofluorescence in the cytoplasm and nucleus was separately measured with ImageJ software (NIH, Bethesda, MD, USA).

Electrophysiology

Sprague-Dawley rats were anaesthetized by inhalation of isoflurane until cessation of breathing. Adrenal glands and carotid bodies were removed and placed in ice-cold saline with low Ca²⁺/Mg²⁺ (137 mM NaCl, 2.8 mM KCl, 2 mM KH₂PO₄, 0.07 mM CaCl₂, 0.05 mM MgCl₂, pH 7.4). Adrenal cortical preparations and carotid bodies were separately placed in saline with low Ca²⁺/Mg²⁺ containing trypsin (0.4 mg ml⁻¹) and collagenase (0.4 mg ml⁻¹) and incubated at 37°C for 25 min. Following trituration, the dispersed cells were resuspended in growth medium (Ham’s F-12, 10%FBS, 23 mM glucose, 4 mM Glutamax-1 (L-alanyl glutamine), 10 kU penicillin/streptomycin and 300 ug ml⁻¹ insulin) for 2 h at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Cells were used within 8 h after plating.

Single channel activity was recorded using a patch clamp amplifier (Axopatch 200B, Molecular Devices, Sunnyvale, CA, USA). Cell-attached patches were formed with gentile suction with sylgard-coated borosilicate glass pipettes with tip resistance of 2 to 3 megaohms. The recorded currents were filtered at 2 KHz and transferred to a computer using the Digidata 1320 interface at a sampling rate of 20 KHz. Single-channel currents were analyzed with the pCLAMP program (version 10). Channel openings were analyzed to obtain channel activity (NP_O) where N is the number of channels in the patch, and P_O is the probability of a channel being open). NP_O was determined from ~20 s of current recording. The pipette solution contained: 140 mM KCl, 1 mM MgCl₂, 5 mM EGTA, 11 mM glucose, and 10 mM Hepes (pH 7.3). The external bath solution contained 117 mM NaCl, 5 mM KCl, 23 mM NaHCO₃, 1 mM MgCl₂, 1 mM CaCl₂, 11 mM glucose, 10 mM Hepes, and pH 7.3. Recording temperature was 34–35°C

Statistics

Data were expressed as means ± SEM unless otherwise noticed. Significance of differences among the groups were assessed with unpaired Student’s t-test or one-way analysis of variance for data with a normal distribution (Shapiro-Wilk test). Otherwise, Mann-Whitney rank sum test or Kruskal-Wallis one-way analysis on ranks was used. A p value <0.05 defined a statistically significant difference. Statistical analysis was performed with Sigma Plot v13.0 software (Systat Software, San Jose, CA, USA).

Results

TASK channel in AC cells

The deletion of genes indicated that TASK3 proteins contribute more significantly to the resting membrane potential in mouse AC cells than TASK1 [26–27]. To explore this notion more directly, single channel currents were recorded from the cell-attached patches from young rat AC cells. The cells were perfused with physiological solution (5 mM K⁺) and the patch pipette was filled with 140 mM K⁺. When the pipette potential was set at 0 mV, inward channel openings were observed in almost all patch membranes (Fig. 1A). These channel currents were absent when K⁺ in the pipette was replaced with Na⁺ (not shown), indicating that they were K⁺ channels. The channel amplitude became close to zero at the pipette potential of −60 to −70 mV, which suggests that the resting membrane potential was near −60 to −70 mV, similar to that reported earlier in AC cells [28–29]. At pipette potentials more negative than the reversal potential, the channel current changed to the outward direction. The amplitude histogram of currents recorded at the pipette potential of 0 mV exhibited two peaks of −1.2 ± 0.1 pA (n = 4) and −2.1 ± 0.1 pA (n = 4) (Fig. 1B). Fig. 1C shows the current-voltage relationships of the two currents, revealing their inwardly rectifying property. Their single channel conductance levels were 16-pS and 36-pS in the inward current direction. The mean open time duration of the 16-pS channel was 1.0 ± 0.1 ms (n = 3), while that of the 36-pS channels was 1.2 ± 0.1 ms (Fig. 3D; n = 3). These properties of 16-pA and 36-pS channels are similar to those of homomeric TASK1 and homomeric TASK3 or heteromeric TASK1–3 exogenously expressed in HeLa cells [10]. Therefore, the 16-pS and 36-pS channels that we recorded in young AC cells most likely represent TASK1 and TASK3 or TASK1–3 channels, respectively. Single channel recordings from 12 cell-attached patches showed that TASK3-like channels are major TASK isoforms present in young AC cells, as the activity of TASK3-like channels was >10-fold higher than that of TASK1-like channels (Fig. 1B).

