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2 and 4 Subunits of BK Channels Confer Differential Sensitivity to Acute Modulation by Steroid Hormones
1Department of Neurobiology and Behavior and 2Department of Biomedical Sciences, Cornell University, Ithaca, New York: and 3Department of Applied Mathematics, University of Texas, San Antonio, Texas
Submitted 22 December 2005; accepted in final form 24 January 2006
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ABSTRACT |
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1 subunits. We show here that 1) multiple members of the family confer sensitivity to multiple steroids on BK channels, 2) that subunits differentiate between steroids, and 3) that different s have distinct relative preferences for particular steroids. Expressed in HEK 293 cells, inside-out patches with channels composed of Slo-
alone showed no steroid sensitivity. Cells expressing 4 exhibited potent, rapid, reversible, and dose-dependent potentiation by corticosterone (CORT; a glucocorticoid), and were potentiated to a lesser degree by other sex and stress steroids. In contrast, 2 channels were potentiated more strongly by dehydroepiandrosterone (DHEA; an enigmatic, stress-related adrenal androgen), and to a lesser extent by CORT, estradiol, testosterone, and DHEA-S. Cholesterol had no effect on any BK channel compositions tested. Conductance–voltage plots of channels composed of plus 2 or 4 subunits were shifted in the negative direction by steroids, indicating greater activation at negative voltages. Thus our results argue that the variety of Slo- subunit coexpression patterns occurring in vivo expands the repertoire of Slo channel gating in yet another dimension not fully appreciated, rendering BK gating responsive to dynamic fluctuations in a multiple of steroid hormones. |
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INTRODUCTION |
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; Makara and Haller 2001; Wehling 1997). Ion channels, G protein–coupled receptors, phospholipases, and protein kinases are modulated acutely, but primary receptors and transducing intermediates are mostly unknown.
Big-conductance calcium- and voltage-activated potassium (BK) channels are prominent in nerve, muscle, endocrine, and exocrine cells. Their functions vary widely with context. In smooth muscle they counteract depolarization and contraction. In presynaptic nerve terminals, they can limit secretion by speeding rapid repolarization. Rapid repolarization and brief afterhyperpolarization mediated by BK current can increase or decrease neuronal repetitive firing rates. These channels can also make interesting and sometimes paradoxical contributions to firing patterns, affecting threshold, spike frequency adaptation, and burst firing patterns (Brayden and Nelson 1992; Fettiplace and Fuchs 1999; Jin et al. 2000; Lingle et al. 1996; McCobb 2004; Van Goor et al. 2001; Vergara et al. 1998). BK channels have a pore formed from a tetramer of -subunits encoded by the Slo gene. subunits encoded by at least four related genes differentially co-occur with Slo and have distinctive and sometimes dramatic effects on channel gating (Orio et al. 2002). 1, prominent in smooth muscle, shifts BK activation to more negative voltages for a given Ca2+ concentration, while slowing activation (Dworetzky et al. 1996; McManus et al. 1995). 2 negatively shifts activation, but also confers rapid inactivation through a "ball-and-chain" mechanism (Uebele et al. 2000; Wallner et al. 1999; Xia et al. 1999). One splice variant of 3 confers extremely rapid but incomplete inactivation (Xia et al. 2000). 4, prevalent in brain, increases the steepness of Ca2+ sensitivity and dramatically slows activation and deactivation kinetics (Behrens et al. 2000; Brenner et al. 2000). Temporal lobe seizures reported in 4 knockout mice show that 4's effects are critical for damping excessive firing frequencies in granule cells of the dentate gyrus (Brenner et al. 2005).
Several steroids modulate BK channels. Estradiol, testosterone, and bile salts, (steroidal anions) can relax smooth muscle by potentiating BK channels (Deenadayalu et al. 2001; Dopico et al. 2002; Rosenfeld et al. 2001; Salom et al. 2002). We have recently shown that adrenal glucocorticoids cortisol and corticosterone (together abbreviated CORT), and their synthetic analog dexamethasone, facilitate BK activation in adrenal chromaffin cells, promoting rapid action potential repolarization, repetitive firing, and presumably augmenting catecholamine secretion under stress (Lovell et al. 2004). Similar effects of CORT on pituitary corticotrope- and somatotrope-like cell lines have also been reported (Huang et al. 2005). Whether CORT modulates BK channels in hippocampus, pituitary, or other native tissues controlling stress responses is not yet known.
