Molecular Components of Large Conductance Calcium-Activated Potassium (BK) Channels in Mouse Pituitary Corticotropes

Michael J. Shipston, Rory R. Duncan, Alan G. Clark1, Ferenc A. Antoni and Lijun Tian

Membrane Biology Group (M.J.S., R.R.D., A.G.C., L.T.) Department of Biomedical Sciences University of Edinburgh Medical School Edinburgh, Scotland, UK, EH8 9AG
Medical Research Council Brain Metabolism Unit (F.A.A.) Edinburgh, Scotland, UK, EH8 7NA


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Large-conductance calcium- and voltage- activated potassium (BK) channels play a fundamental role in the signaling pathways regulating mouse anterior pituitary corticotrope function. Here we describe the cloning and functional characterization of the components of mouse corticotrope BK channels. RT-PCR cloning and splice variant analysis of mouse AtT20 D16:16 corticotropes revealed robust expression of mslo transcripts encoding pore-forming {alpha}-subunits containing the mouse homolog of the 59-amino acid STREX-1 exon at splice site 2. RT-PCR and functional analysis, using the triterpenoid glycoside, DHS-1, revealed that native corticotrope BK channels are not functionally coupled to ß-subunits in vivo. Functional expression of the STREX-1 containing {alpha}-subunit in HEK 293 cells resulted in BK channels with calcium sensitivity, single-channel conductance, and inhibition by protein kinase A identical to that of native mouse corticotrope BK channels. This report represents the first corticotrope ion channel to be characterized at the molecular level and demonstrates that mouse corticotrope BK channels are composed of {alpha}-subunits expressing the mouse STREX-1 exon.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The excitability of anterior pituitary cells is tightly controlled by G-protein-linked receptor-mediated regulation of multiple ion channels in the plasma membrane of target cells (1, 2, 3). Although pharmacological and electrophysiological analysis has revealed the nature of some of these ion channels, molecular components of anterior pituitary ion channels are poorly characterized.

In mouse AtT20 corticotropes large conductance calcium- and voltage- activated potassium (BK) channels play a pivotal role in the regulation of ACTH secretion (4, 5). In this system the stimulatory neuropeptide, CRH, potently inhibits BK channels through protein kinase A (PKA)-mediated phosphorylation of the BK channel complex (5, 6). Importantly, glucocorticoids prevent PKA inhibition of BK channels, and this action of the steroid is pivotal to early glucocorticoid inhibition of ACTH secretion in this system (4, 5, 6).

In contrast to the multiple gene families of other voltage-dependent cation channels, the pore-forming {alpha}-subunits of BK channels are encoded by a single gene, Slo (7, 8), that undergoes extensive, hormonally regulated, alternative RNA splicing (9, 10). These splice variants give rise to channels with different functional properties, including calcium sensitivity, single-channel conductance, and regulation by intracellular signaling pathways (11, 12, 13, 14). In addition, association of {alpha}-subunits with accessory subunits may result in differential channel properties or modify their cellular distribution (15, 16, 17, 18).

Mouse corticotrope BK channels display considerably higher calcium sensitivity (6) than previously identified {alpha}-subunits cloned from mouse brain. Furthermore, in common with native BK channels from several cell types of the anterior pituitary (5, 19, 20), mouse corticotrope BK channels are inhibited by PKA-mediated phosphorylation, whereas the majority of brain BK channels are activated by PKA (21, 22).

Elucidation of the molecular components of BK channels in specific cell types is beginning to reveal the molecular basis for their functional role in fundamental physiological processes (23, 24). To further address the functional role, and molecular regulation, of BK channels in mouse corticotrope cell function, we have 1) characterized and cloned the BK channel {alpha}-subunit splice variants expressed in mouse AtT20 D16:16 corticotropes, and 2) directly compared the functional characteristics (calcium sensitivity and regulation by PKA) of these identified subunits expressed in HEK 293 cells with native AtT20 D16:16 BK channels.

This report represents the first corticotrope ion channel to be functionally characterized at the molecular level. We demonstrate that mouse corticotrope BK channels are composed of {alpha}-subunits expressing the mouse homolog of the previously described cysteine-rich STREX-1 exon (10, 25). Furthermore, the STREX-1 {alpha}-subunit variants expressed in HEK 293 cells are inhibited by PKA-mediated phosphorylation.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning and Splice Variant Analysis of Mouse Corticotrope BK Channel {alpha}-Subunits
Cloned RT-PCR products of the entire open reading frame (ORF) of BK channel {alpha}-subunits from AtT20 D16:16 corticotropes had sequence identical to previously published mouse brain mslo BK channels (8, 26) with variations at known mammalian splice sites (25) (Fig. 1AGo). The majority of full-length cDNA clones, as well as RT-PCR products spanning {alpha}-subunit splice sites expressed in mouse AtT20 D16:16 corticotropes, contained the mouse variant of the previously described cysteine-rich, 59-amino acid insert at splice site 2, termed STREX-1 (10, 25). In approximately 25% of clones, either the IYF motif or null (ZERO) insert was expressed at splice site 2 (Fig. 1AGo). No evidence for inserts at splice sites 1, 3, or 4 was found (Fig. 1AGo). A STREX-1 splice site 2 insert identical to that characterized in AtT20 D16:16 corticotropes was also expressed in adult mouse anterior pituitary.



