Pflugers Arch - Eur J Physiol (2006) 452: 756–765 DOI 10.1007/s00424-006-0081-6
SK ELETA L MUSCLE
Igor I. Krivoi . Tatiana M. Drabkina . Violetta V. Kravtsova . Alexander N. Vasiliev . Misty J. Eaton . Serguei N. Skatchkov . Frederic Mandel
On the functional interaction between nicotinic acetylcholine receptor and Na+,K+-ATPase Received: 28 October 2005 / Revised: 24 February 2006 / Accepted: 23 March 2006 / Published online: 25 April 2006 # Springer-Verlag 2006
Abstract Previous studies have shown that nanomolar acetylcholine (ACh) produces a 2 to 4-mV hyperpolarization of skeletal muscle fibers putatively due to Na+,K+ATPase activation. The present study elucidates the involvement of the nicotinic ACh receptor (nAChR) and of Na+,K+-ATPase isoform(s) in ACh-induced hyperpolarization of rat diaphragm muscle fibers. A variety of ligands of specific binding sites of nAChR and Na+,K+-ATPase were used. Dose–response curves for ouabain, a specific Na+,K+-ATPase inhibitor, were obtained to ascertain which Na+,K+-ATPase isoform(s) is involved. The ACh dose–response relationship for the I. I. Krivoi (*) . V. V. Kravtsova . A. N. Vasiliev Department of General Physiology, St. Petersburg State University, 7/9 University emb., St. Petersburg, 199034, Russia e-mail:
[email protected] Tel.: +7-812-3233842 Fax: +7-812-3232454 T. M. Drabkina A.A. Ukhtomski Institute of Physiology, St. Petersburg State University, 7/9 University emb., St. Petersburg, 199034, Russia M. J. Eaton Department of Biochemistry, University Central del Caribe, Bayamon, PR 00960, USA S. N. Skatchkov Department of Biochemistry and Department of Physiology, University Central del Caribe, Bayamon, PR 00960, USA F. Mandel Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA Present address: F. Mandel Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0576, USA
hyperpolarization was also determined. The functional relationship between these two proteins was also studied in a less complex system, a membrane preparation from Torpedo electric organ. The possibility of a direct ACh effect on Na+,K+-ATPase was studied in purified lamb kidney Na+,K+-ATPase and in rat red blood cells, systems where no nAChR is present. The results indicate that binding of nAChR agonists to their specific sites results in modulation of ouabain-sensitive (most probably α2) isoform of Na+,K+-ATPase, leading to muscle membrane hyperpolarization. In the Torpedo preparation, ouabain modulates dansyl-C6-choline binding to nAChR, and vice versa. These results provide the first evidence of a functional interaction between nAChR and Na+,K+ATPase. Possible interaction mechanisms are discussed. Keywords Na+,K+-ATPase isoforms . Nicotinic acetylcholine receptor . Skeletal muscle . Diaphragm . Torpedo californica membrane preparation
Introduction The Na+,K+-ATPase and the nicotinic acetylcholine (ACh) receptor (nAChR) are essential for physiological functioning of excitable cells. The Na+,K+-ATPase maintains the steep Na+ and K+ gradients across the cell plasma membrane that generate the resting membrane potential (RMP), provide electrical excitability, and furnish the driving force for numerous other transport mechanisms. The Na+,K+-ATPase exists as a heteromer, composed of equimolar amounts of αcatalytic and β-glycoprotein subunits. Four isoforms of the α subunit are known to exist in tissues of vertebrates. It is generally accepted that the ubiquitous α1 isoform plays the main “house-keeping” role while the other isoforms expressing in a cell- and tissue-specific manner possess additional regulatory functions that are still poorly understood [3, 32]. In skeletal muscle fibers, the α1 and α2 subunits are expressed [18, 23, 33]. Human skeletal muscle also expresses the α3 isoform [21]. An extracellular domain of the α-subunit serves as a specific receptor for plant cardiac