Fig. 1.

Single channel recordings from a cell-attached patch membrane of young AC cells

(A) condensed and expanded traces of single channel recordings from a cell-attached patch membrane at the patch membrane potential of 0 to −120 mV. The cell was perfused in 5 mM K⁺-containing external solution and the patch pipette was filled with 140 mM K⁺-containing solution. (B) Amplitude histogram of current recorded at the patch membrane of 0 mV. Bin size is 0.05pA. (C) Current-voltage relationships for single channel currents with small conductance (16 pS) and large conductance (36 pS). The voltage on the x-axis represents the pipette potential. Each point is the mean ± SD of 4 values. (D) Duration histogram for 36-pS channel activity at the patch membrane of 0 mV. Bin size is 0.5 ms.

Fig. 3.

Immunostaining for IF₁ in adrenal cortical and chromaffin cells of newborn rats

(A and B) Confocal images of IF₁- and chromogranin A (CgA) or CYP11A1-like immunoreactive (IR) material in dissociated adrenal cortical (AC) and adrenal medullary chromaffin (AMC) cells of newborn rats, respectively. Upper row in each panel shows confocal images of IF₁- and CgA- or CYP11A1-like immunofluorescence; lower row shows a differential interference contrast (DIC) image and merge of fluorescence and DIC images. IF₁- and CgA- or CYP11A1-like IR material were visible as FITC- and rhodamine-like fluorescence, respectively. Crude adrenal preparations were treated with collagenase and then cells were mechanically dissociated with needles in a glass-bottomed dish. Arrows represent lipid droplets.

Effects of mitochondrial inhibition on TASK channel activity in AC cells

The single channel analysis revealed that TASK channels are active and therefore contribute to the resting membrane potential in young rat AC cells. Thus, the effects of mitochondrial inhibitors on TASK channel activity in a cell-attached patch membrane were investigated. As shown in Fig. 2, A and B, the extracellular application of complex IV inhibitors (0.3 mM NaCN or 0.1 mM NaHS) for 90–100 s had no significant effect on TASK channel activity recorded in the cell-attached patch membrane of AC cells. The single channel recordings at the expanded scale showed that the single channel conductance and the opening frequency were not affected by the mitochondrial inhibitors. Channel activities were 0.063 ± 0.01 (control) and 0.058 ± 0.016 (NaCN) (n = 4) (p > 0.1) and 0.054 ± 0.014 (control) and 0.051 ± 0.0133 (NaHS) (n = 4) (p > 0.1). In contrast, addition of NaCN or NaHS to the bath solution produced reversible inhibition of TASK channel activity in the cell-attached patches from CB glomus cells. Channel activities in glomus cells were 0.36 ± 0.03 (control) and 0.06 ± 0.01 (NaCN) (n = 4) (p < .01) and 0.56 ± 0.04 (control) and 0.011 ± 0.003 (NaHS) (n = 4) (p < 0.01). Thus, TASK activity under control basal conditions was much higher in glomus cells than in AC cells, suggesting that TASK channel density is higher in glomus cells compared to that in AC cells. The effects of mitochondrial inhibitors on TASK are similar to those reported previously in rat glomus cells [10, 17].

Fig. 2.

Effects of mitochondrial inhibitors on single channel activity recorded from cell-attached membranes of AC and carotid body glomus cells

(A, B, C, and D) Condensed and expanded traces of single channel recordings from cell-attached patch membranes of young AC cells (A and B) and young carotid body glomus cells (C and D) at the pipette potential of 0 mV. NaCN (A and C) or NaHS (B and D) was bath applied to the cells during the indicated periods. a and b in the condensed traces correspond to a and b in the expanded traces.