View larger version (29K):
[in this window]
[in a new window]
 
Figure 1. BK Channel {alpha}-Subunit Splice Variants Expressed in Mouse AtT20 Corticotropes

A, Schematic of mammalian splice site inserts characterized in mouse AtT20 D16:16 corticotropes by RT-PCR analysis (see Materials and Methods) with predicted inserts represented by single amino acid codes. B, Amino acid sequence alignment of mammalian STREX-1 inserts characterized to date: mouse (AtT20 D16:16 cell and normal mouse pituitary, this study); rat chromaffin cells (25 ) and rat anterior pituitary (this study); rabbit kidney tubules (29 ); and human pancreatic islets (27 ). Boxed amino acids indicate differences from the mouse STREX-1 insert. Note: the STREX-2 insert (10 ) that contains additional amino acids at the N terminus of the STREX-1 exon have not been included in the alignment. Alignment positions are relevant to the start proline (P) amino acid of the respective splice insert. C, Western blot analysis of BK channel {alpha}-subunit expression in partially purified membrane fractions using the C-terminally directed {alpha}slo(913-926) antibody as described in Materials and Methods. This antibody recognizes the VNDTNVQFLDQDDD motif in the C-terminal tail (indicated by * in panel A) conserved in all mslo variants (28 ). Membranes were isolated from: HEK 293 cells stably expressing the mouse AtT20 corticotrope STREX-1 variant (10 µg/lane, STREX-1); native AtT20 cells (40 µg/lane, AtT20); and mock transfected HEK 293 cells (40 µg/lane, HEK).

 
The mouse corticotrope splice site 2 insert (STREX-1) was similar, at the amino acid level, to recently identified STREX-1 exons in other mammalian endocrine cells including rat chromaffin cells (25) and human pancreatic islets (27) (Fig. 1BGo). Western blot analysis was performed using the C-terminally directed {alpha}-subunit antibody {alpha}slo(913-926) (28) that recognizes all mslo variants. Immunoblotting of partially purified plasma membranes from native mouse AtT20 corticotropes and HEK 293 cells stably expressing the entire coding region of the cloned STREX-1 {alpha}-subunit revealed expression of an approximately 125-kDa immunoreactive protein band that was not detectable in mock transfected HEK 293 cells: the predicted molecular mass of the STREX-1 variant from translation of the ORF is 131 kDa.

Functional Characterization of Mouse Corticotrope {alpha}-Subunits Expressed in HEK 293 Cells
Expression of the STREX-1 and ZERO variants of mouse AtT20 corticotrope BK channel {alpha}-subunits in HEK 293 cells resulted in large outward macropatch voltage- and calcium-activated potassium currents (Fig. 2Go). In mock transfected HEK 293, under identical recording conditions, outward macropatch calcium- or voltage-activated potassium currents were not observed (Fig. 2AGo). Furthermore, RT-PCR (not shown) and Western blot (Fig. 1DGo) analysis did not reveal endogenous BK channel expression in this cell line. Transfection of cloned mouse AtT20 corticotrope BK channel {alpha}-subunits lacking the proposed initiator methionine (8, 26) in HEK 293 cells did not result in functional BK channel expression (M. J. Shipston, R. Duncan, and L. Tian, unpublished data).



View larger version (25K):
[in this window]
[in a new window]
 
Figure 2. Functional Characterization of STREX-1 and ZERO {alpha}-Subunits Expressed in HEK 293 Cells

A, Representative isolated inside-out macropatch recordings from mock transfected HEK 293 cells and cells expressing the mouse AtT20 D16:16 corticotrope STREX-1 {alpha}-subunit. The intracellular face of patches was exposed to 0.1 µM [Ca2+]i as described in Materials and Methods in the absence of ATP under physiological potassium gradients. Patches were held at -50 mV and depolarized for 500 msec to the respective potential (-40 to +80 mV). B, Relative conductance-voltage plots for the STREX-1 (open square, n = 9) and ZERO (closed square, n = 4) variants determined from macropatch recordings under the conditions in panel A. The lines show the best fit of a single Boltzmann function (see Materials and Methods). C, Representative current recordings at + 40 mV demonstrating the effect of [Ca2+]i (<10 nM (0), 0.1 µM, and 0.5 µM) on outward STREX-1 macropatch currents under the conditions in panel A with 1 mM ATP in the intracellular solution. D, Plot of unitary current amplitude as a function of activation voltage determined at 0.1 µM [Ca2+]i as in panel A for the STREX-1 (open square, n = 8) variant expressed in HEK 293 cells and native AtT20 BK channels (open circle, n = 8). The inset shows representative unitary current records from patches expressing the STREX-1 variant in HEK 293 cells at + 40 mV (top trace) and 0 mV (bottom trace), respectively. All data are means ± SEM with error bars within the symbol size unless otherwise indicated.

 
The half-maximal activation voltage (V50) for the STREX-1 variant exposed to 0.1 µM [Ca2+]i in physiological potassium gradients was 30.7 ± 1.6 mV (n = 9) compared with 96.1 ± 3.8 mV (n = 4) for the ZERO variant (Fig. 2BGo). Exposure of the intracellular face of ZERO channels to 10 µM [Ca2+]i resulted in a V50 of 15.3 ± 2.4 mV (n = 3). The V50 for the STREX-1 variant is similar to that observed for the majority (> 90%) of native mouse AtT20 BK channels (26.3 ± 5.8 mV, n = 11, not shown) under identical recording conditions. Furthermore, the negative shift in V50 is similar in magnitude to that observed between other mammalian STREX-1 and corresponding ZERO BK {alpha}-subunit variants expressed in mammalian cell lines (25, 29). Increasing [Ca2+]i at the intracellular face of patches expressing the STREX-1 variant resulted in outward macropatch currents with faster activation kinetics and larger steady-state current amplitude, demonstrating the calcium sensitivity of the channel (Fig. 2CGo). It is likely that native AtT20 BK channels (< 10%) with low calcium sensitivity are composed of ZERO {alpha}-subunit variants.