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glycosides and for bufadienolides as well as for circulating endogenous Na+,K+-ATPase ligands [3, 4, 28]. In rodents, the α1 isoform is atypical as it is highly ouabain-resistant, whereas the α2 isoform is ouabain-sensitive [3, 32]. The nAChR is well known as a ligand-operated ion channel that mediates fast synaptic transmission. The nAChRs are pentamers of homologous subunits. In vertebrate skeletal muscle, they have the composition α2βɛδ in adult muscle, while in fetal muscle and in Torpedo electrocytes, the composition is α2βγδ [14, 27]. The ligand binding sites are situated at the intersection between pairs of subunits, αδ and either αɛ or αγ. These sites bind both agonists such as ACh, carbamylcholine and nicotine as well as competitive antagonists such as αbungarotoxin and members of the curare family. In addition, noncompetitive antagonists such as proadifen can bind at and block the receptor channel. It has been suggested that the Na+,K+-ATPase (in particular its regulatory isoforms α2–α4) may functionally interact with several neighboring proteins [1, 5, 20, 37]. Although, several previous studies have suggested that agonists of the nAChR affect the Na+,K+-ATPase, little is known with regard to a functional relationship between the Na+,K+-ATPase and the nAChR itself. Wang et al. [35], showed that chronic nicotine exposure selectively affects the α2 Na+,K+-ATPase in rat brain. In C2C12 myotubes, it has been shown that chronic exposure to carbamylcholine (CCh) induces membrane hyperpolarization due to an increase in α2 Na+,K+-ATPase abundance mediated by the nAChR [15, 17]. In adult skeletal muscle, steady-state concentrations (up to ∼50 nM) of endogenous nonquantal ACh are continuously present in the synaptic cleft. These concentrations produce a 2 to 4-mV hyperpolarization at the end-plate zone in contrast to membrane depolarizations observed with micromolar concentrations of ACh. Previous studies [10, 22, 34] suggested that this hyperpolarization is due to Na+,K+-ATPase activation. However, no attempts were made to assess the Na+,K+-ATPase isoforms responsible. Furthermore, it remains obscure whether the nAChR itself is involved in the ACh-induced hyperpolarization (AIH). It is difficult to elucidate these problems because of multiple highly interrelated molecular events operating at the synapse that might affect the postsynaptic events and complicate quantification of the ACh effects. In the present work, we used rat diaphragm muscle to study the hyperpolarizing effect of low concentrations (≤100 nM) of exogenous ACh in nonsynaptic membranes that contain both the Na+,K+-ATPase and extrasynaptic nAChR. Such an approach provides a far simpler experimental model. To test the involvement of the nAChR and the Na+,K+-ATPase in AIH, a variety of ligands of specific binding sites of these proteins were used. A dose–response curve for ouabain, a specific inhibitor of Na+,K+-ATPase, was obtained to ascertain which Na+,K+-ATPase isoform(s) is involved in the AIH. In addition, the putative functional relationship between the nAChR and the Na+,K+-ATPase was studied in a much less complex system, a membrane preparation from the electric organ of Torpedo californica. The effect of ouabain
on the binding of the cholinergic ligand dansyl-C6-choline (DCC) to nAChR was studied using the stopped-flow technique. Finally, we tested the possibility of a direct ACh effect on Na+,K+-ATPase activity in two other systems where no nAChR is present: purified lamb kidney Na+,K+ATPase and rat red blood cells Na+,K+-ATPase. The present study establishes that the binding of nAChR agonists to their specific nAChR sites results in the modulation of Na+,K+-ATPase leading to muscle membrane hyperpolarization. The results also suggest that in rat skeletal muscle, it is the ouabain-sensitive α2 isoform of Na+,K+-ATPase that is involved. Lastly, we observed that in the Torpedo membrane preparation, ouabain modulates the binding of DCC to nAChR and vice versa. In summary, our results suggest a functional interaction between the nAChR and the Na+,K+-ATPase.