Immunostaining in newborn AMC cells

The finding that mitochondrial inhibition had no effect on TASK channel activity in AC cells led us to investigate the expression of the mitochondrial IF₁ in newborn rat AC and AMC cells. The reason is that newborn rat AMC cells are capable of sensing a decrease in O₂ tension with suppression of K⁺ channels [5] and secrete catecholamine in response to acute hypoxia or mitochondrial inhibitors [1]. When crude adrenal medullary preparations were subjected to enzymatic digestion and the subsequent mechanical dissociation, isolated cells obtained consisted of AMC and AC cells, which were identified by the absence and presence of lipid droplets in the cytoplasm, respectively. When dissociated cells were stained for chromogranin A (CgA), a marker protein for AMC cells [24], cells with lipid droplets exhibited no CgA-like immunofluorescence. As shown in Fig. 3A, IF₁-like immunoreactive (IR) material was detected in lipid droplet-containing cells (AC cells), which were negative for CgA, and the cells (AMC cells) positive for CgA showed no immunoreactivity against IF₁. If IF₁-like immunofluorescence represents IF₁, its IR material is expected to be present in mitochondria [19]. This was examined by double staining for IF₁ and CYP11A1, which is an enzyme involved in adrenal steroid synthesis in mitochondria [30]. As is expected, IF₁-like immunofluorescence almost completely coincided with CYP11A1-like that (Fig. 3B). These findings show that the expression of IF₁ in newborn AMC cells is very low, compared with that in newborn AC cells (see below; Fig. 4)

Fig. 4.

Developmental change in IF₁ expression in AMC cells, but not in AC cells

(A and B) Confocal images of immunostaining for IF₁ in AMC and AC cells of newborn and adult rats, respectively. Arrows represent lipid droplets. (C) Summary of fluorescence intensity of IF₁ in the cytoplasm of AMC and AC cells of newborn and adult rats. Data represent means ± SEM (n = 25 and n = 12 for newborn AMC and AC cells, respectively; n = 26 and n = 7 for adult AMC and AC cells, respectively). ns stands for not significant.

Developmental change in IF₁ expression

To assess quantitatively the expression levels of IF₁ in AMC and AC cells, the fluorescence intensities in the cytoplasm and the nucleus of cells were separately measured and the latter was subtracted from the former to obtain the intensity of IF₁-specific fluorescence. The fluorescence intensity obtained in such a manner in newborn AMC cells that were identified by the absence of lipid droplets was markedly smaller than that in AC cells with lipid droplets (Fig. 4). In contrast to newborn adrenal cells, there was no difference in fluorescence intensity between AMC cells and AC cells of adult rats (Fig. 4). These results unambiguously indicate that among AMC and AC cells of newborn and adult rats, the expression level of IF₁ is very low only in newborn AMC cells and that of IF₁ is maintained at a constant level in AC cells irrespective of the developmental stage.

Discussion

TASK in adrenal cells

Our single channel recordings in adult rat AC cells show that 16-pS and 36-pS K⁺ channels are active at rest and thus contribute to the resting membrane potential in AC cells. The kinetic properties of these K⁺ channels are very similar to those of homomeric TASK1 (16 pS) and homomeric TASK3 (36 pS) or heteromeric TASK1–3 channels (36 pS) exogenously expressed in HeLa cells [10]. Therefore, the 16-pS and 36-pS channels most likely represent homomeric TASK1 and homomeric TASK3 and/or heteromeric TASK1–3 channels, respectively. In AC cells, the activity of TASK3-like channels was high compared with that of TASK1-like channels. The present experiment, however, could not determine what fractions of the 36-pS K⁺ channels were represented by the homomeric and heteromeric TASK3. The mRNAs subtracted from rat AC cells were found to encode TASK3, but not TASK1 [31], and deletion of the Task3 gene [26] resulted in a much larger depolarization in mouse AC cells, compared with that of the Task1 gene [27].

Furthermore, our previous immunocytochemical study suggested that the predominant type of TASK channels at the plasma membrane of AC cells is homomeric TASK3. First, TASK3-like IR material was mainly located at the cell periphery of rat AC cells, whereas the majority of TASK1-like IR material was present in the cytoplasm [32]. Secondly, this different distribution of TASK1 and TASK3 was reproduced by exogenous expression of GFP-TASK proteins in H295R cells [32], a cell line that originated from a human adrenal cortical carcinoma [33]. When the present findings are combined with earlier reports [26–27, 31–32]. It appears that homomeric TASK3 channels predominantly contribute to the resting membrane potential in rodent AC cells [34].

It is worth to note that heteromeric TASK1–3 channels are the major isoform contributing to the resting membrane potential in rat [10] and mouse [11] CB glomus cells. TASK1 and TASK3 proteins are expressed in both AC [27, 31, 35] and glomus cells [36–37]. In addition, p11, which plays an important role for heteromeric channel formation of TASK1 and TASK3 [23, 38], is also expressed in both cells [32, 37]. In fact, a sizeable fraction of TASK1 proteins in rat AC cells was found to form heteromeric channels with TASK3, which were mainly located in the cytoplasm. The difference in TASK channel subtype between rat AC cells and CB glomus cells may be explained at least in part by the notion that the expression level of TASK1 relative to that of TASK3 in AC cells is lower than that in glomus cells. Thus, excess TASK3, which escapes from the trapping by p11 through TASK1 in the endoplasmic reticulum [23, 39], can be trafficked to the cell membrane as a homomeric TASK3 channel in CB cells.

