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(Circulation Research. 1995;76:335-342.)
© 1995 American Heart Association, Inc.

Articles

Stable Expression and Coupling of Cardiac L-Type Ca²⁺ Channels With ß₁-Adrenoceptors

Atsuko Yatani, Minoru Wakamori, Tetsuhiro Niidome, Satoshi Yamamoto, Isao Tanaka, Yasuo Mori, Kouich Katayama, Stuart Green

From the Departments of Pharmacology and Cell Biophysics (A.Y., M.W., S.Y., Y.M.) and Medicine (Pulmonary) (A.Y., S.G.), University of Cincinnati (Ohio) College of Medicine, and Tsukuba Research Laboratories (T.N., I.T., K.K.), Eisai Co Ltd, Ibaraki, Japan.

Correspondence to Dr A. Yatani, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0575.

	Abstract

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Abstract A number of neurotransmitters modulate cardiac dihydropyridine-sensitive L-type Ca²⁺ channels through several homologous G protein–coupled receptors. Previous studies that have examined receptor–Ca²⁺ channel interactions have suffered because of the coexpression of various receptor subtypes in native cells. To study the functional coupling of a particular receptor subtype to these channels, rabbit cardiac Ca²⁺ channel ₁ and skeletal ß and ₂/ subunits were stably expressed in baby hamster kidney cells. In this stable cell line, Ca²⁺ channels remained at high levels (>1000 fmol/mg protein, or 2700 channels per cell) over extended times. The expressed recombinant Ca²⁺ channels displayed the voltage dependence of activation and inactivation, unitary conductance, and pharmacology characteristic of native cardiac L-type Ca²⁺ channels. Subsequent coexpression of the ß₁-adrenoceptors (150 to 300 fmol/mg protein) with the Ca²⁺ channels resulted in cell responsiveness to the extracellular application of isoproterenol. These results indicate that heterogeneous expression in mammalian cells provides a useful system for studying both biophysical analysis of Ca²⁺ channel properties and receptor-coupled regulatory processes.

Key Words: Ca²⁺ channels • ß₁-adrenoceptors • patch clamp • baby hamster kidney cells

	Introduction

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The dihydropyridine (DHP)-sensitive L-type Ca²⁺ channel is one of the main targets of various physiological modulators in cardiac myocytes. ß-Adrenergic agonists such as isoproterenol (Iso) increase L-type Ca²⁺ channel currents through cytoplasmic cAMP-dependent phosphorylation and also via a membrane-delimited direct interaction with the stimulatory G protein (G_s).^{1 2 3 4 5 6} Evidence from radioligand binding and functional studies indicates that both ß₁- and ß₂-adrenoceptors (ß₁AR and ß₂AR, respectively) coexist in heart preparations of many mammalian species including human and are functionally involved in the regulation of contractile force and/or heart rate.⁷ A combination of subtype-specific agonists and antagonists have been used to analyze the cardiotropic effects of individual ßAR subtypes.^{8 9} However, quantitative studies of the ßAR subtype–specific interactions with cardiac Ca²⁺ channels are complicated by the coexistence of mixed populations of subtypes in native myocytes, since truly selective activation or blockade of one subtype without effect on the other is difficult to attain.¹⁰ Systematic analysis of individual ßAR effects should therefore ideally compare identical cells expressing pure populations of receptor subtypes in a constant biochemical environment.

Structurally, the L-type Ca²⁺ channel is a complex of four proteins: the ₁ subunit, which contains the binding sites for Ca²⁺ channel blockers and forms the ion-conducting pore; the ₂/ subunit, a disulfide-linked dimer; the intracellularly located ß subunit; and the transmembrane subunit.¹¹ cDNAs for each of the four subunits have been cloned from skeletal muscle. By use of these cDNAs as probes, distinct gene products encoding analogous subunits have been cloned from other tissues, including cardiac muscle. These studies have revealed that the subunit is expressed only in skeletal muscle, whereas the ₂/ subunit is highly conserved in most tissues including heart. Expression studies in several systems have also demonstrated that the ₁ subunit is the functional component and determines the pore, gating, and much of the pharmacology of the channel.¹²