Dehydroepiandrosterone (DHEA) is another steroid that has been proposed to regulate BK channels in vivo. DHEA, with its sulfated form DHEA-S, is the most abundant, but perhaps least understood, steroid hormone. Produced in the adrenal cortex and elsewhere, including the brain, serum DHEA levels are increased by stress in humans (Kroboth et al. 1999; Oberbeck et al. 1998; Zinder and Dar 1999). An age-related decline inserum levels has prompted widespread therapeutic use, despite very limited understanding of its natural role(s) or mechanisms of action (Allolio and Arlt 2002; Vermeulen 1995; Wolkowitz et al. 1997). There is evidence for neuroprotective effects of DHEA in hippocampus (Bastianetto et al. 1999; Kimonides et al. 1998). DHEA or DHEA-S modulates CNS GABAA receptors and calcium channels (Ffrench-Mullen and Spence 1991; Majewska et al. 1990). Importantly, BK channel activation in pulmonary vascular smooth muscle is acutely potentiated by DHEA, reducing vasoconstriction under hypoxia (Farrukh et al. 1998; Peng et al. 1999).
Coexpression of 1 and Slo in Xenopus oocytes or HEK-293 cells confers both estradiol binding and channel facilitation (Behrens et al. 2000; Valverde et al. 1999). The response mimics that of BK channels in vascular smooth muscle cells. Here we test the possibility that other related subunits mediate modulation of BK channels by other steroids. We report that 4, prominent in hippocampus and other brain regions (Brenner et al. 2000), confers particular sensitivity to CORT. On the other hand, inactivation-conferring 2, expressed in lung, adrenal medulla, and brain, confers preferential sensitivity to DHEA. We submit that distinct, multisteroidal interaction profiles add another nuance to subunit-related diversification of BK channel function in vivo.
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METHODS |
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A human BK channel -subunit cDNA (hSlo; accession no. U13913) was excised as a HindIII-SalI fragment and cloned into the expression vector pcDNA3.1 (Invitrogen) under the control of a cytomegalovirus promoter. The human BK channel 1 subunit cDNA (accession no. NM004137) was excised with BamHI, and NotI and subcloned into pcDNA3.1. The human 2 subunit cDNA (American Type Culture Collection; accession no. AF099137) was excised with HindIII and XhoI and subcloned into pcDNA3.1. 2NI, a noninactivating 2 mutant lacking the inactivation-conferring amino terminal residues FIW in positions 2–4 (Xia et al. 2003), was created by incorporating a HindIII site followed by an intial methionine and residues 5–9 into the forward primer, and XhoI in the reverse primer, and amplifying from the full length cDNA using a proof-reading polymerase (Pfu). The amplicon was digested with HindIII and XhoI, gel purified, and cloned into the corresponding sites in pcDNA3.1(+). Rat 4 subunit was amplified by RT-PCR from rat adrenal RNA, and was TA cloned into pCR2.1, excised with HindIII and XhoI, and subcloned into pcDNA3.1. Directional orientation and sequence of the inserts was determined by DNA sequencing at the BioResource Center (Cornell University).
HEK-293 cells (American Type Culture Collection: CRL-1573) were transiently transfected using lipofectamine 2000 (Gibco) following the manufacturer's protocols. Briefly, HEK cells were grown to 70–80% confluency in DMEM +2 mM glutamine supplemented with 10% fetal bovine serum. The cells were grown at 37°C in a humidified atmosphere of 5% CO2 and 95% air. After DNA-lipid complexes were allowed to form; cells were transfected with either cDNAs encoding Slo- alone (1 µg DNA) or cotransfected with cDNAs for Slo- and Slo- subunits in a 1:1 ratio. An additional plasmid containing a modified green fluorescent protein (GFP; pBFP-N1; Clontech, Palo Alto, CA) was also cotransfected into the cells for identification. Cells were plated on 35-mm culture dishes (Falcon) coated with collagen (0.53 mg/ml; Vitrogen, Palo Alto, CA) and lifted with a solution containing (in mM) 0.5 EDTA, 38 sucrose, 34 D-glucose, 156 NaCl, 5 KCl, 4 NaHCO3, and 9 HEPES, pH 7.2. Transfected cells were patched between 2 and 5 days after replating.
Chromaffin cell isolation and culture
Rat chromaffin cells were isolated and cultured as described previously (Lovell et al. 2000). Briefly, Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were anesthetized with CO2 and killed by cervical spinal dislocation. Adrenomedullary tissue was dissected from surrounding cortical tissue, minced, washed in sterile saline, and incubated for 60 min in collagenase B (Boehringer Mannheim, Indianapolis, IN; 1.5 mg/ml; pH 7.0), at 37°C. Tissue was washed in Ca2+- and Mg2+-free saline, and incubated for 30 min at 37°C in trypsin (Gibco; 0.125% in Hank's Ca2+- and Mg2+-free saline). Chromaffin cells were triturated through fire-polished pipettes and plated in sterile culture medium (Gibco; RPMI 1640 with 10% horse serum, 5% fetal calf serum, 2 U/ml penicillin-G, 2 µg/ml streptomycin sulfate, 100 U/ml nystatin) in glass-bottom dishes coated with poly-D-lysine (Sigma, St. Louis, MO; 0.01% in ddH2O). Cell cultures were maintained in a 5% CO2 atmosphere at 37°C for 1–4 days.
