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Secretagogue-induced Exocytosis Recruits G Protein-gated K+ Channels to Plasma Membrane in Endocrine Cells

1999; Elsevier BV; Volume: 274; Issue: 12 Linguagem: Inglês

10.1074/jbc.274.12.7969

ISSN

1083-351X

Autores

Ken‐ichirou Morishige, Atsushi Inanobe, Yukiko Yoshimoto, Hirohisa Kurachi, Yuji Murata, Yoshimitu Tokunaga, Toshihiro Maeda, Yoshio Maruyama, Yoshihisa Kurachi,

Tópico(s)

Neuroscience and Neuropharmacology Research

Resumo

Stimulation-regulated fusion of vesicles to the plasma membrane is an essential step for hormone secretion but may also serve for the recruitment of functional proteins to the plasma membrane. While studying the distribution of G protein-gated K+ (KG) channels in the anterior pituitary lobe, we found KG channel subunits Kir3.1 and Kir3.4 localized on the membranes of intracellular dense core vesicles that contained thyrotropin. Stimulation of these thyrotroph cells with thyrotropin-releasing hormone provoked fusion of vesicles to the plasma membrane, increased expression of Kir3.1 and Kir3.4 subunits in the plasma membrane, and markedly enhanced KG currents stimulated by dopamine and somatostatin. These data indicate a novel mechanism for the rapid insertion of functional ion channels into the plasma membrane, which could form a new type of negative feedback control loop for hormone secretion in the endocrine system. Stimulation-regulated fusion of vesicles to the plasma membrane is an essential step for hormone secretion but may also serve for the recruitment of functional proteins to the plasma membrane. While studying the distribution of G protein-gated K+ (KG) channels in the anterior pituitary lobe, we found KG channel subunits Kir3.1 and Kir3.4 localized on the membranes of intracellular dense core vesicles that contained thyrotropin. Stimulation of these thyrotroph cells with thyrotropin-releasing hormone provoked fusion of vesicles to the plasma membrane, increased expression of Kir3.1 and Kir3.4 subunits in the plasma membrane, and markedly enhanced KG currents stimulated by dopamine and somatostatin. These data indicate a novel mechanism for the rapid insertion of functional ion channels into the plasma membrane, which could form a new type of negative feedback control loop for hormone secretion in the endocrine system. G protein-gated potassium channel inwardly rectifying potassium channel thyroid-stimulating hormone (thyrotropin) thyrotropin-releasing hormone adrenocorticotropic hormone reverse transcriptase-polymerase chain reaction membrane capacitance membrane conductance membrane current The G protein-gated K+(KG)1 channel, a member of the inwardly rectifying K+ (Kir) channel family, is directly activated by pertussis toxin-sensitive G proteins. This system was first discovered in the muscarinic deceleration of the heartbeat (1Kurachi Y. Am. J. Physiol. 1995; 269: C821-C830Crossref PubMed Google Scholar). More recently, it is considered to play an essential role in the hormone-mediated inhibitory regulation of neural excitability (1Kurachi Y. Am. J. Physiol. 1995; 269: C821-C830Crossref PubMed Google Scholar, 2Hille B. Neuron. 1992; 9: 187-195Abstract Full Text PDF PubMed Scopus (385) Google Scholar). Electrophysiological studies have revealed that a variety of inhibitory receptors, including M2-muscarinic, A1-purinergic, D2-dopamine, α2-adrenergic, serotonin, γ-aminobutyric acid type B, opioid, and somatostatin receptors in the brain, are coupled to KG channels (3North R.A. Br. J. Pharmacol. 1989; 98: 13-28Crossref PubMed Google Scholar). In endocrine organs such as the anterior pituitary lobe and pancreatic islet, it was also reported that some neurotransmitters including dopamine and somatostatin hyperpolarize the membrane by activating KG channels, which results in the inhibition of hormone secretion (4Einhorn L.C. Oxford G.S. J. Physiol. (Lond.). 1993; 462: 563-578Crossref Scopus (34) Google Scholar, 5Yatani A. Codina J. Sekura R.D. Birnbaumer L. Brown A.M. Mol. Endocrinol. 1987; 1: 283-289Crossref PubMed Scopus (133) Google Scholar, 6Pennefather P.S. Heisler S. McDonald J.F. Brain Res. 1988; 444: 346-350Crossref PubMed Scopus (49) Google Scholar, 7Rorsman P. Bokvist K. Ammala C. Arkhammar P. Berggren P.O. Larsson O. Wahlander K. Nature. 