Materials and methods Recording the RMP of rat diaphragm muscle fibers The experiments were performed on diaphragm muscles rapidly dissected from adult male Wistar rats (150–180 g) anesthetized by ether and killed by cervical dislocation. A 10 to 15-mm wide strip of muscle with nerve stump (∼20 mm long) was dissected from the left hemidiaphragm muscle. Muscles were placed in a Plexiglas chamber and superfused at 28°C with a physiological solution consisting of (in mM): 137 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 24 NaHCO3, 1 NaH2PO4, and 11 glucose (bubbled with 95% CO2, 5% O2; pH 7.4). The muscles were equilibrated in the solution for 1 h before recordings were initiated. The RMP was recorded intracellularly using standard microelectrode techniques. All recordings were made in nonsynaptic regions of the muscle fibers (about 2 mm from visually identified terminal branches of the phrenic nerve). The RMP was amplified, digitized with a 12-bit analog-to-digital converter, and stored and analyzed using a PC computer. A special computer program was used for electronic correction of the electrode-to-solution potential (zero balance) and for sampling the recorded signal. The zero potential of preamplifier input was automatically adjusted by a signal from the computer keyboard before impalement. Approximately 1 s after impalement, a signal for RMP measurement was generated. Due to the automation of the protocol, a maximum of 10 s was required to impale the fiber, collect data, and move the electrode to another fiber. Thus, the total recording time for 30–35 fibers was about 5 min. Two standard protocols were used. (1) As described above, in most experiments, the RMP was recorded in rapid succession from the same muscle strip. These measurements were repeated every 15–30 min during the course of the experiment (up to 2–3 h). The entire protocol was repeated in four to six muscle strips from different animals to obtain RMP values from at least 130–170 fibers at each data point. (2) In the second set of experiments that measured the short-term dynamics of ACh action, initial RMP was recorded as described above. Then, the
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subsequent recordings were made in different fibers as rapidly as possible during 15 min of ACh exposure and for 15 min during washout. The RMP determinations obtained during each 1-min time interval were then averaged for all the muscles studied. In these experiments, the complete exchange of the bath solution took no more than 30 s. Rapid stopped-flow fluorescent spectroscopy in Torpedo membrane preparations Membranes containing both the nAChR and the Na+,K+-ATPase were isolated from T. californica electric organ by differential sucrose ultracentrifugation as described by Pedersen et al. [24]. Although this preparation involves both continuous and discontinuous sucrose centrifugations, it still contains other proteins besides the nAChR including the Na+,K+ATPase [7]. The most highly purified fraction utilized in this study typically contained 1.0–1.6 nmol of nAChR binding sites per milligram of total protein as determined by either [3H]-ACh binding or [125I] α-bungarotoxin binding and 0.15–0.25 nmol of Na+,K+-ATPase binding sites per milligram of total protein as determined by [3H]ouabain binding. Membranes were stored in 37% sucrose/0.2% NaN3 at −80°C under argon. Immediately before use, the membranes were treated with diisopropyl fluorophosphate to inactivate acetylcholinesterase that is typically present in the membrane preparations. The rapid association of the fluorescent cholinergic ligand dansyl-C6-choline, DCC, to membrane preparations was monitored by changes in fluorescence intensity at 557 nm using a stopped-flow instrument from KinTek (Model SF-2001, Austin, TX, USA). To maximize the signal to noise ratio, the experiments were done under conditions of energy transfer from the nAChR tryptophans to DCC [26, 29]. Fluorescence was excited at 290 nm with a 4-nm slit width using a 75-W Xenon lamp. The emission was collected through a 495-nm cut-off filter. DCC association assays were conducted either in Torpedo physiological saline buffer, HTPS (in mM): 250 NaCl, 5 KCl, 3 CaCl2, 2 MgCl2, and 20 Hepes (pH 7.0) or in a buffer containing only 250 mM NaCl, and 20 mM Hepes, pH 7.0, as indicated. The latter solution was used to elucidate the effect of Mg2+ and ouabain on DCC binding in the absence of other complicating factors (e.g., K+ and Ca2+). As DCC ligand binding to nAChR is a function of ionic strength [30], the buffer chosen for these experiments was similar to HTPS in ionic strength. Furthermore, because Ca2+ and K+ are known to modulate both the nAChR and the Na+,K+-ATPase, we felt that experiments performed in a buffer lacking those cations would have fewer independent variables and, hence, be more definitive. To measure association kinetics, nAChR-rich membranes were incubated with various concentrations of ouabain for 1 h and then loaded into one syringe, while 1 μM DCC was loaded into the other. The association kinetics were determined by rapidly mixing the solutions in a volume ratio of 1:1. The concentration of nAChR used was typically 100 or 200 nM ACh binding sites (approximately 0.06–0.20 mg/ml). The fluorescence intensity was monitored between ∼1 ms and 15 s. Each