The ₁ and ß subunits of the purified skeletal muscle Ca²⁺ channel are phosphorylated by cAMP-dependent protein kinase (PKA), with the ₁ subunit appearing to be the primary substrate. In cardiac muscle, however, the target(s) for phosphorylation by PKA is unclear.¹³ For example, in Xenopus oocytes, cardiac Ca²⁺ channel currents expressed by recombinant ₁ subunits are not increased by cAMP unless the ß subunit is coexpressed.¹⁴ In contrast, Ca²⁺ channel currents mediated by the ₁ subunit expressed in Chinese hamster ovary (CHO) cells are increased by cAMP-dependent phosphorylation.^{15 16} These differences may result from the lack of appropriate regulatory components in oocytes. However, the oocyte membrane does contain various amounts of a ß subunit–responsive endogenous Ca²⁺ channel,^{17 18} and these are modulated by phosphorylation.¹⁹ Such discrepancies have served to render the oocyte expression system particularly problematic for studies of Ca²⁺ channel regulation. In addition, the presence of extensive infoldings of the oocyte membrane and highly lipophilic compartments within the oocytes reduces effective exchange with bulk solutions used in electrophysiological measurements.²⁰

To circumvent these complications, we stably coexpressed the cDNAs encoding rabbit cardiac ₁,²¹ skeletal ß,²² and skeletal ₂/²¹ subunits in baby hamster kidney (BHK) cells²³ and studied adrenergic regulation of the resulting recombinant cardiac Ca²⁺ channel. BHK cells were selected for study since these cells do not contain detectable endogenous Ca²⁺ channels or ß-adrenoceptors. In this stable cell line, designated BHKC112, Ca²⁺ channels remain at high levels without diminution over extended periods (>60 passages) and display gating, conductance, and pharmacology of native L-type cardiac Ca²⁺ channels. We further show that coexpression of the ß₁AR in BHKC112 cells results in Iso-dependent modulation of the expressed Ca²⁺ currents.

	Materials and Methods

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Constructs, Transfections, and Cell Culture
To express a functional cardiac DHP-sensitive Ca²⁺ channel in a mammalian cell, the following constructs were derived. cDNA encoding the rabbit cardiac ₁ subunit excised from the plasmid pCARD1²¹ was ligated along with the dihydrofolate reductase selection gene derived from pAdD26SV(A)²⁴ into pKCR,²⁵ yielding the plasmid pK4KC. cDNA encoding the rabbit skeletal muscle ₂ subunit derived from pSPCA1²¹ was ligated into pKNH²⁶ to form pCAA2, which includes a neomycin resistance gene. cDNA encoding the rabbit skeletal muscle ß subunit was reverse-transcribed from rabbit skeletal muscle total RNA and then amplified by polymerase chain reaction (PCR) using primers corresponding to nucleotides 80 to 102 (sense) and 1809 to 1835 (antisense) of the rabbit sequence.²² PCR was performed for 35 cycles at 94°C for 1 minute, 68°C for 1 minute, and 72°C for 3 minutes. The 1.8-kb PCR product obtained was subcloned into M13mp18 and mp19 for confirmatory dideoxy sequencing before ligation into pKCRH2,²⁷ yielding the plasmid pCABE.

Stable transfections were performed on BHK cells²³ by modified calcium phosphate precipitation (CellPhect, Pharmacia) by using 5 µg each of pK4KC, pCAA2, and pCABE. BHKC112 cells were selected in DMEM containing 600 µg/mL G418 (a neomycin analogue) and 250 nmol/L methotrexate; cells were subsequently maintained in 5% fetal calf serum, 30 µg/mL streptomycin, 30 U/mL penicillin, 80 µg/mL G418, and 250 nmol/L methotrexate. Confirmation of expression of a DHP-sensitive Ca²⁺ channel was performed by Northern analysis, radioligand binding, and electrophysiological techniques as described below.