1991; 349: 77-79Crossref PubMed Scopus (110) Google Scholar). The main subunit of KG channels has been cloned from the heart and designated GIRK1/Kir3.1 (8Kubo Y. Reuveny E. Slesinger P.A. Jan Y.N. Jan L.Y. Nature. 1993; 364: 802-806Crossref PubMed Scopus (545) Google Scholar, 9Dascal N. Schreibmayer W. Lim N.F. Wang W. Chavkin C. DiMagno L. Labarca C. Kieffer B.L. Gaveriaux-Ruff C. Trollinger D. Lester H.A. Davidson N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10235-10239Crossref PubMed Scopus (337) Google Scholar). In the brain and heart, Kir3.1 forms functional KG channels by assembling with other Kir3.0 subunits such as GIRK2/Kir3.2, GIRK3/Kir3.3 (10Karschin C. Dissmann E. Stuhmer W. Karschin A. J. Neurosci. 1996; 16: 3559-3570Crossref PubMed Google Scholar, 11Ponce A. Bueno E. Kentros C. Vega-Saenz de Miera E. Chow A. Hillman D. Chen S. Zhu L. Wu M.B. Wu X. Rudy B. Thornhill W.B. J. Neurosci. 1996; 16: 1990-2001Crossref PubMed Google Scholar), and GIRK4/CIR/Kir3.4 (12Iizuka M. Tsunenari I. Momota Y. Akiba I. Kono T. Neuroscience. 1997; 77: 1-13Crossref PubMed Scopus (24) Google Scholar). Because Kir3.1 mRNA was detected in the anterior pituitary lobe (13Karschin C. Schreibmayer W. Dascal N. Lester H. Davidson N. Karschin A. FEBS Lett. 1994; 348: 139-144Crossref PubMed Scopus (80) Google Scholar) and pancreatic islet (14Ferrer J. Nichols C.G. Makhina E.N. Salkoff L. Bernstein J. Gerhard D. Wasson J. Ramanadham S. Permutt A. J. Biol. Chem. 1995; 270: 26086-26091Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), this subunit may also contribute to the formation of KG channels in endocrine cells. The anterior pituitary lobe contains several kinds of endocrine cells: lactotrophs, somatotrophs, corticotrophs, and thyrotrophs. Although electrophysiological experiments have shown that somatostatin or dopamine activate KG currents in lactotrophs (4Einhorn L.C. Oxford G.S. J. Physiol. (Lond.). 1993; 462: 563-578Crossref Scopus (34) Google Scholar) and also in several cell lines derived from the anterior pituitary lobe, such as GH3 (5Yatani A. Codina J. Sekura R.D. Birnbaumer L. Brown A.M. Mol. Endocrinol. 1987; 1: 283-289Crossref PubMed Scopus (133) Google Scholar) and AtT20 (6Pennefather P.S. Heisler S. McDonald J.F. Brain Res. 1988; 444: 346-350Crossref PubMed Scopus (49) Google Scholar), the cellular and subcellular localizations of KG channels in in vivo pituitary endocrine cells have not been examined. In this study, using a polyclonal antibody specific to Kir3.1, we found that this subunit was expressed only in thyrotroph cells and, surprisingly, was localized predominantly on intracellular secretory vesicles. Kir3.4 was co-localized on the vesicles with Kir3.1. Thyrotropin-releasing hormone (TRH) stimulation of thyrotrophs caused fusion of the vesicles to the cell membrane, increase of cell capacitance, and enhancement of dopamine- or somatostatin-induced KG current. These data indicate a novel mechanism for the rapid insertion of functional ion channels into the plasma membrane, which could form a new type of negative feedback control loop for thyrotrophs by tuning up their inhibitory regulatory signaling system in response to the stimulatory signal. The polyclonal antibody for Kir3.1 (aG1C-1) was raised in rabbit against a synthetic peptide corresponding to amino acid residues 488–501 (LPAKLRKMNSDRFT) of Kir3.1/GIRK1 (15Morishige K. Inanobe A. Takahashi N. Yoshimoto Y. Kurachi H. Miyake A. Tokunaga Y. Maeda T. Kurachi Y. Biochem. Biophys. Res. Commun. 1996; 220: 300-305Crossref PubMed Scopus (31) Google Scholar, 16Inanobe A. Morishige K. Takahashi N. Ito H. Yamada M. Takumi T. Nishina H. Takahashi K. Kanaho Y. Katada T. Kurachi Y. Biochem. Biophys. Res. Commun. 1995; 212: 1022-1028Crossref PubMed Scopus (87) Google Scholar). We have also developed the antibody to Kir3.4 (aG4N-10) in rabbit using the antigenic peptide, DSRNAMNQDMEIGV, which corresponds to amino acids 4–17 of Kir3.4 (17Krapivinsky G. Gordon E.A. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (754) Google Scholar). These antibodies have been successfully used for immunoprecipitation and Western blotting analyses of Kir3.1 protein (15Morishige K. Inanobe A. Takahashi N. Yoshimoto Y. Kurachi H. Miyake A. Tokunaga Y. Maeda T. Kurachi Y. Biochem. Biophys. Res. Commun. 