Electrophysiological methods
Single channel and macroscopic currents were recorded in the inside-out configuration. Patch electrodes (3–5 M
) were pulled from borosilicate glass and coated with silicone elastomer (Sylgard 184, Dow Corning, Midland, OH). Data were collected using a List EPC-9 patch clamp amplifier (Heka Electronik, Lambrecht, Germany), Bessel filtered at 10 kHz, and stored on a Power Macintosh G3 using Pulse 8.5 software (Heka Electronik, Lambrecht, Germany). Off-line analysis was done with custom software written for Igor Pro (Wavemetrics, Lake Oswego, OR).
Experiments were conducted at 20–22°C. After attaining gigaohm seals, calcium-free saline was perfused on the cell and inside-out patches were pulled. Exchange of solutions was accomplished with eight computer-controlled, gravity-fed lines converging near the tip of a large perfusion pipette.
For inside-out patches, standard patch-clamp recording techniques were used. HEK cells were typically held at –80 mV and stepped to +80 mV for 450 ms after a prepulse to –140 mV for 450 ms, with a 2-s interval between sweeps. For the determination of the voltage dependence of BK channels, a series of increasing voltage steps was applied in 20-mV increments from –140 to +140 mV for 350 ms. For the voltage dependence of inactivation, the steady-state current after activation was measured by a second voltage step to +80 mV for 350 ms.
Solutions
To eliminate a potassium driving force and allow DC offset to be cancelled at 0 mV, symmetrical K+ solutions were used. The pipette saline and perfusion solutions were comprised of (in mM) 160 KCl, 10 HEPES, 1 HEDTA, and 0.0375 CaOH; pH was adjusted with KOH to 7.2 to make 
500 nM to 1 µM free [Ca2+]. Calculations of free [Ca2+] were made through MaxChelator software (WebMaxC v2.10). Zero-Ca2+ solution contained 5 mM EGTA (Sigma). Rodent Ringer contained (in mM) 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.2. The osmolarity of all solutions before the addition of steroids was measured by dew point osmometry and adjusted to 300 ± 10 mosM with ddH20 or NaCl. Steroids were dissolved in 100% dimethylsulfoxide (DMSO), stored in small aliquots, and added fresh to recording salines. DMSO vehicle in the control saline (0.1%, same concentration as steroid solution) did not alter BK channel activity.
Data and statistical analysis
Single and multichannel currents were linear leak subtracted and ensemble currents fit using a Levenberg-Marquardt search algorithm to estimate kinetic parameters. Percent changes in steroids were measured by comparing the maximum peak amplitudes of the current traces before and after treatment with steroid. Ensemble currents were usually averages of 15–30 individual current traces taken 30–60 s after switching perfusion lines to allow patches to stabilize. Patches where BK current was not stable after 3 min were excluded from analysis. Patches used in analysis showed stable BK currents throughout the experiment (Figs. 2B and 4B). Dose–response curves were generated with mostly patches exposed to only one concentration of steroid. In a few cases, patches were included where complete washout was observed after the previous application of a lower concentration of steroid.
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RNA extraction and RT-PCR
Total RNA was harvested from quick-frozen tissues using the Qiagen RNeasy kit. Two micrograms of RNA was added to each standard 20-µl reverse transcription reaction (Allolio and Arlt 2002) with Superscript II reverse transcriptase (GibcoBRL) and 10 µM oligo-(dT). RT product (1.5 µl) was transferred to each 30-µl PCR reaction with Taq DNA polymerase. Primers for rat 2 (r2s: 5'-AATCACACTGCTGCGCTCATACAT-3'; r2as: 5'-TTCTGTGTGGTAGAGGAGGAGC-3') gave a predicted product of 319 bp (accession no. AF209747). Primers for 4 (r41s: 5'-CGGCTCGGCTTGTTCCTCA-3'; r42as: 5'-GCTGGTGCTGGTCGCTGT-3') gave a predicted product of 265 bp (accession no. AF207992). After 3 min at 95°C, 30 cycles were run with 30 s at 95 and 55°C and 45 s at 72°C. Six microliters of PCR product was run on a 2% agarose gel. Ethidium bromide stained gels were UV transilluminated and images captured with a Cohu camera and an LG-3 digitizer, controlled using modified National Institutes of Health image software (Scion, Frederick, MD).
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RESULTS |
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alone or in combination with 1, 2, or 4 cDNA, in addition to GFP. Inside-out patches were pulled from green-fluorescing cells after 48–72 h (Fig. 1). Representative ensemble-averaged current traces obtained in the presence of 500 nM [Ca2+]i show fast activating and deactivating BK current from cells expressing the Slo subunit alone. Ca2+ and voltage-sensitivity and kinetics were altered by coexpression with subunits. Thus 1 and 4 slow the activation and the deactivation kinetics of the channel, and rapid inactivation is conferred by coexpression with 2.