The slope conductance of homomeric unitary STREX-1 channels [127.2 ± 4.2 picosiemens (pS), n = 8] in physiological potassium gradients was not significantly different from the slope conductance of native mouse AtT20 corticotrope channels recorded under identical conditions (125.2 ± 3.6 pS, n = 8, Fig. 2DGo). In contrast, the slope conductance of the homomeric ZERO channel variant was significantly greater (145.3 ± 3.1 pS, n = 7) than either STREX-1 or native mouse AtT20 corticotrope channels (not shown).

ß-Subunits Are Not Integral Components of BK Channels in Mouse AtT20 Corticotropes
Taken together, these data suggest that native mouse AtT20 corticotrope BK channels are largely composed of the {alpha}-subunit STREX-1 variant. However, in some systems, including vascular smooth muscle cells (24), BK channel {alpha}-subunits functionally associate with regulatory ß-subunits that confer enhanced calcium sensitivity compared with the {alpha}-subunit expressed alone (16, 30). To establish whether the enhanced calcium sensitivity of native mouse AtT20 corticotrope is a result of expression of the STREX-1 exon rather than association of ZERO variants with ß1- or ß2-subunits, we assayed the sensitivity of native mouse AtT20 BK channels for the triterpenoid glycoside, DHS-1 (16, 30). Previous studies have shown that submicromolar concentrations of DHS-1 activates BK channels only when the ß1 (or ß2)-subunit is functionally coupled to {alpha}-subunits (16, 25, 30). Coexpression of the ZERO {alpha}-subunit variant with ß1-subunit in HEK 293 cells resulted in a leftward shift of the half-maximal activation potential by approximately 60 mV to 35.3 ± 7.4 mV, (n = 3) at 0.1 µM [Ca2+]i compared with the ZERO variant alone (see Fig. 2BGo). Application of 100 nM DHS-1 to the intracellular face of patches containing ZERO + ß1-subunit resulted in robust activation (259.0 ± 86.7%, n = 4) of channel activity in all patches tested (Fig. 3Go, A and B) that reversed to control upon washout. In contrast, under identical recording conditions, application of 100–500 nM DHS-1 to the intracellular face of patches from native mouse AtT20 BK channels or HEK 293 cells expressing the STREX-1 {alpha}-subunit variant or ZERO subunits alone (Fig. 3BGo) had no significant effect on BK channel activity. Previous studies (25) have reported that ß1-subunits increase the sensitivity of the STREX-1 variant to [Ca2+]i and DHS-1. Furthermore, RT-PCR analysis using degenerate primers based on the published mouse, human, and bovine ß1-subunit sequences (16, 31) support our functional data that native mouse AtT20 corticotropes do not express endogenous ß1-subunits (Fig. 3CGo). As a positive control for the RT-PCR screen, RNA from HEK 293 cells cotransfected with the ZERO {alpha}-subunit and ß1-subunit showed robust expression (Fig. 3CGo). Thus, as DHS-1 has no effect on endogenous BK channel activity, ß1-subunit transcripts are not expressed, and native AtT20 BK channels do not inactivate (5, 6, 32), our data suggests that the previously described ß1- or ß2-subunits are not integral components of native AtT20 corticotrope BK channels (16, 30).



View larger version (28K):
[in this window]
[in a new window]
 
Figure 3. Mouse Corticotrope BK Channel {alpha}-Subunits Are Not Functionally Coupled to ß-Subunits

A, Representative traces of the effect of 100 nM DHS-1 applied to the intracellular face of a patch excised from HEK 293 cells coexpressing the ZERO mouse AtT20 corticotrope BK channel {alpha}-subunit and bovine ß1-subunit (ß1) in HEK 293 cells. Traces from the same patch are for control (Control), 5 min after DHS-1 application (+ DHS-1), and 5 min after DHS-1 washout (wash). B, Summary of effect of DHS-1 on coexpressed channels as in A (ZERO + ß1, n = 4, 100 nM DHS-1); native mouse AtT20 corticotrope BK channels (AtT20, n = 5, 100–500 nM DHS-1); and mouse AtT20 corticotrope STREX-1 subunits expressed alone in HEK 293 cells (STREX-1, n = 3, 100–500 nM DHS-1). Data are expressed as the mean % activation (percentage increase in channel NPo 5 min after DHS-1 application relative to pretreatment control) ± SEM. **, P < 0.01 (ANOVA) compared with STREX-1 or AtT20 group. C, Representative 1% agarose gel of ethidium bromide-stained PCR products generated as described in Materials and Methods from: pcDNA3 plasmid containing bovine ß1 subunit construct (pBKß); reverse transcription (RT) product from HEK 293 cells transiently coexpressing the mouse AtT20 ZERO corticotrope BK {alpha}-subunit variant and bovine ß1 subunit (ZERO + ß1); RT product from mock transfected HEK 293 cells (HEK); and RT product from native mouse AtT20 corticotropes (AtT20). Molecular weight markers (mw: 100-bp ladder) are shown; arrow indicates position of full-length ß1-subunit transcript.