1996; 220: 300-305Crossref PubMed Scopus (31) Google Scholar, 16Inanobe A. Morishige K. Takahashi N. Ito H. Yamada M. Takumi T. Nishina H. Takahashi K. Kanaho Y. Katada T. Kurachi Y. Biochem. Biophys. Res. Commun. 1995; 212: 1022-1028Crossref PubMed Scopus (87) Google Scholar) and Kir3.4 protein 2A. Inanobe and Y. Kurachi, unpublished results. in the brain and heart. Furthermore, in the rat heart, the immunoreactivities were detected with the antibodies only in the atrium but not in the ventricle (data not shown). This finding coincides with the expression of the cardiac KG channel, which is composed of the subunits Kir3.1 and Kir3.4 in the heart atrium (13Karschin C. Schreibmayer W. Dascal N. Lester H. Davidson N. Karschin A. FEBS Lett. 1994; 348: 139-144Crossref PubMed Scopus (80) Google Scholar). Immunohistochemistry was performed according to a method described elsewhere (15Morishige K. Inanobe A. Takahashi N. Yoshimoto Y. Kurachi H. Miyake A. Tokunaga Y. Maeda T. Kurachi Y. Biochem. Biophys. Res. Commun. 1996; 220: 300-305Crossref PubMed Scopus (31) Google Scholar). Rats were anesthetized with sodium pentobarbital and perfused transcardially with 4% (w/v) paraformaldehyde, 0.5% (w/v) glutaraldehyde, and 0.2% (w/v) picric acid, 0.1 m phosphate buffer (pH 7.4). The anterior pituitary lobe was removed, postfixed in a fixative containing 4% paraformaldehyde and 0.2% picric acid for 48 h at 4 °C, and transferred to 0.1 m phosphate buffer containing 15% (w/v) sucrose and 0.1% (w/v) sodium azide at 4 °C. Sections were cut with a cryostat at 20 μm of thickness and stored at 4 °C in the same solution until used. After rinsing, the sections were placed in free-floating states into aG1C-1 that had been diluted (1:10,000) in phosphate-buffered saline containing 0.3% (v/v) Triton X-100 and 1% (w/v) bovine serum albumin and incubated for 3 days at 4 °C. After incubation with biotinylated goat anti-rabbit IgG, staining was accomplished using the avidin-biotin complex method (Vectastain Elite kit, Vector Laboratories, Burlingame, CA), with nickel-diaminobenzidine as the chromogen. Control procedures consisted of preabsorbing aG1C-1 (1:10,000 dilution) by incubating with a saturating concentration of the antigenic peptide. For double immunofluorescent staining, sections were first incubated with aG1C-1 and a monoclonal antibody for each pituitary hormone,i.e. anti-prolactin monoclonal antibody (QED Bioscience Inc., San Diego, CA), anti-growth hormone monoclonal antibody (Quartett, Berlin, Germany), anti-luteinizing hormone monoclonal antibody (Quartett), anti-adrenocorticotropic hormone (ACTH) monoclonal antibody (YLEM, Rome, Italy), or anti-thyrotropin (TSH) monoclonal antibody (Quartett). The sections were washed thoroughly and incubated with fluorescein isothiocyanate-labeled anti-rabbit IgG (E. Y. Laboratories, San Mateo, CA) and Texas Red-labeled anti-mouse IgG (Protos ImmunoResearch, San Francisco, CA). These sections were examined with a confocal microscope (MRC-1024, Bio-Rad Laboratories, Hertfordshire, U. K.). For electron microscopy, sections were processed according to the method described elsewhere (18Tadokoro C. Yoshimoto Y. Sakata M. Imai T. Yamaguchi M. Kurachi H. Oka Y. Maeda T. Miyake A. Biochem. Biophys. Res. Commun. 1995; 214: 1211-1218Crossref PubMed Scopus (22) Google Scholar). TRH stimulation involved the injection of 10 μg of TRH into the femoral vein 10 min before the perfusion, which was enough to stimulate the thyrotroph in vivo (19Martin J.B. Reichlin S. Science. 