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4 confers sensitivity to corticosterone on Slo channels
Inside-out patches with varying numbers of BK channels were pulled from HEK 293 cells expressing Slo- and 4 subunits. In control saline (500 nM [Ca2+]i), large-conductance (300 pS) openings were often discernible in individual sweeps at +80 mV (450-ms duration; Fig. 2A). BK currents were allowed to stabilize for 3–5 min before application of steroid. Ensemble traces best show the average relative increase in current amplitude produced by the steroid. Channel opening events increased in number within tens of seconds after the application of steroid with concentrations as little as 10 nM CORT (Fig. 2B). Although responses at 10 nM were not observed for every patch, washoff and repeat application in specific cases provided compelling proof of the effect at this low dose. Effects at 100 nM, while still variable, were statistically significant on average, as described below. The potentiation did not diminish appreciably with time over 30 min or more of continuous exposure, and could be at least partially reversed by washout, typically fully at lower concentrations. CORT (1 µM) had minimal effect on alone.
CORT effects could not be explained by a change in single channel conductance. Single channel current amplitude at +80 mV in control saline and 1 µM CORT averaged 25.2 ± 0.5 and 24.4 ± 0.7 pA, respectively (n = 9 each; P = 0.4, Student's t-test). Reversibility was observed to be more complete at lower concentrations of CORT: at 0.01, 0.1, and 1 µM, cases of complete washout of steroid effect (100%) were observed with an average washout of 81.8 ± 8.0 (n = 13), 81.6 ± 24.9 (n = 7), and 63.7 ± 10.6% (n = 7), respectively. At concentrations of 10 and 100 µM CORT, an average washout of 53.1 ± 8.6 (n = 12) and 63.0 ± 6.8% (n = 15) was observed, respectively. To rule out effects of switching between perfusion lines, switches between two lines with control saline were performed. This had no significant effect on peak current amplitudes (mean change = 1.7 ± 1.2%, n = 16, data not shown).
CORT was found to increase BK current measured at test potentials of +80 mV in a roughly dose-dependent manner in inside-out patches expressing 4 channels (Fig. 2C). Mean increases in current elicited with steps to +80 mV were measured for CORT concentrations covering six orders of magnitude. Increases were 10.2 ± 5.5 (±SE; n = 10), 20.2 ± 4.3 (n = 21), 38.4 ± 4.9 (n = 15), 88.2 ± 28.2 (n = 18), 101.9 ± 38.4 (n = 23), and 88.4 ± 20.0% (n = 21) for 0.001, 0.01, 0.1, 1, 10, and 100 µM CORT, respectively. Responses to 0.01, 0.1, 1, 10, and 100 µM CORT were significantly different from switching between control solutions. Additionally, 1, 10, and 100 µM were significantly different from 0.001 and 0.01 µM CORT (1-way ANOVA, F statistic with 6 and 122 degrees of freedom for between treatment and within treatment variation, F6,122 = 20.13; P < 0.001, means compared with Tukey's pairwise post hoc comparisons, family error rate of 0.05). Cholesterol (10 µM), a steroid precursor, had a significantly smaller effect on 4 channels than 10 µM CORT (2.0 ± 9.3%, n = 5, P < 0.05, Student's t-test). The dose–response curve was fit by a Hill equation with a Hill coefficient of 1.4 and a half-maximal effect at 182 nM. Under the whole cell recording configuration, 1 µM CORT also potentiated 4 BK channels within seconds of application (Fig. 2D, left). In the example shown (Fig. 2D, right), 1 µM CORT potentiated BK current by 57.3%. In whole cell mode, the average potentiation by 1 µM CORT was 52.3 ± 5.1% (n = 2).
For a subset of patches, the effects of steroids were tested with a full series of test potentials (Fig. 3A), revealing that steroids produce a simple parallel shift of the G-V curve in the negative direction. In the example shown in Fig. 3A, 1 µM CORT shifted V1/2 by –10.0 mV. On average, V1/2 was shifted by –13.0 ± 2.8 mV by 1 µM CORT (Fig. 3B, n = 4; note [Ca2+]i = 1 µM in these experiments). The average V1/2 was significantly different in control conditions than in 1 µM CORT (V1/2 = 83.4 ± 7.3 and 70.4 ± 5.5 mV in control and in 1 µM CORT, n = 4 and 4, respectively, P = 0.019, Student's t-test). At other concentrations, V1/2 was also shifted to more negative potentials. At 10 and 100 µM CORT (with [Ca2+]i = 500 nM), V1/2 was shifted on average by –10.8 ± 0.8 and –17.4 ± 4.1 mV, n = 5 and 4, respectively).
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1.0 and half-maximal response values of 183, 278, and 360 nM, respectively.