resultant trace consisted of 1,000 points. To reduce noise, approximately 20 individual mixing traces (the maximum buffer capacity of the KinTek stopped-flow) were averaged to obtain a single “averaged curve”. For each set of experimental conditions (e.g., control), one or more “averaged curves” were obtained. Then, experiments with the next set of conditions (e.g., in the presence of 20 nM ouabain) were performed. After all the different sets of experiments were completed, the same sequences of experiments were repeated one or two more times to make sure that any differences observed were not due to elapsed time. The curves shown in Figs. 6 and 7 are representative curves of results obtained on different days with the same or different preparations. Each curve shown is the average of two or more “averaged curves” obtained as above and, hence, the average of more than 40 individual traces. The results obtained at the beginning of the day and those at the end were not significantly different indicating the absence of any drift during the course of the experiments. Measurement of the Na+,K+-ATPase activity Transport activity of the Na+,K+-ATPase was determined by the ouabain-sensitive uptake of nonradioactive Rb+ into rat red blood cells using emission flame photometry as in Longo et al. 2001 [19]. The red blood cells were obtained from 2 ml of whole blood, washed with a fourfold volume of cold (4°C) solution consisting of 145 mM NaCl and 10 mM TRIS (pH 7.4), and centrifuged at 600 g for 3 min. The supernatant and the upper layer containing leukocytes were removed and the red blood cells washed again. This procedure was repeated four times; after which, the precipitated red blood cells were diluted back to 2 ml with an incubation solution consisting of (in mM): 145 NaCl; 1 CaCl2; 1 NaH2PO4; 2 MgCl2; 10 TRIS and 10 glucose (pH 7.4). Each sample consisted of 0.1 ml of red blood cell suspension diluted with 0.9 ml of the incubation solution either containing ACh or not (control). After 1 h preincubation of the red blood cells with 5 μM armin (diethoxy-p-nitrophenylphosphate), an organophosphate acetylcholinesterase inhibitor, ACh was added. Fifteen minutes later, 0.1 ml of 50 mM RbCl was added to all samples. The samples were subsequently centrifuged for 3 min at 800 g and then washed four times with a 4°C solution of 93 mM MgCl2. For the hemolysis of the precipitated red blood cells, 1 ml of distilled water was added to each sample. The samples were then stirred and kept for 24 h at 4°C. Concentrations of Rb+, K+, and Na+ were measured using a Perkin Elmer 306 atomic absorption spectrophotometer. The transport activity of the Na+,K+ATPase was taken as the difference between the Rb+ influx in the presence and in the absence of 1 mM ouabain. The activity of purified Na+,K+-ATPase from lamb kidney was estimated using the coupled pyruvate kinase/ lactic dehydrogenase linked-enzyme system as in Farr et al. 2001 [12]. The decrease of NADH absorbance was measured at 340 nm (Beckman DU-7 spectrophotometer). The specific Na+,K+-ATPase activity was taken as the
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difference between the activity in the presence and in the absence of 1 mM ouabain (10 min preincubation). Materials T. californica was purchased from Aquatic Research Consultants, San Pedro, California. The DCC was a gift from Dr. Steen Pedersen (Baylor College of Medicine). Diisopropyl fluorophosphate was obtained from Sigma-Aldrich (St Louis, MO, USA), α-bungarotoxin was from Molecular Probes (Eugene, OR, USA) and [3H]-ouabain was obtained from Amersham (Piscataway, NJ, USA), armin was from the Institute of Organic Chemistry, Moscow, Russia. The specific Na+,K+-ATPase inhibitor marinobufagenin isolated from Bufo marinus, was a gift from Dr. Alexei Bagrov (National Institutes of Health). Purified Na+,K+-ATPase from lamb kidney was a gift from Dr. J. Ball (University of Cincinnati Medical Center). Other chemicals were from Sigma Chemical. Data analysis Data are presented throughout the text and figures as mean values±SEM. The statistical significance of the differences between means was calculated by Student’s t test. Statistics and binding parameters were calculated using ORIGIN 6.1 or SIGMAPLOT 9.0.
Results The effect of nAChR agonists on the RMP of rat diaphragm muscle fibers In control experiments, the RMP at the beginning of the experiments was −78.0±0.5 mV (six muscles, 177 fibers), and did not change significantly over a period of 3 h (Fig. 1a). In our preparations, normal parameters of postsynaptic potentials as well as action potentials were observed (Fig. 1a, insert) verifying that the muscle fibers were in their normal physiologically functioning states. In the first set of experiments, 100 nM ACh was added for 15 min. After 15 min of exposure to ACh, the RMP was hyperpolarized from −78.1±0.4 mV (eight muscles, 264 fibers) to −82.3±0.4 mV (282 fibers). Thus, the AIH was 4.2±0.5 mV (p