1970; 168: 1366-1368Crossref PubMed Scopus (36) Google Scholar). The vibratome sections were dehydrated in a graded series of ethanol and incubated in a mixture of ethanol and LR Gold resin (London Resin, Berkshire, U. K.) followed by flat embedment in fresh LR Gold containing 0.1% (v/v) benzyl on silicon-coated glass slides. The embedded sections were polymerized for 6 h in an ultraviolet cryochamber (Pelco, Ted Pella Inc., Redding, CA) at −20 °C. Small blocks containing anterior pituitary lobe were cut out and glued to black resin. Ultrathin sections were cut using an ultramicrotome (Ultracut UCT, Leika, Vienna, Austria) and mounted on collodion and carbon-coated nickel grids (thin bar grid, Nishin EM Co. Ltd., Tokyo, Japan). The nickel grids with ultrathin sections were incubated with 3% (v/v) normal goat serum in 0.1 mphosphate-buffered saline containing 0.2% bovine serum albumin, 0.2% (w/v) saponin, and 0.05% (w/v) NH4Cl for 30 min at room temperature. The grids were then incubated with aG1C-1 (1:100) or aG4N-10 (1:50) in the same buffer for 2 h and then with a 15-nm immunogold conjugated goat anti-rabbit IgG (British BioCell International Inc., Cardiff, U. K., diluted 1:40) for 1.5 h at room temperature. In the case of double immunolabeling, the grids were incubated with aG1C-1 (1:100) or aG4N-10 (1:50), and anti-TSH monoclonal antibody (1:10) followed by incubation with immunogold-conjugated anti-rabbit IgG (15-nm gold particles) and immunogold-conjugated anti-mouse IgG (10 nm gold particles). The grids were washed with 0.1 m phosphate-buffered saline and then with distilled water before staining with uranyl acetate and Reynold's lead citrate. These sections were examined with an electron microscope (H7100TE, Hitachi, Hitachinaka, Japan). Anterior pituitary glands were dissected from adult female Wistar rats. Cells were dispersed enzymatically using a non-trypsin dissociation protocol modified from that used by Einhorn and Oxford (4Einhorn L.C. Oxford G.S. J. Physiol. (Lond.). 1993; 462: 563-578Crossref Scopus (34) Google Scholar). In brief, the glands were removed and minced in sterile Hanks' balanced salt solution that was free of calcium and magnesium (Hanks' CMF). After washing, the fragments were incubated in a shaking water bath for 1 h in 37 °C in Hanks' CMF containing trypsin inhibitor (Sigma, 0.1 mg/100 ml), collagenase (Worthington, 0.3%), and DNase I (Sigma, 1.0 mg/100 ml). Fragments were then mechanically dispersed by trituration with a siliconized Pasteur pipette, washed with Dulbecco's modified Eagle's medium, filtered through nylon mesh (20 μm), and harvested via centrifugation. Cells were incubated in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Whole cell recordings were made utilizing the gigaohm seal patch-clamp technique (20Ishii M. Horio Y. Tada Y. Hibino H. Inanobe A. Ito M. Yamada M. Gotow T. Uchiyama Y. Kurachi Y. J. Neurosci. 1997; 17: 7725-7735Crossref PubMed Google Scholar). Recordings were made using a patch-clamp amplifier (EPC-7, List Electronics, Darmstadt, Germany) and recorded on videocassette tapes with a PCM converter system (RP-880, NF Electronic Circuit Design, Yokohama, Japan), reproduced and low pass-filtered at 1 kHz (−3 db) by a filter with Bessel characteristics (48 db/octave slope attenuation) for an analysis and illustration. Experiments were performed at 22–24 °C in a bath solution composed (in mm) of the following: NaCl (91), KCl (50), MgCl2 (1Kurachi Y. Am. J. Physiol. 1995; 269: C821-C830Crossref PubMed Google Scholar), glucose (5.5), and Hepes buffer (5Yatani A. Codina J. Sekura R.D. Birnbaumer L. Brown A.M. Mol. Endocrinol. 1987; 1: 283-289Crossref PubMed Scopus (133) Google Scholar), pH 7.2. The composition of the pipette filling solution was as follows (in mm): KCl (140), MgCl2 (8Kubo Y. Reuveny E. Slesinger P.A. Jan Y.N. Jan L.Y. Nature. 1993; 364: 802-806Crossref PubMed Scopus (545) Google Scholar), EGTA (0.1), CaCl2 (0.0415), GTP (0.1), and Hepes (5Yatani A. Codina J. Sekura R.D. Birnbaumer L. Brown A.M. Mol. Endocrinol. 1987; 1: 283-289Crossref PubMed Scopus (133) Google Scholar), pH 7.2. Cytoplasmic RNAs of a dissociated single pituitary cell, which was aspirated and transferred into a microcentrifuge tube with a glass tip used for patch-clamp experiments, were extracted, and cDNAs were synthesized as described elsewhere (21Sucher N.J. Deitcher D.L. Neuron. 1995; 14: 1095-1100Abstract Full Text PDF PubMed Scopus (58) Google Scholar). To eliminate the contamination of genomic DNAs, we treated RNA samples with DNase I (Takara) followed by cDNA synthesis with reverse transcriptase (Life Technologies, Inc.). To ascertain that cytoplasmic RNAs were correctly aspirated, samples were conducted to RT-PCR of β-actin by the β-actin PCR kit (CLONTECH). We could detect the β-actin transcripts in all of the Type I cells, 16 Type II cells, 45 Type III cells, and 45 cells after capacitance measurement. Using the β-actin detectable samples, nested PCR was done for Kir3.1, Kir3.2, and Kir3.4. Primers for Kir3.1 PCR corresponded to nucleotides −10 to 10 and 760–781 (first PCR) and −10 to 10 and 736–757 (second PCR) of rat Kir3.1 cDNA (8Kubo Y. Reuveny E. Slesinger P.A. Jan Y.N. Jan L.Y. Nature. 