 
STREX-1 {alpha}-Subunits Are Inhibited by cAMP-Mediated Protein Phosphorylation
BK channels from mouse AtT20 corticotropes are potently inhibited by cAMP-dependent PKA activity intimately associated with the channel protein (5, 6). BK channels in other anterior pituitary cell types are also inhibited by PKA-dependent phosphorylation (19, 20). Previous studies have shown that expression of the human BK channel {alpha}-subunit homolog of the ZERO variant in Xenopus oocytes results in channel activation by PKA (33). Thus, to investigate whether the mouse STREX-1 variant could be the functional target for PKA-mediated inhibition in mouse AtT20 corticotropes, we examined the effect of activating endogenous PKA activity in excised inside-out patches from HEK 293 cells expressing the STREX-1 variant.

Application of 1 mM cAMP to the intracellular face of patches, in the presence of Mg-ATP, resulted in a robust inhibition of channel activity at both the macropatch and unitary current level (Figs. 4Go and 5Go) in all patches tested. In macropatch recordings maximal inhibition (mean percentage change in pretreatment steady-state outward current, Io, was -47.5 ± 3.6%, n = 4, P < 0.01 determined at + 40 mV) was observed 10 min after application of cAMP to the intracellular face of the patch at all potentials examined (Fig. 4Go, A–C). The inhibitory effect of cAMP was completely blocked by the specific PKA inhibitor peptide, PKI(5-24) (mean percentage change in Io was 5.5 ± 13.2%, n = 3). Furthermore, in the absence of ATP, cAMP alone had no significant inhibitory effect. Indeed, removal of ATP resulted in a small, but significant, increase in steady-state outward current by 24.6 ± 11.9%, n = 4, P < 0.05 after 10 min (not shown). In an inside-out patch containing a single STREX-1 BK channel, application of cAMP to the intracellular face of the patch resulted in a robust inhibition of single-channel mean open probability (Po) with a time course similar to that observed for macropatch records (Fig. 5Go, A–C). Furthermore, in three other patches in which unitary currents could be resolved, cAMP significantly inhibited NPo (P < 0.01), an effect that was blocked by PKI(5-24) (n = 2 patches; not shown).



View larger version (22K):
[in this window]
[in a new window]
 
Figure 4. Activation of PKA Inhibits Macropatch STREX-1 BK Currents

A, Representative outward macropatch currents from HEK 293 cells stably transfected with the STREX-1 variant before (control) and 10 min after application of 1 mM cAMP to the intracellular face of the patch. Macropatch currents were determined as in Fig. 2Go by depolarization from -50 mV to the respective potential (-40 to + 80 mV) with 0.1 µM [Ca2+]i and 1 mM ATP. B, Current-voltage relationship for the traces shown in panel A above. Peak steady-state current (Io) was determined 450 msec into the pulse and plotted as a function of membrane potential. C, Effect of cAMP on Io 10 min after application of 1 mM cAMP to the intracellular face of macropatches (cAMP, n = 4) or after preincubation with the specific PKA inhibitor, PKI(5-24), and subsequent 10 min application of cAMP (cAMP + PKI, n = 3). Data were determined as in panel A with percentage change in Io determined at + 40 mV and expressed as mean ± SEM; P < 0.01 (t test) compared with cAMP + PKI group

 


View larger version (21K):
[in this window]
[in a new window]
 
Figure 5. Closely Associated PKA Inhibits Single STREX-1 BK Channels

A, Representative unitary current records from an isolated inside-out patch containing a single BK channel from HEK 293 cells stably expressing the STREX-1 variant before (Control) and 10 min after (+ cAMP) application of 1 mM cAMP to the intracellular face of the patch. Patches were exposed to 0.5 µM [Ca2+]i and 1 mM ATP and depolarized to 0 mV for 60 sec at the respective time point. B, Diary plots of mean single-channel open probability (Po) determined every second (vertical solid bars) 3 min before and 10 min after the application of cAMP. C, Mean Po determined from 60 sec of continuous recording and plotted as a function of time for the above. cAMP (1 mM) was applied at 0 min for the duration of the experiment (horizontal open bar).

 
Taken together, these results demonstrate that cAMP-dependent activation of PKA closely associated with the STREX-1 BK channel complex inhibits STREX-1 channel activity expressed in HEK 293 cells. As native BK channels in AtT20 mouse corticotrope cells are potently inhibited by PKA closely associated with the channel complex (6), our data suggest that the STREX-1 {alpha}-subunit, or a protein that functionally associates with the STREX-1 {alpha}-subunit in corticotropes and HEK 293 cells, is the functional target for PKA-mediated inhibition.

Mouse Corticotrope BK Channels Are Predominantly Composed of the STREX-1 {alpha}-Subunit Variant
The data presented in this report provide the first molecular and functional characterization of an ion channel expressed in anterior pituitary corticotropes. Several lines of evidence suggest that mouse AtT20 corticotrope BK channels are composed of {alpha}-subunits containing the mouse homolog of the previously described 59-amino acid, cysteine-rich, STREX-1 exon at splice site 2 (10, 25).