CORT also reduced the rate of current deactivation (
deact estimated by a single exponential fit to the tail current). On average, the time constant of deactivation at a test potential of +80 mV was increased significantly from 3.3 ± 0.3 ms in control saline to 10.6 ± 2 ms in 1 µM CORT (n = 15 and 8, respectively, P = 0.008; Fig. 3D). This effect was observed at several CORT concentrations and several test potentials. The dose response of this effect could also be well approximated by a Hill function with a coefficient of 1.0 and a half-maximal point <500 nM.
2 confers sensitivity to DHEA on Slo channels
Inside-out patches with varying numbers of channels were pulled from cells expressing Slo plus 2 subunits. In control saline (500 nM [Ca2+]i), large-conductance (260 pS) openings were often discernible in individual sweeps at +80 mV (450-ms duration) after a brief prepulse to –140 mV to relieve all inactivation (Fig. 4A). Ensemble averages of 25 traces show the average relative increase in current amplitude produced by the steroid. As with CORT on 4 channels, channel openings increased in number within tens of seconds after application of 10 µM DHEA (Fig. 4B). Mean responses were measured at concentrations covering four orders of magnitude (Fig. 4C). The average increase at 100 nM DHEA was not significant (7.9 ± 3.6%; n = 7). However, 1 µM DHEA significantly increased current compared with control saline by an average of 17.9 ± 4.5% (n = 17); 10 and 100 µM DHEA increased current by 36.9 ± 6.8 and 42.5 ± 12.2%, respectively (n = 18 and 11; F4,64 = 19.18; P < 0.001). Responses at 10 and 100 µM were significantly greater than at 1 µM but not significantly different from each other. The dose–response curve was fit with a Hill equation having a coefficient of 1.03 and a half-maximal concentration at 2.2 µM. Responses at all concentrations were quite variable in size among patches. However, in individual patches, higher concentrations typically produced bigger responses (Fig. 4G).
Conductance–voltage (G-V) plots were constructed from peak amplitudes of currents elicited by an incremental series of depolarizing steps before and after application of one or more concentrations of DHEA (Fig. 4D, bottom). In the example in Fig. 4D, 10 µM DHEA shifted V1/2 by –15.1 mV. Similar shifts were seen in an additional five cases, with an average V1/2 shift of –14.3 ± 3.5 mV. DHEA (10 µM) also produced a negative shift in the voltage dependence of steady-state inactivation (Vinact; Fig. 4D, bottom). In this example, Vinact was shifted –13.9 mV. The average shift was –10.5 ± 1.9 mV (n = 3). In all patches, DHEA increased the rate of inactivation at relatively negative voltages. The rate was sharply voltage-dependent, particularly at potentials in the range of +20 to +60 mV (Fig. 4E; estimated from a single-exponential fit to the current decay during steps from the –140 mV prepulse). At +40 mV, control traces had an average of 72.9 ± 3.8 mV, whereas in 10 µM DHEA, the average was 58.3 ± 1.9 ms (n = 6 patches; P = 0.01, Student's t-test; Fig. 4F).
To rule out any possible confounding effects of inactivation on apparent steroid sensitivity, we removed the inactivation domain. Deletion of the amino terminal residues FIW in positions 2–4 from the 2 construct (Xia et al. 2003) completely eliminated functional inactivation. The effects of DHEA on noninactivating 2NI BK current were very similar to those seen with the fully intact 2 construct (Fig. 4G). From inside-out patches, at 1 µM DHEA, peak current at +80 mV was increased by 17.9 ± 4.5% (n = 17) for 2 channels and 21.9 ± 6.3% (n = 12) for 2NI channels (P = 0.60, Student's t-test). At 10 µM DHEA, peak current was increased by 37.0 ± 6.8% (n = 13) for 2 channels and 29.8 ± 14.2% (n = 7) for 2NI channels (P = 0.61, Student's t-test). At 100 µM DHEA, peak current was increased by 42.5 ± 12.2% (n = 8) for 2 channels and 45.7 ± 27.1% (n = 7) for 2NI channels (P = 0.91, Student's t-test).