1993; 364: 802-806Crossref PubMed Scopus (545) Google Scholar). Primers for Kir3.2 PCR corresponded to nucleotides 171–190 and 893–912 (first PCR) and 285–305 and 879–897 (second PCR) of rat Kir3.2 cDNA (22Lesage F. Duprat F. Fink M. Guillemare E. Coppola T. Lazdunski M. Hugnot J.P. FEBS Lett. 1994; 353: 37-42Crossref PubMed Scopus (268) Google Scholar). Primers for Kir3.4 PCR corresponded to nucleotides 904–923 and 1276–1294 (first PCR) and 989–1006 and 1246–1265 (second PCR) of rat Kir3.4 cDNA (17Krapivinsky G. Gordon E.A. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (754) Google Scholar). For TSHβ PCR, primers corresponded to nucleotides 101–121 and 378–398 of rat TSHβ cDNA (23Croyle M.L. Bhattacharya A. Gordon D.F. Maurer R.A. DNA (N. Y.). 1986; 5: 299-304Crossref PubMed Scopus (33) Google Scholar). PCR amplification was performed for 30 cycles at 94 °C for 45 s, at 58 °C (Kir3.1), 60 °C (Kir3.2), 55 °C (Kir3.4), or 56 °C (TSHβ) for 1 min, and at 72 °C for 1 min and then at 72 °C for 8 min. Because the products from the single pituitary cell were insufficient for visualization, second cycles of reactions using the second PCR primers and the same amplification conditions were performed with a part of the first PCR products. The nucleotide sequence of the PCR products was confirmed with the dye primer method and DNA sequencer (A-381, Perkin-Elmer) after TA cloning (Invitrogen). Simultaneous measurements of changes in membrane capacitance (Cm), conductance (Gm), and membrane current (Im) were carried out using a patch-clamp amplifier (EPC-7, List Electronics) and a two-phase lock-in amplifier (NF5610B, NF Electronic Circuit Design) as described previously (24Maruyama Y. J. Physiol. (Lond.). 1996; 492: 807-814Crossref Scopus (15) Google Scholar). The capacitance transients originating from the capacitance of the plasma membranes were minimized by adjusting the capacitance and the time constant of the capacitance cancellation circuit in the patch-clamp amplifier while applying voltage pulses with an amplitude of 10 mV. This adjustment gave whole cell capacitance values of 9.0–13.4 picofarads and series resistance values of 5–9 megaohms. After the cancellation, a 600-Hz, 3.4-mV peak to peak sine-wave voltage was superimposed on the holding potential of −100 mV. The resulting current output was fed into the lock-in amplifier. The phase offset of the lock-in amplifier was adjusted so that when the capacitance of the cancellation circuit was modified to calibrate Cm, there was no change in the output for Gm (closed circles at theCm trace in Fig. 3 A). KG channels are heteromultimeric proteins composed of the subunits Kir3.1 + Kir3.4 in the heart (17Krapivinsky G. Gordon E.A. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (754) Google Scholar) and Kir3.1 + Kir3.2 in the brain (25Lesage F. Guillemare E. Fink M. Duprat F. Heurteaux C. Fosset M. Romey G. Barhanin J. Lazdunski M. J. Biol. Chem. 1995; 270: 28660-28667Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 26Liao Y.J. Jan Y.N. Jan L.Y. J. Neurosci. 1996; 16: 7137-7150Crossref PubMed Google Scholar). The Kir3.1 subunit may therefore be common to the KG channels of various tissues (8Kubo Y. Reuveny E. Slesinger P.A. Jan Y.N. Jan L.Y. Nature. 1993; 364: 802-806Crossref PubMed Scopus (545) Google Scholar, 9Dascal N. Schreibmayer W. Lim N.F. Wang W. Chavkin C. DiMagno L. Labarca C. Kieffer B.L. Gaveriaux-Ruff C. Trollinger D. Lester H.A. Davidson N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10235-10239Crossref PubMed Scopus (337) Google Scholar). We examined the distribution of Kir3.1 immunoreactivity in sections of rat pituitary using a specific polyclonal antibody (aG1C-1) (15Morishige K. Inanobe A. Takahashi N. Yoshimoto Y. Kurachi H. Miyake A. Tokunaga Y. Maeda T. Kurachi Y. Biochem. Biophys. Res. Commun. 