First, cDNA cloning and RT-PCR splice site analysis revealed robust expression of STREX-1 containing {alpha}-subunits in mouse AtT20 corticotropes. Importantly, an identical STREX-1 exon is expressed in normal adult mouse anterior pituitary gland and a highly homologous exon (2 amino acid differences out of 59) is expressed in rat anterior pituitary cells. Second, functional expression of the STREX-1 {alpha}-subunit in HEK 293 cells revealed channels with almost identical calcium sensitivity, unitary conductance, and inhibition by PKA as for the majority (> 90%) of native AtT20 D16:16 BK channels under identical recording conditions. In contrast, expression of the ZERO {alpha}-subunit variant resulted in channels with higher single-channel slope conductance and significantly lower calcium sensitivity compared with the STREX-1 {alpha}-subunit or native AtT20 BK channels. Finally, RT-PCR analysis and functional assays using the triterpenoid glycoside, DHS-1, revealed that the high calcium sensitivity of native mouse AtT20 corticotrope BK channels is not a result of association of {alpha}-subunits with previously described ß-subunits (16, 30).

Importantly, the STREX-1 {alpha}-subunit variant, as for endogenous BK channels in mouse AtT20 corticotropes (6), is inhibited by PKA-dependent protein phosphorylation. Indeed, several putative PKA consensus phosphorylation sites can be assigned from primary sequence data that are distributed across the C-terminal intracellular domain. However, we cannot exclude that the STREX-1 {alpha}-subunit associates with unidentified regulatory subunits, in native AtT20 D16:16 corticotropes or HEK 293 cells, that confer sensitivity to PKA inhibition.

Increasing evidence suggests that the STREX-1 or STREX-2 (STREX) exon (10, 25) is widely expressed in neuroendocrine cells including pancreatic ß-cells and adrenal chromaffin cells (25, 27) as well as anterior pituitary corticotropes (this study). Thus, the STREX exon may be a common feature of neuroendocrine cells in which BK channel {alpha}-subunits retain a high sensitivity to calcium in the absence of functional interaction with ß-subunits, as described for vascular smooth muscle cells (24). Intriguingly, the rat STREX exons expressed in adrenal chromaffin cells is hormonally regulated by the stress axis and has been proposed to be expressed in excitable cells associated with enhanced repetitive action potential firing (10, 23). Thus, the STREX variants in other components of the stress axis, including anterior pituitary cells, may also be under dynamic long-term regulation as well as involved in the short-term regulation of corticotrope function by CRH and other signaling pathways (5, 6, 32). Elucidation of the molecular components of mouse corticotrope BK channels reported in this study should allow us to define further the functional role, and molecular regulation, of these important multiple coincidence detectors in anterior pituitary corticotropes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
AtT20 D16:16 cells were maintained as previously described (5). Briefly, cells were maintained in DMEM containing 10% FCS (Harlan Seralab, Crawley Down, UK) in a humidified atmosphere of 95% air-5% CO2 at 37 C. Cells were routinely passaged every 7 days using 0.25% trypsin in HBSS containing 0.1% EDTA after reaching 80% confluency. HEK 293 cells, a generous gift from David N. Sheppard, University of Edinburgh, were maintained as for AtT20 D16:16 cells except cells were passaged every 3–4 days.

Isolation of cDNAs Encoding Mouse Corticotrope BK Channels
An RT-PCR strategy was used to amplify cDNAs encoding the entire ORF of mouse anterior pituitary AtT20 D16:16 corticotrope BK channel {alpha}-subunits. Briefly, total cellular RNA from approximately 107 mouse AtT20 D16:16 corticotropes was isolated using standard techniques (34) and mRNA purified using poly (A) quik columns (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. One microgram of this RNA was used as template in a first strand cDNA synthesis directed from an anchored deoxyoligo d(T) primer (5'-TTCTAGAATTCAGCGGCCGC(T)30N1N2), using Superscript II reverse transcriptase (Life Technologies, Inc., Paisley, UK). The resultant cDNA was diluted and used in a PCR to amplify the entire ORF using forward (spanning initiator methionine: 5'-GAT GGA TGC GCT CAT CAT MCC G) and reverse (spanning stop codon: 5'-CTG GGA TAG GCA TTA TCC GGC TCA) deoxynucleotides, with Expand polymerase (Roche Diagnostics Ltd, Lewes, UK). PCR product(s) were ligated to a T/A vector (pCR2.1, Invitrogen, San Diego, CA) and completely sequenced on both strands (OSWEL DNA Services, Southampton, UK). The STREX-1 variant sequence has been deposited in Genbank, accession number: AF156674.

Splice Variant Analysis
First-strand cDNA from mRNA isolated from AtT20 D16:16 cells and adult mouse anterior pituitary glands was generated as described above. Analysis of splice sites was performed using primers encompassing splice sites 1–3: forward (5'-CAG AGT CAA GAT AGA GTC AGC); reverse (5'-AAG TGG CAT CAC CAG GTT CCG) and for splice site 4: forward (5'-GAT ACT TCG CTT CAG GAC AAG G); reverse (5'-AAT GTC TGC GGA GTG CTG TAG C). In some experiments a nested PCR approach, with external primers that spanned the pore region and splice sites 1–4 in the first round amplification, followed by a second round amplification with the above internal primer sets, was used to confirm splice variants (not shown). PCR products were characterized by gel electrophoresis and restriction digestion, and products of interest were ligated into the T/A vector pCR2.1 (Invitrogen) and sequenced on both strands.

For RT-PCR analysis of ß1-subunit expression, degenerate primers were designed against the published bovine, human, and mouse ß1-subunits (16, 31). Forward primer: 5'-ATG GKR AAG AAG CTG GTG ATG GCC; reverse primer: 5'-TCT GRG CCG CCA GGA TGG; and PCR products analyzed as above.