subunits confer distinct steroid sensitivities
Steroid responses were different for different channel subunit compositions. With a test potential of +80 mV, the application of 1 or 10 µM CORT on HEK cells expressing only the Slo- subunit had little effect on BK currents. On average, 1 or 10 µM CORT altered current amplitude in channels by 14.3 ± 3.2 and 12.4 ± 9.8% and (n = 3 and 7, respectively). 2 channels (both the full construct and noninactivating construct) were typically potentiated by 1 and 10 µM CORT (0.2 ± 5.5%, n = 5 and 17.6 ± 4.8%, n = 16, respectively), but to a lesser extent than 4 channels (88.2 ± 28.2%, n = 18 for 1 µM CORT and 101.9 ± 38.4%, n = 23 for 10 µM CORT; Fig. 5A). Cells expressing 4 channels were significantly more responsive to 1 µM CORT than -only or 2 channels (F2,23 = 16.46, P < 0.01). At 10 µM CORT, 4 channels responded significantly more than 2 or -only channels (F2,43 = 8.21, P < 0.01; Fig. 5A). The application of 1 or 10 µM DHEA also had little effect on channels, current was altered by 4.6 ± 6.2% (n = 5) for 1 µM DHEA and –2.1 ± 8.8% (n = 12) for 10 µM DHEA (Fig. 5B). Patches expressing 2 channels were potentiated to a greater extent by 1 or 10 µM DHEA (22.2 ± 6.2%, n = 12, and 36.3 ± 6.5%, n = 19, respectively) than patches expressing 4 channels (–6.5 ± 16.5%, n = 6 for 1 µM DHEA and 9.6 ± 10.4%, n = 15 for 10 µM DHEA; Fig. 5B). At 1 µM DHEA, 2 channels were significantly more sensitive to DHEA than 4 channels (F2,20 = 4.82, P < 0.05). At 10 µM DHEA, 2 channels responded significantly more than 4 or -only channels (F2,43 = 12.71, P < 0.01; Fig. 5B).
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2 and 4 confer sensitivity to multiple steroids
We examined the possibility that several other related steroids might also modulate BK channels (Fig. 6A). For 2 channels, mean percentage increases in peak current amplitude at 10 µM were 5.3 ± 2.5% for cholesterol, 36.3 ± 6.5% for DHEA, 18.2 ± 4.9% for DHEA-S, 17.6 ± 4.8% for corticosterone, 5.6 ± 8.9% for androstenedione, 19.9 ± 9.5% for testosterone, and 29.6 ± 5.1% for 17-estradiol (Fig. 6B, top; n = 5, 19, 14, 16, 3, 6, and 6 patches, respectively). Effects of DHEA, CORT, and estradiol were significantly different from control saline. In addition, the effects of DHEA and estradiol were significantly different from cholesterol (F7,73 = 6.42; P < 0.001). For 4 channels, mean percentage increases in 10 µM steroid were 2.0 ± 9.3% for cholesterol, 9.6 ± 10.4% for DHEA, 17.7 ± 4.9% for DHEA-S, 101.9 ± 38.4% for corticosterone, 18.1 ± 4.3% for progesterone, 30.9 ± 8.3% for testosterone, and 40.6 ± 3.2% for 17-estradiol (Fig. 6B, bottom; n = 5, 15, 9, 23, 11, 8, and 7 patches, respectively). Effects of CORT, testosterone, and estradiol were significantly different from control saline, effects of DHEA and CORT were significantly different from each other, and effects of estradiol and CORT were significantly different from 10 µM cholesterol (F7,82 = 7.89; P < 0.001).
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We recently showed that glucocorticoids modulate BK gating and chromaffin cell excitability in rat and bovine chromaffin cells (Lovell et al. 2004). The gating effects of CORT and DHEA on heterologously expressed channels described above are very similar. Inside-out patches were pulled from bovine chromaffin cells and stepped from –80 to +80 mV. BK currents were recorded before and after application of 10 µM CORT (Fig. 7A). CORT (10 µM) caused a rapid increase in BK current (in the trace shown, a 103.2% increase). An average increase of 48.5 ± 10.8% was observed in five additional patches. While full characterization of net effects of DHEA on native channel gating and its consequences remain to be investigated, we have made an initial test to determine whether DHEA has any effect on rat chromaffin BK channels. In this study, complications from inactivation were minimized by stepping for 450 ms to –140 mV before the test step to +80 mV, as was done with HEK-293 patches. The magnitude of ensemble current from inside-out patches from dissociated cells was robustly increased by DHEA. Peak current in this case (Fig. 7A, bottom) was increased by 40.2%. Current was increased similarly in six of six patches (mean increase of 42.5 ± 13.2%). Both inactivating and sustained components of the current were increased similarly, compared with control experiments where patches were reperfused with control saline (P < 0.05, Student's t-test).