1996; 220: 300-305Crossref PubMed Scopus (31) Google Scholar, 16Inanobe A. Morishige K. Takahashi N. Ito H. Yamada M. Takumi T. Nishina H. Takahashi K. Kanaho Y. Katada T. Kurachi Y. Biochem. Biophys. Res. Commun. 1995; 212: 1022-1028Crossref PubMed Scopus (87) Google Scholar) (Fig.1). The Kir3.1-positive cells were scattered in the anterior pituitary lobe and made up ∼3% of the anterior pituitary cells (Fig. 1 A). Under the light microscope Kir3.1 immunoreactivity was detected diffusely in the cytosol of each positive cell (Fig. 1, A and B). The Kir3.1-positive cells had a polygonal or stellate shape between 20 and 30 μm in size. To identify which types of pituitary cells express Kir3.1, sections were double-stained for Kir3.1 and various pituitary hormones. Fig. 1, B and C, shows that Kir3.1 immunoreactivity (green) was detected only in the cells that were stained with anti-TSH antibody (red). In 176 TSH-positive cells (i.e. thyrotrophs), 149 cells (∼85%) were also Kir3.1-positive. No Kir3.1 immunoreactivity was detected in cells stained with anti-prolactin, anti-growth hormone, anti-luteinizing hormone, or anti-ACTH antibodies. Prominentorange-yellow signals generated by the double staining ofgreen (Kir3.1) and red (TSH) suggest that they may be localized in close proximity in thyrotrophs (Fig.1 D). The subcellular localization of Kir3.1 was examined using immunoelectron microscopy (Fig. 1, E, F, and H). The immunoreactive signals (gold particles) were detected predominantly on secretory vesicles and rarely on the plasma membrane of Kir3.1-positive pituitary cells (Fig. 1 E). The cells were ∼30 μm in diameter and polygonal in shape. The reactive intracellular secretory vesicles were ∼100 nm in diameter. These morphological features of Kir3.1-positive cells are those of the thyrotroph (27Ozawa H. Kurozumi K. Anat. Embryol. 1989; 180: 207-212Crossref PubMed Scopus (16) Google Scholar). Furthermore, with double immunolabeling under the electron microscope, we found that Kir3.1 (detected by 15-nm gold particles) and TSH (10-nm gold particles) were localized in the same secretory vesicles (Fig. 1 E, inset). In contrast, the adjacent cell, which contained large 200–300-nm-diameter electron dense vesicles, did not show any immunoreactivity to aG1C-1. Fig. 1, F and H, depicts examples of Kir3.1 immunoreactivity in the thyrotrophs obtained from TRH-administered rats. TRH stimulation caused an increase in the detection of gold particles on the plasma membrane (Fig. 1 F,arrows). The number of gold particles detected on the plasma membrane in the control was 0.25 ± 0.25% (mean ± S.E.,n = 15 cells) of the total number of particles that existed within 1.5 μm of the plasma membrane. This number increased to 7.56 ± 1.80% (n = 15 cells) after TRH stimulation (Fig. 1 G). Furthermore, as indicated by thearrowheads in Fig. 1 H, after TRH stimulation various stages of fusion of the gold particle-positive vesicles with the plasma membrane were detected. These results suggest that the KG channels on the secretory vesicles of thyrotrophs could be translocated to the plasma membrane during TRH-induced exocytosis. It is known that dopamine-D2 and somatostatin receptors can couple to KG channels via pertussis toxin-sensitive G proteins in pituitary cells (2Hille B. Neuron. 1992; 9: 187-195Abstract Full Text PDF PubMed Scopus (385) Google Scholar). Therefore, TRH-induced recruitment of the KG channels to the plasma membrane of thyrotrophs may in turn facilitate the effect of inhibitory transmitters such as dopamine and somatostatin. To examine this possibility, electrophysiological techniques were applied to dissociated pituitary cells (Fig. 2). From 82 cells examined, we observed three distinct types of cells in terms of their responses to bromocriptine, somatostatin, and TRH (Types I–III). Fig.2 A depicts a representative cell current record of the response of Type I cells (n = 6). The dopamine receptor agonist bromocriptine induced a small inwardly rectifying K+ (Kir) current, which was markedly enhanced upon the addition of TRH to the bath. The enhanced bromocriptine-induced Kir current was inhibited by sulpiride, an antagonist for the dopamine receptor. After ∼10 min of wash-out of bromocriptine and sulpiride, somatostatin induced a small