Construction of Expression Plasmids and Expression in HEK 293 Cells
Subcloning of the entire ORF of AtT20 BK channel subunits into pcDNA3.1 resulted in low expression of channels in HEK 293 cells compared with channel constructs containing additional 5'- and 3'-untranslated region (UTR) (14). To improve channel expression HindIII-NheI restriction fragment(s) from AtT20 BK channel clones, spanning splice sites 1–3, were ligated into the HindIII-NheI site of the mbr5 mslo (8) variant (Genbank accession number: L16912). The HindIII-NheI sites are conserved in all mslo variants, and constructs were generated from the mbr5-BSmxt plasmid construct, a generous gift from Dr Lawrence Salkoff (Washington University, St. Louis, MO) (8). The mbr5 clone is identical in sequence to characterized mouse corticotrope clones except that splice sites 1–4 do not contain inserts and mbr5 contains additional 5'- and 3'-UTR. Thus mbr5 is identical to the ZERO variant (see Results) isolated from AtT20 D16:16 corticotropes. The KpnI-XbaI fragment of mbr5 encompassing the entire ORF and additional 5'- and 3'-UTR and containing either the AtT20 null splice site 2 (ZERO, see Results) or containing the AtT20 STREX-1 splice site 2 insert (STREX-1) were subcloned into the mammalian expression vector pcDNA3.1+ or pcDNA3.1+ zeo respectively (Invitrogen BV, Leek, The Netherlands) for expression in HEK 293 cells.

The bovine tracheal smooth muscle cell BK channel ß1-subunit 1) cDNA was kindly provided by Dr. Reid J. Leonard (Merck Research Laboratories, Rahway, NJ) (16). The EcoRI-NotI fragment encoding the entire ORF was subcloned into the mammalian expression vector pcDNA3.1+ zeo (Invitrogen BV).

For transient transfections, HEK 293 cells were seeded onto glass coverslips 24 h before transfection at 40% confluency and transfected with 1 µg of the respective expression plasmid using lipofectamine (Life Technologies, Inc.) essentially as described by the manufacturer. For cotransfections, ß1-subunit DNA was transfected at a 5-fold excess over the {alpha}-subunit. Cells were used 24–72 h after the start of transfection at between 40–80% confluency. Stable cell lines were created as above by seeding in 24-well cluster dishes (Costar, Cambridge, MA), and stable transformants were selected for zeocin resistance using 0.2 mg/ml zeocin (Invitrogen).

Electrophysiology
BK channels in AtT20 D16:16 corticotropes or cloned channels expressed in HEK 293 cells were analyzed under voltage-clamp in the excised inside-out configuration of the patch clamp technique at room temperature (20–24 C) using physiological potassium gradients essentially as previously described (6, 14). The pipette solution (extracellular) contained (in millimolar concentration): 140 NaCl, 5 KCl, 0.1 CaCl2, 5 MgCl2, 20 glucose, 10 HEPES, pH 7.4. The bath solution (intracellular) contained (in millimolar concentration): 140 KCl, 5 NaCl, 2 MgCl2, 1 or 5 (1,2-bis-O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 30 glucose, 10 HEPES, pH 7.35, with free calcium [Ca2+]i buffered to the concentration indicated in the respective figure legend and calculated as previously described (35). For [Ca2+]i greater than, or equal to, 1 µM, dibromo-BAPTA was used as the calcium buffer.

Data acquisition and voltage protocols were controlled by an Axopatch 200 A or B amplifier and pCLAMP6 software (Axon Instruments Inc., Foster City, CA). All recordings were sampled at 10 kHz and filtered at 2 kHz. For patches in which unitary currents could be resolved, channels were voltage clamped at the potential indicated in the respective figure legend. Mean steady-state single-channel open probability (Po) was determined from at least 30 sec of continuous recording under each experimental condition. For macropatch recordings, outward BK currents were evoked by 500-msec step depolarizations (-50 to + 80 mV), and the steady-state current amplitude, averaged from five consecutive depolarizations 450 msec into the pulse, was determined at each potential. For macropatch recordings with seal resistances > 10 G{Omega} leak subtraction was not routinely applied. Pipettes were manufactured from Garner no. 7052 glass, coated with sylgard, and had typical resistances of between 2–10 M{Omega} in bath solution after fire polishing. Patches containing a single BK channel (verified at + 80 mV and 10 µM [Ca2+]i) were extremely rare (<0.5% of total patches) even with pipette resistance values > 10 M{Omega}.

Additional analysis and curve fitting was performed using Igor Pro3.1 (Wavemetrics Inc., Lake Oswego, OR). Conductance values (G) were calculated from peak current (I) measured at 450 msec into the voltage pulse using the relationship G = I/(V-Ek) where V is the activation potential and Ek is the calculated potassium reversal potential. Conductance-voltage curves were fit with a single Boltzmann function, G(V) = Gmax/(1+ exp(V50 - V)/k), where Gmax corresponds to the maximal conductance, V50 is the voltage for half-maximal activation, V is the activation potential, and k is the slope factor reflecting the voltage dependence of conductance.