IF1 expression in adrenal cells

IF₁-like IR material was well expressed in adult rat AMC cells, but minimally expressed in newborn AMC cells, whereas it was well expressed in both newborn and adult AC cells. The anti-IF₁ antibody used in this experiment recognized a 10 KDa band of IF₁ [40] in the homogenates of adult rat adrenal medullae [22] and IF₁-like immunofluorescence coincided with immunofluorescence for the mitochondrial resident protein CYP11A1, which is involved in adrenal steroid synthesis [30]. These results indicate that IF₁-like IR material represents IF₁ and its expression level in newborn rat AMC cells is very low, compared with that in newborn and adult AC cells.

There are several reasons to assume that the practical absence of IF₁ expression in newborn rat AMC cells is relevant to the capability of the cells to secrete catecholamine in response to hypoxia and mitochondrial inhibitors [1]. First, the rank of order of mitochondrial inhibitors in inducing catecholamine secretion in guinea-pig AMC cells was found to be the same as that in producing a decrease in intracellular ATP [18]. Secondly, the expression level of IF₁ in the adrenal medullae of adult guinea pigs where exposure to hypoxia or mitochondrial inhibitors resulted in significant catecholamine secretion [41] was significantly lower than that in the adrenal medullae of adult rats [22] where mitochondrial inhibitors produced minimal catecholamine secretion [22]. Thirdly, TASK channel activity in the inside-out patch membrane of rat CB glomus cells depends on [ATP] at the cytoplasmic side of the patch membrane [17]. Thus, the reversible inhibition of TASK channel activity by extracellular application of mitochondrial inhibitors in glomus cells was suggested to be due to a reversible decrease in cellular ATP [17]. These earlier findings, together with our results presented here, suggest that hypoxia or mitochondrial inhibitors induce catecholamine secretion in AMC cells through a decrease in cellular ATP, which may be mainly due to the reverse operation of F₁F₀-ATPase because of the relative absence of IF₁ [19, 22]. The decrease in cellular ATP would inhibit TASK activity, causing cell depolarization and elevation of cytosolic [Ca²⁺], resulting in catecholamine secretion. This finding would suggest that the IF₁ level is very low in glomus cells from adult animals. Further studies are needed to prove this point.

The role of IF₁ is further supported by the findings that TASK channel activity recorded from cell-attached patches in adult AC cells where IF₁ is well expressed did not decrease when NaCN or NaHS was applied to the cells. In contrast to AC cells, K⁺ channel activity recorded in adult CB glomus cells markedly decreased following application of the mitochondrial inhibitors (Fig. 4) [42].

IF₁ was well expressed in both newborn and adult rat AC cells, indicating that IF₁ is expressed in AC cells throughout postnatal development. The reason could be that adrenal cortical steroids are essential for lung maturation [43] and maintenance of life [47]. If IF₁ were not expressed in AC cells, the cells would be deteriorated in ischemia or during exposure to hypoxia, in particular at the time of birth when the fetus is exposed to hypoxia.

Summary

The present study provides further evidence for functional relevance of IF₁ expression in excitability in AMC cells in response to hypoxia and mitochondrial inhibitors. The hypoxia detection mechanisms in O₂-sensing cells may be multiple and complex [8], and different levels of IF₁ expression in various cell types could explain some of the difference in the response to hypoxia and mitochondrial inhibitors. The relative absence of IF₁ expression may be responsible for a rapid decrease in cellular ATP in O₂-sensing cells, affecting the signaling to ion channels such as TASK channels in the plasma membrane.

Highlights.

1. IF₁ was well expressed in adult rat adrenal medullary chromaffin (AMC) cells.

2. IF₁ expression was minimal in newborn AMC cells.

3. IF₁ was well expressed in both newborn and adult adrenal cortical (AC) cells.

4. 36-pS TASK3-like channels were active at rest in young AC cells.

5. Mitochondrial inhibitors did not affect TASK3-like channels in young AC cells.

Abbreviations:

AMC

adrenal medullary chromaffin

AC

adrenal cortical

CB

carotid body

TASK

TWIK-related acid-sensitive K⁺

CgA

chromogranin A

IR

immunoreactive

IF₁

inhibitor factor 1