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2 and 4 mRNA in adrenal medulla and a small sample of other tissues. Both were robustly represented in RNA acutely extracted from medullary tissue (Fig. 7B). Both were expressed in hippocampus and both anterior and posterior pituitary, although 2 signal was weak in anterior pituitary, possibly reflecting contamination from posterior tissue. The results were consistent with the hypothesis that these subunits participate in transducing DHEA responses of native chromaffin BK channels. |
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DISCUSSION |
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1 and 4 subunits to potentiate BK channel gating (Behrens et al. 2000; Dick and Sanders 2001; Valverde et al. 1999), suggest that all products of the family of mammalian Slo- subunit genes should be considered candidate receptors for rapid BK modulation by multiple steroids. We show that physiologically relevant concentrations of corticosterone facilitate gating of BK channels in HEK-293 cells comprised of plus 4 subunits, acting within tens of seconds of application to cell-free inside-out patches and under whole cell conditions. CORT acts in a rapid and reversible manner, ruling out transcriptional mechanisms. Its effects were dose-dependent and consistent with a Hill coefficient of 1.0 (no co-operativity between effector sites) and a half-maximal dose <400 nM. Percentage increases in current amplitude depend on test potential, reflecting the fact that CORT acts primarily by shifting the voltage dependence of gating in the negative direction, under conditions of constant [Ca2+]i. CORT's effects on 4 channels were significantly different from on BK channels with 2-encoded subunits, showing that subunits differentiate between steroids. The difference was not an artifact of 2 channel inactivation; a noninactivating mutant 2 also conferred only weak sensitivity to CORT. While 2 channels did not respond as strongly to any of the steroids yet tested, robust, rapid, and dose-dependent potentiation was elicited by exposure to DHEA, an androgen-related steroid produced in large quantities by the adrenal cortex. The DHEA sensitivity conferred by 2 was significantly greater than that conferred by 4. Both channel types showed modest potentiation by several other steroids, as tested with test potentials of +80 mV, but no response to the common precursor steroid cholesterol. BK channels comprised of only subunits were completely insensitive to any steroids at all levels tested.
The results indicate that the previously known modifying effects of the various subunits on BK gating seen in the absence of steroids must represent only part of their biological significance; the shared ability of subunits to confer sensitivity to changes in steroid concentrations broadens their potential relevance to cellular excitability in many contexts. Moreover, while subunits tend to react to more than one steroid, they clearly discriminate between steroids; different members of the family confer distinct profiles of relative affinity for specific steroids. This heterogeneity implies a greater dynamic repertoire of BK functional nuances than previously postulated, warranting further study in vivo.
Increasing concentrations of CORT (from 10–9 to 10–4 M) and DHEA (from 10–7 to 10–4 M) had progressively stronger effects, suggesting a wide dynamic range of steroid modulation, encompassing physiologically relevant concentrations. Nevertheless, variability in the percentage increase in current was substantial at all concentrations. Some of this can be attributed to the stochastic nature of BK channel activity, given their relatively small number in patches and large unit conductance, and the irregular bursting and mode-shifting behaviors for which BK channels are known. Variation in steroid sensitivity per se also probably arises from variation in the number of subunits associated with each Slo- tetramer (1–4); variation in this stoichiometry is suggested by variation in the kinetics of inactivation, a property also conferred by 2 (Wang et al. 2002). We observed a modest positive correlation between the DHEA effect and the rate of inactivation (reciprocal of the inactivation time constant). Additional heterogeneity almost certainly derives from unknowns in both and functional status, including phosphorylation, glycosylation, redox state, and accessory protein associations (Gong et al. 2000; Jin et al. 2002a,b; McCobb 2004; Tang et al. 2001).
Previous research has shown that BK channel activation by DHEA or estrogens was not mediated by the production of cAMP and cGMP or by the activation of cyclic nucleotide–dependent protein kinases (Dick and Sanders 2001; Farrukh et al. 1998; Peng et al. 1999). The demonstration that expression of 1 by itself confers estradiol binding on either oocytes or HEK-293 cells argued that estradiol-binding capacity is inherent in the structure (Valverde et al. 1999). Most of our experiments were conducted on inside-out patches (to control [Ca2+]i); steroids are highly lipophilic and can easily pass through the membrane. However, application of CORT to the outside of the cell in whole cell experiments also resulted in rapid potentiation of 4 BK channels. Thus our results are not inconsistent with action at binding sites in extracellular domains of the subunits, as has been suggested for estradiol, xenoestrogens, and fatty acids (Clarke et al. 2002; Dick et al. 2001; Valverde et al. 1999). However, the steroids could also act at a site that depends on the presence of both and (Korovkina et al. 2004) or to an intermediate membrane protein that interacts with the subunit to confer a conformational change in the channel (Dick and Sanders 2001). To formally eliminate the possibility that the subunit induces a steroid-binding capacity that is extrinsic to the subunit itself, direct binding of steroids to purified subunits must be shown. The observation that similar but distinct binding specificities to multiple steroids are conferred by different s, which have 21–43% amino acid identity (Behrens et al. 2000; Brenner et al. 2000; Uebele et al. 2000; Wallner et al. 1999; Xia et al. 1999), further supports the idea that binding per se is inherent in the subunits. Several segments of conserved residues interspersed with numerous nonconserved regions in the extracellular domains of the 2 and 4 subunits could be responsible for overlapping but distinct steroid sensitivities. Cross-comparisons and targeted mutagenesis experiments with these gene products should elucidate relationships between steroid and protein structural and functional interactions.