Western Blotting
Partially purified membrane homogenates from AtT20 D16:16 cells were prepared by homogenizing approximately 107 cells on ice in the following (in millimolar concentration): 150 KCl, 5 EGTA, 2 MgCl2, pH 10.6, containing 12 U/ml aprotinin, 5 µg/ml leupeptin, 6 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 4 mM Pepstatin A followed by two freeze thaw cycles. After centrifugation for 5 min 1,000 x g at 4 C, the resultant supernatant was pelleted at 40,000 x g to give the crude membrane fraction. Protein samples (10–40 µg) were separated on a 10% SDS gel and electroblotted to Immobilon polyvinylidene fluoride membranes. Membranes were blocked for 2 h at room temperature with PBS containing 0.1 mM EDTA, 0.1% Triton X-100, pH 7.4 (PBS-TE), and 5% (wt/vol) low-fat milk (Marvel). Blots were incubated overnight at 4 C with a 1:2000 dilution of the affinity-purified antibody {alpha}slo(913-926) [directed toward residues 913–926 of the pore-forming {alpha}-subunit of mouse brain BK channels (28)] in PBS-TE containing 1% wt/vol Marvel. Blots were washed five times with PBS-TE and incubated for 45 min at room temperature with horseradish peroxidase-conjugated antirabbit IgG (Amersham Pharmacia Biotech, Piscataway, NJ; 1:5000 final dilution) in PBS-TE containing 5% (wt/vol) Marvel. After five washes in PBS-TE, blots were incubated with enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech) according to the manufacturer’s protocol and blots were exposed to ECL film in the linear response range (Amersham Pharmacia Biotech).

Reagents
ATP magnesium or potassium salt, was from Sigma-Aldrich Co. (St. Louis, MO) and stored as buffered 1 M stock solutions at -20 C before use. ATP and cAMP were buffered in bath solution to pH 7.35 and applied to the intracellular patch by gravity perfusion with 10 volumes of bath solution at a flow rate of 1–2 ml/min. PKI5-24, BAPTA, dibromo-BAPTA, and Fura-2 were obtained from Calbiochem (Nottingham, UK). DHS-1 was a generous gift of Dr. Owen McManus (Merck Research Laboratories, Rahway, NJ). The C-terminal {alpha}slo(913-926) antibody was a generous gift of Dr. Hans-Guenther Knaus (University of Innsbruck, Innsbruck, Austria). All other reagents, unless otherwise stated, were of the highest analytical grade available from Sigma-Aldrich Co. Ltd., Poole, UK, or Merck Ltd., Poole, UK.


    ACKNOWLEDGMENTS
 
We thank M. C. Lim, A. K. Long, D. McDonald, and Z. Higgs for some of the preliminary RNA isolation and RT-PCR. We are grateful for helpful discussions with other members of the Membrane Biology Group.


    FOOTNOTES
 
Address requests for reprints to: Michael J Shipston, Ph.D., Membrane Biology Group, Department of Biomedical Sciences, University of Edinburgh, Medical School, Teviot Place, Edinburgh, Scotland, UK, EH8 9AG.

This work was supported by The Wellcome Trust (Ref: 046787/Z) and The Physiological Society.