Kir current similar to that induced by bromocriptine under control conditions. The somatostatin-induced Kir current was also enhanced by the addition of TRH. In the six Type I cells, TRH increased the bromocriptine-induced KG current by 6.0 ± 0.5-fold (mean ± S.E.) and the somatostatin-induced KG current by 7.0 ± 0.8-fold, measured at the command pulse to −100 mV. TRH alone, in the absence of bromocriptine or somatostatin, did not induce any appreciable Kir current (data not shown). Both bromocriptine- and somatostatin-induced currents reversed at approximately −30 mV close to the equilibrium potential for K+ with 50 mm extracellular K+ and exhibited clear inward rectification (Fig.2 A, lower right panel). The currents activated slowly during hyperpolarizing command voltage pulses, which is a feature of KG channels containing Kir3.1 subunits (Fig.2 A, lower left panels) (17Krapivinsky G. Gordon E.A. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (754) Google Scholar, 25Lesage F. Guillemare E. Fink M. Duprat F. Heurteaux C. Fosset M. Romey G. Barhanin J. Lazdunski M. J. Biol. Chem. 1995; 270: 28660-28667Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). In RT-PCR analysis of cytoplasmic RNA aspirated from the cell presented in Fig.2 A, we detected transcripts of Kir3.1 and TSHβ as shown inlane 1 of Fig. 2 D. The same results were obtained in the remaining five Type I cells (including the result shown inlane 4 of Fig. 2 D). Thus, Type I cells are thyrotrophs that may possess KG channels containing the Kir3.1 subunit. Because it is known that homomeric KG channels composed of Kir3.1 are not functional (17Krapivinsky G. Gordon E.A. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (754) Google Scholar) but that heteromers of Kir3.1 and another Kir3.0 subunit form functional KG channels, we have tried to identify the Kir3.0 subunit that may assemble with Kir3.1 in the vesicular KG channels. Kir3.3 was not detected by RT-PCR in the mRNA from whole anterior pituitary lobe (data not shown). Therefore, the expression of Kir3.2 and Kir3.4 subunits was examined by single-cell RT-PCR. All six Type I cells expressed a Kir3.4 transcript, and one of them also expressed Kir3.2. Furthermore, we detected Kir3.4 immunoreactivity (15-nm gold particles) on the vesicles containing TSH (10-nm particles) with electron microscopic immunocytochemistry (Fig. 2 E). Kir3.2 immunoreactivity was not detected on the vesicles (data not shown). These results suggest that Kir3.4 may be the subunit that makes up the vesicular KG channel with Kir3.1. In Type II cells (n = 21), bromocriptine or somatostatin induced a very small Kir current, which was not affected by TRH (Fig. 2 B). Lanes 2 and 5 in Fig. 2 D depict examples of RT-PCR analysis of Type II cell mRNA; lane 2 is from the cell shown in Fig.2 B. In the cytoplasmic RNAs of 10 cells of this type, we detected transcripts of Kir3.2 and/or Kir3.4 but not of Kir3.1. TSHβ mRNA was detected only in the cell shown in lane 2 but not in the remaining nine Type II cells. In Type III response cells (n = 55), bromocriptine or somatostatin did not induce any Kir current, and TRH showed no appreciable effect (Fig.2 C). We could not detect any transcripts of Kir3.1, Kir3.2, or TSHβ in 10 Type III cells examined (lane 3 in Fig.2 D is from the cell shown in Fig. 2 C). Two of the 10 Type III cells expressed Kir3.4 mRNA. In conclusion, TRH enhances bromocriptine or somatostatin activation of KGcurrent specifically in thyrotrophs, which express Kir3.1 mRNA. To examine whether the TRH-induced augmentation of KGchannel activity in Type I thyrotrophs is derived from the exocytosis of secretory vesicles, as was suggested by the electron microscopic examination (Fig. 1, E–H), we simultaneously measuredCm, Gm, and Imin Type I cells (Fig. 3 A). The cells were bathed in 5 mm K+ bathing solution and held at −100 mV. A 600-Hz sine wave with a peak to peak amplitude of 3.4 mV was applied to the cell under voltage clamp. The application of bromocriptine caused some increase of Gm and of the small inward K+ current without any significant effect on Cm. When TRH was added in the continued presence of bromocriptine, the inward K+ current was markedly enhanced in parallel with the simultaneous