BHKC112 cells were transiently transfected with cDNA encoding the human ß₁AR by calcium phosphate precipitation similar to that previously described.²⁸ For these transfections, cells were maintained in DMEM supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. cDNA encoding the human ß₁AR excised from the plasmid pBC12BI²⁸ was ligated into the plasmid pcDNA-1/Neo (Invitrogen) under the control of the CMV early promoter, yielding the plasmid pcNeoß₁. Plasmid DNA (20 to 40 µg) was used for each transfection. Cells were maintained in DMEM supplemented with 10% fetal calf serum and antibiotics and assayed when confluent (24 to 48 hours). Preliminary data indicated that BHKC112 cells transfected in this manner expressed detectable ß₁AR for up to 5 days.

Northern Analysis
Total RNA was isolated from nontransfected BHK cells and from BHKC112 cells by the single-step method of Chomczynski and Sacchi.²⁹ Total RNA (5 µg) was electrophoresed through 1% agarose/formaldehyde gels and transferred to nylon membranes by standard techniques. Double-stranded cDNA probes for the Ca²⁺ channel ₁, ₂, and ß subunits were prepared from the following sources: ₁, 2.1-kb EcoRI/EcoRV fragment of pCARD3²¹ ; ₂, 2.4-kb HindIII fragment of pSPCA1²¹ ; and ß, 0.8-kb Pst I/Sca I fragment of pCaB2.³⁰ Each probe was then labeled by nick translation. Hybridization was carried out at 42°C overnight, membranes were washed, and autoradiographs were exposed for 72 hours at -70°C.

Radioligand Binding
Ca²⁺ channel radioligand binding studies were performed by methods similar to those previously described.³¹ After the cells were washed with Ca²⁺-free phosphate-buffered saline (PBS), confluent BHKC112 cells in tissue culture dishes were used to prepare crude membranes by mechanical disruption in 5 mmol/L Tris (pH 7.4) and 2 mmol/L EDTA buffer and centrifugation at 39 000g for 10 minutes at 4°C. Saturation binding studies were performed by suspending membranes in assay buffer (50 mmol/L MOPS, pH 7.4) at 30 µg/mL and incubating for 2 hours at 22°C with 0 to 10 nmol/L [³H]PN200-110 in a final volume of 500 µL. Nonspecific binding was defined by 1 µmol/L nitrendipine. Reactions were stopped by dilution and rapid filtration over glass fiber filters, which were then counted in a scintillation counter.

For ßAR radioligand binding studies, membranes were prepared as described above and suspended in 75 mmol/L Tris (pH 7.4), 12.5 mmol/L MgCl₂, and 2 mmol/L EDTA at a final concentration of 0.1 mg/mL before incubating with 400 pmol/L [¹²⁵I]cyanopindolol (ICYP) for 2 hours at 22°C. Reactions were stopped by dilution and rapid filtration over glass fiber filters. Nonspecific binding was defined by 1 µmol/L propranolol. Competition assays were performed similarly, except that incubations were performed in the presence of (final concentrations) 100 µmol/L GppNHp, 30 pmol/L ICYP, and varying amounts of competing ligand as indicated. Both saturation and competition data were analyzed by nonlinear techniques as described previously²⁸ ; Hill coefficients were typically close to 1.0. For all radioligand binding assays, total counts bound were typically <10% of counts added. Protein was measured by a copper bicinchoninic assay,³² with bovine serum albumin used as standard.

cAMP Determinations
Whole-cell cAMP determinations were assessed by radioimmunoassay (RIA) similar to that previously described.³³ BHKC112 cells were transfected with ß₁AR cDNA as described above; when confluent (24 hours), these were detached with 0.5% trypsin/EDTA and seeded into 24-well culture dishes. Twenty-four to 48 hours later, cells were washed three times with PBS and allowed to warm at 37°C for 10 minutes in 450 µL PBS supplemented with 0.1 mmol/L ascorbic acid. Iso was then added at varying concentrations into duplicate wells, and reactions were terminated 10 minutes later by adding 50 µL of 1N HCl. Aliquots of supernatant were acetylated, and cAMP content was determined by RIA as previously described.³³