1 Present address: Organon Laboratories Ltd., Newhouse, Lanarkshire, ML1 5SH, Scotland, UK. Back

Received for publication April 22, 1999. Revision received June 9, 1999. Accepted for publication June 28, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Mason WT, Rawlings SR, Cobbett P, Sikdar SK, Zorec R, Akerman SN, Benham CD, Berridge MJ, Cheek T, Moreton RB 1988 Control of secretion in anterior pituitary cells linking ion channels, messengers and exocytosis. J Exp Biol 139:287–316[Abstract]
  2. Ozawa S, Sand O 1986 Electrophysiology of excitable endocrine cells. Physiol Rev 66:887–952[Free Full Text]
  3. Ritchie AK, Kuryshev YA, Childs GV 1996 Corticotropin-releasing hormone and calcium signalling in corticotropes. Trends Endocrinol Metab 7:365–369[CrossRef]
  4. Lim MC, Shipston MJ, Antoni FA 1998 Depolarization counteracts glucocorticoid inhibition of adenohypophysial corticotroph cells. Br J Pharmacol 124:1735–1743[Abstract]
  5. Shipston MJ, Kelly JS, Antoni FA 1996 Glucocorticoids prevent protein kinase A inhibition of calcium-activated potassium channels. J Biol Chem 271:9197–9200[Abstract/Free Full Text]
  6. Tian L, Knaus H-G, Shipston MJ 1998 Glucocorticoid regulation of calcium-activated potassium channels mediated by serine/threonine protein phosphatase. J Biol Chem 273:13531–13536[Abstract/Free Full Text]
  7. Atkinson N, Robertson G, Ganetzky B 1991 A component of calcium-activated potassium channels encoded by the Drosophila slo locus. Science 253:551–555[Medline]
  8. Butler A, Tsunoda S, McCobb DP, Wei A, Salkoff L 1993 mSlo, a complex mouse gene encoding ’maxi’ calcium-activated potassium channels. Science 261:221–224[Medline]
  9. Vergara C, Latorre R, Marrion N, Adelman J 1998 Calcium-activated potassium channels. Curr Opin Neurobiol 8:321–329[CrossRef][Medline]
  10. Xie J, McCobb DP 1998 Control of alternative splicing of potassium channels by stress hormones. Science 280:443–446[Abstract/Free Full Text]
  11. Adelman J, Shen K, Kavanaugh M, Warren R, Wu Y, Lagrutta A, Bond C, North R 1992 Calcium-activated potassium channels expressed from cloned complimentary DNAs. Neuron 9:209–216[Medline]
  12. Lagrutta A, Shen K-Z, North RA, Adelman JP 1994 Functional differences among alternatively spliced variants of Slowpoke, a Drosophila calcium-activated potassium channel. J Biol Chem 269:20347–20351[Abstract/Free Full Text]
  13. Tseng-Crank J, Foster C, Krause J, Mertz R, Godinot N, DiChiara T, Reinhart P 1994 Cloning, expression, and distribution of functionally distinct Ca2+-activated K+ channel isoforms from human brain. Neuron 13:1315–1330[Medline]
  14. Clark A, Hall S, Shipston M 1999 ATP inhibition of a mouse brain large-conductance K+ (mslo) channel variant by a mechanism independent of protein phosphorylation. J Physiol 516:45–53[Abstract/Free Full Text]
  15. Dworetzky S, Boissard C, Lum-ragan J, McKay M, Post-Munson D, Trojnacki J, Chang C, Gribkoff V 1996 Phenotypic alteration of a human BK (hslo) channel by hslo ß subunit co-expression: changes in blocker sensitivity, activation/relaxation kinetics, and protein kinase A modulation. J Neurosci 16:4543–4550[Abstract/Free Full Text]
  16. McManus O, Helms L, Pallanck L, Ganetzky B, Swanson R, Leonard R 1995 Functional role of the ß-subunit of high-conductance calcium-activated potassium channels. Neuron 14:645–650[Medline]
  17. Schopperle W, Holmqvist M, Zhou Y, Wang J, Wang Z, Griffith L, Keselman I, Kusinitz F, Dagan D, Levitan I 1998 Slob, a novel protein that interacts with the slowpoke calcium-dependent potassium channel. Neuron 20:565–573[Medline]
  18. Xia X, Hirschberg B, Smolik S, Forte M, Adelman J 1998 Dslo interacting protein 1, a novel protein that interacts with large-conductance calcium-activated potassium channels. J Neurosci 18:2360–2369[Abstract/Free Full Text]
  19. Sikdar SK, McIntosh RP, Mason WT 1989 Differential modulation of Ca2+-activated K+ channels in ovine pituitary gonadotrophs by GnRH, Ca2+ and cyclic AMP. Brain Res 496:113–123[CrossRef][Medline]
  20. White RE, Schonbrunn A, Armstrong DL 1991 Somatostatin stimulates Ca2+-activated K+ channels through protein dephosphorylation. Nature 351:570–573[CrossRef][Medline]
  21. Reinhart PH, Levitan IB 1995 Kinase and phosphatase-activities intimately associated with a reconstituted calcium-dependent potassium channel. J Neurosci 15:4572–4579[Abstract]
  22. Levitan IB 1994 Modulation of ion channels by protein phosphorylation and dephosphorylation. Annu Rev Physiol 56:193–212[CrossRef][Medline]
  23. Ramanathan K, Michael TH, Jiang GJ, Hiel H, Fuchs PA 1999 A molecular mechanism for electrical tuning of cochlear hair cells. Science 283:215–217[Abstract/Free Full Text]
  24. Tanaka Y, Meera P, Song M, Knaus HG, Toro L 1997 Molecular constituents of maxi KCa channels in human coronary smooth muscle: predominant {alpha} + ß subunit complexes. J Physiol 502:545–557[Abstract]
  25. Saito M, Nelson C, Salkoff L, Lingle CJ 1997 A cysteine-rich domain defined by a novel exon in a Slo variant in rat adrenal chromaffin cells and PC12 cells. J Biol Chem 272:11710–11717[Abstract/Free Full Text]
  26. Pallanck L, Ganetzky B 1994 Cloning and characterization of human and mouse homologs of the Drosophila calcium-activated potassium channel gene, slowpoke. Hum Mol Genet 3:1239–1243[Abstract]
  27. Ferrer J, Wasson J, Salkoff L, Permutt M 1996 Cloning of human pancreatic islet large conductance Ca2+-activated K+ channel (hslo) cDNAs: evidence for high levels of expression in pancreatic islets and identification of a flanking genetic marker. Diabetologia 39:891–898[CrossRef][Medline]
  28. Knaus H-G, Schwarzer C, Koch R, Eberhart A, Kaczorowski G, Glossman H, Wunder F, Pongs O, Garcia M, Sperk G 1996 Distribution of high-conductance Ca 2+-activated K+ channels in rat brain: targeting to axons and nerve terminals. J Neurosci 16:955–963[Abstract]
  29. Morita T, Hanaoka K, Morales MM, Montrose-Rafizadeh C, Guggino WB 1997 Cloning and characterization of maxi K+ channel {alpha}-subunit in rabbit kidney. Am J Physiol 273:F615–F624
  30. Wallner M, Meera P, Toro L 1999 Molecular basis of fast inactivation in voltage and Ca2+-activated K+ channels: a transmembrane ß-subunit homolog. Proc Natl Acad Sci USA 96:4137–4142[Abstract/Free Full Text]
  31. Jiang Z, Wallner M, Meera P, Toro L 1999 Human and rodent MaxiK channel ß-subunit genes: cloning and characterization. Genomics 55:57–67[CrossRef][Medline]
  32. Tian L, Philp J, Shipston M 1999 Glucocorticoid block of protein kinase C signalling in mouse pituitary corticotroph AtT20 D16:16 cells. J Physiol 516:757–768[Abstract/Free Full Text]
  33. Nara M, Dhulipala PD, Wang YX, Kotlikoff MI 1998 Reconstitution of ß-adrenergic modulation of large conductance, calcium-activated potassium (maxi-K) channels in Xenopus oocytes. Identification of the cAMP-dependent protein kinase phosphorylation site. J Biol Chem 273:14920–14924[Abstract/Free Full Text]
  34. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[CrossRef][Medline]
  35. Tsien R 1980 New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry 19:2396–2404[Medline]