increase of bothCm and Gm. The increase ofCm may reflect the fusion of secretory vesicles to the plasma membrane induced by TRH (28Neher E. Marty A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6712-6716Crossref PubMed Scopus (796) Google Scholar). In the continued presence of TRH, Cm, Gm, andIm fluctuated in parallel and slowly returned toward their basal levels. The decrease of Cm may represent endocytosis of membrane containing KG channels. This TRH-induced response was observed in 5 of 45 cells examined. In these 5 cells, expression of mRNAs of Kir3.1, Kir3.4, and TSHβ was confirmed by retrospective single-cell RT-PCR (data not shown). The remaining 40 cells did not express any Kir3.1transcript. The simultaneous measurement of Cm,Gm, and Im indicates that TRH-induced augmentation of KG channel activity in Type I cells is associated with the exocytosis of secretory vesicles. TRH-induced TSH secretion may be composed of voltage-dependent and voltage-independent components, as is the case for prolactin secretion by TRH in the lactotroph (29Fomina A.F. Levitan E.S. J. Neurosci. 1995; 15: 4982-4991Crossref PubMed Google Scholar). The increase in Cm measured in Fig. 3 A must represent a voltage-independent component because these experiments were conducted under voltage clamp. The TRH-induced voltage-independent fusion of secretory vesicles causes an increase in the number of KG channels on the plasma membrane. The increased KG channels should then facilitate the hyperpolarization induced by dopamine and somatostatin, which may suppress the depolarization-induced secretion. TRH-induced recruitment of vesicular KG channels to the plasma membrane may act as a feedback control mechanism to avoid excessive stimulation of the cell (Fig.3 B). At the crustacean neuromuscular junction, repetitive stimulation of motor nerves resulted in an increase of the number of active zones containing a voltage-dependent Ca2+ channel at the axonal terminus, which was implicated in the long term facilitation of neurotransmitter release (30Wojtowicz J.M. Marin L. Atwood H.L. J. Neurosci. 1994; 14: 3688-3703Crossref PubMed Google Scholar). The increase of active zones is thought to be the result of the translocation to the plasma membrane of a voltage-dependent Ca2+ channel localized on secretory vesicles (31Passafaro M. Rosa P. Sala C. Clementi F. Sher E. J. Biol. Chem. 1996; 271: 30096-30104Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Similarly, it was shown that the number of postsynaptic γ-aminobutyric acid type A receptors was rapidly increased by insulin (32Wan Q. Xiong Z.G. Man H.Y. Ackerley C.A. Braunton J. Lu W.Y. Becker L.E. McDonald J.F. Wang Y.T. Nature. 1997; 388: 686-690Crossref PubMed Scopus (461) Google Scholar) or in an experimental model of temporal lobe epilepsy (33Nusser Z. Hajos N. Somogyi P. Mody I. Nature. 1998; 395: 172-177Crossref PubMed Scopus (399) Google Scholar). The stimulation-induced increase of γ-aminobutyric acid type A receptors may underlie the long term modification of synaptic transmission in hippocampal inhibitory synapses (32Wan Q. Xiong Z.G. Man H.Y. Ackerley C.A. Braunton J. Lu W.Y. Becker L.E. McDonald J.F. Wang Y.T. Nature. 1997; 388: 686-690Crossref PubMed Scopus (461) Google Scholar, 33Nusser Z. Hajos N. Somogyi P. Mody I. Nature. 1998; 395: 172-177Crossref PubMed Scopus (399) Google Scholar). These previous studies require a mechanism that enables stimulation to translocate ion channels from an intracellular pool to the plasma membrane. This study provides, for the first time, clear evidence to indicate that the localization of ion channels on secretory vesicles actually plays a role in the physiological regulation of membrane excitability. Further studies are needed to elucidate the molecular mechanisms localizing ion channels on secretory vesicles. This line of study may provide novel insights into the stimulation control of cellular excitability, including long term modification of synaptic transmission. We thank Dr. Ian Findlay (University of Tours, Tours, France) for critical reading of this manuscript. We also thank Kiyomi Okuto for technical assistance and Keiko Tsuji for secretarial support.

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