The proximity of chromaffin cells to the source of adrenal steroid synthesis exposes them to at least episodic pulses of steroids at much higher concentrations than in systemic blood. Systemic CORT levels may not greatly exceed 1 µM, but adrenal blood concentrations may reach concentrations as high as 100 µM (Betito et al. 1992, 1994). Androgen levels in adrenal blood are not known, but systemic DHEA and DHEA-S levels are on the order of 10 nM and 10 µM, respectively (Baulieu 1996). BK channels shape the intrinsic excitability of chromaffin cells, particularly repetitive firing properties, and thus shape catecholamine secretory responses to input (Lingle 1996; Lovell and McCobb 2001; Lovell et al. 2004). In addition to data presented here, molecular and functional expression studies suggest that 2 (Behrens et al. 2000; Ding and Lingle 2002; Solaro and Lingle 1992; Xia et al. 1999) and 4 (Behrens et al. 2000; Brenner et al. 2000) are expressed in chromaffin cells. Channel inactivation very similar to that conferred by 2 is seen in both rat and bovine cells, although viewed across the respective chromaffin populations, there are striking species differences (Lovell et al. 2000). Thus inactivation is more common and tends to be faster and more complete in rat than bovine cells. We hypothesize that 2 and 4 subunits constitute at least part of the mechanism for both DHEA and corticosterone effects on chromaffin BK channels (Lovell et al. 2004). It seems probable that differential expression within one gland, between individuals, and between species, also confers differential stress-related tuning on cell excitability.
DHEA (50 µM) acutely facilitates BK channel activation in patches from ferret pulmonary vascular smooth muscle cells by shifting the voltage dependence of activation 20–25 mV in the negative direction (Farrukh et al. 1998; Peng et al. 1999). In addition to 1, 2 is strongly expressed in rat lung, if perhaps less strongly in human (Behrens et al. 2000; Wallner et al. 1999; Xia et al. 1999). 4 is apparently expressed in human lung as well (Behrens et al. 2000). Given the similarities between the ferret lung and our patch-clamp results, we postulate that 2 and/or 4 are involved and speculate that stress-evoked rises in DHEA promote vasodilation to facilitate oxygen uptake.
Roles played by glucocorticoids in stress responses have been studied in some detail. The ability of CORT to both reduce neuronal firing rate in celiac ganglion cells and enhance firing rate in cardiovascular neurons located in the rostral ventrolateral medulla (Hua and Chen 1989; Rong et al. 1999) shows the importance of rapid steroid modulation in neuronal excitability. Most recently, the acute application of dexamethasone has been shown to increase BK channel activity in pituitary GH3 and AtT-20 cells and reduce the firing of action potentials in GH3 cells (Huang et al. 2005). However, rapid modulation of BK channels and repetitive firing properties of adrenomedullary chromaffin cell by steroids synthesized in the adrenal cortex had not been considered until recently (Lovell et al. 2004). The "strategic" significance of DHEA and adrenal androgens in relation to stress responses or other aspects of physiology remains enigmatic. Chronic effects of DHEA on alternative splicing of the Slo- subunit at the STREX site in chromaffin cells were recently reported to be directly opposite those of glucocorticoids (Lai and McCobb 2002). In contrast, acute effects of the two classes of steroids on BK gating seem to run in parallel (Lovell et al. 2004). This sort of multidimensional complexity of steroid functionality (elegantly reviewed by Sapolsky et al. 2000) ultimately bears on the complexities of organismal survival. Differential expression of Slo- subunits has already been suggested to spawn species differences in nuanced BK gating and cell excitability (Lovell et al. 2000; Solaro et al. 1995; Wang et al. 2002). We suggest that differences in the multisteroidal sensitivity conferred by the variants allow for the fine tuning of BK dynamics by different steroids, and provide yet another level of flexibility to the coupling between excitatory input to the adrenal medulla and catecholamine output.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. P. McCobb, Dept. of Neurobiology and Behavior, W153 Mudd Hall, Cornell Univ., Ithaca, NY 14853 (E-mail: dpm9{at}cornell.edu)
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ABSTRACT
INTRODUCTION
6 channels, BK currents were activated by a test pulse to +80 mV from a holding potential of –80 mV. Application of 1 µM corticosterone (CORT) to the patch rapidly increased the number of BK channels open during successive sweeps. Ensemble averages of 30 such sweeps are shown below. B: time scale plot from an inside-out patch showing acute potentiation of BK current (within 30 s) at 10 nM CORT, reproducibility, and reversibility of steroid effect. Each point on the time scale represents a running average of 5 trace samples. C: dose-dependent increases are evident in different concentrations of CORT covering 6 orders of magnitude (means ± SE). Current amplitudes were measured from tails at –80 after a test potential of +80 mV. A significant increase compared with perfusion of control saline (*P < 0.001, Kruskal-Wallis test) was seen at concentrations of 0.01, 0.1, 1, 10, and 100 µM CORT. Effects at 1, 10, and 100 µM were significantly greater than at 0.001 and 0.01 µM CORT; 10 µM CORT had a significantly greater effect on 