Electrophysiology
For electrophysiological measurements, cells were seeded onto glass coverslips and incubated in culture medium with fetal calf serum for 1 to 3 days. Cells prepared in this manner were spherical and had diameters of 10 to 20 µm when measured with an eyepiece micrometer at x40 (x600 total magnification). The surface areas of cells were thus between 314 and 1256 µm², assuming that the cells maintained a smooth spherical shape. Whole-cell and single-channel currents were recorded by patch-clamp techniques as previously described.³⁴ Patch electrodes were coated with Sylgard and had resistances of 2 to 5 M. Whole-cell currents were low-pass–filtered (-3 dB) at 0.2 to 1 kHz and digitized at 0.5 to 5 kHz. Leak and capacitative currents were corrected by using a P/4 subtraction procedure unless noted otherwise. The external solution contained (mmol/L) BaCl₂ 2, MgCl₂ 1, tetraethylammonium chloride 135, 4-aminopyridine 5, glucose 10, and HEPES 10 (pH 7.3 with Tris base). The pipette solution was (mmol/L) cesium aspartate 110, CsCl 20, MgCl₂ 2, ATP 2, GTP 0.5, EGTA 5, and HEPES 5 (pH 7.3 with Tris base). The membrane capacitance of the cells was measured by using voltage ramps of 0.8 V/s from a holding potential of -50 mV. The conductance-voltage relation (G/G_max) was determined by using an interactive nonlinear regression fitting procedure to the Boltzmann equation: G/G_max=1/{1+exp[(V_0.5-V_m)/k]}, where V_m is the membrane potential, V_0.5 is the midpoint potential, and k is the slope factor. Similarly, voltage-dependent inactivation (I/I_max) was determined by using the Boltzmann equation: I/I_max= 1/{1+exp[(V_m-V_0.5)/k]}.

Single-channel currents were low-pass–filtered at 1 to 2 kHz, digitized at 5 to 10 kHz, and analyzed as previously described.³⁴ The patch electrodes contained (mmol/L) BaCl₂ 90, glucose 10, and HEPES 10 (pH 7.3 with Tris base). Cells were bathed in a high-KCl depolarizing solution containing (mmol/L) KCl 140, MgCl₂ 2, EGTA 5, and HEPES 5 (pH 7.3 with KOH). Curve fitting was performed by using a maximum likelihood estimator.

Solution changes were made by using a modification of Y-tube techniques described by Nakagawa et al.³⁵ A microcapillary tube (0.1-mm diameter) was set 200 µm away from the cell being recorded. Solutions were then delivered by a negative pressure of -40 cm Hg. After the pressure was released, external solution was emitted from the outlet of the Y tube by gravity. Complete exchange of the solution surrounding the recording cell can be achieved within 50 milliseconds. To avoid accumulation of test substances in the bulk solution, the chamber was continuously perfused at 5 mL/min.

All experiments were performed at room temperature (20°C to 21°C). Mean±SEM values are given in the text. Comparisons between conditions were evaluated by using Student's t test, with significance imparted at the P<.05 level.

Materials
All radioligands were from New England Nuclear. BHK cells were from American Type Culture Collection. DMEM and fetal calf serum were from JRH Bioscience. G418 was from GIBCO BRL. pcDNA-1/Neo was from Invitrogen. ICI 118551 and nitrendipine were from Research Biochemicals, Inc. CGP20712 was a gift from CIBA-GEIGY. All other reagents were from Sigma Chemical Co.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Properties of Expressed Ca²⁺ Channels
Cotransfection of BHK cells with cDNAs encoding the rabbit cardiac ₁ and skeletal ₂ and ß subunits resulted in easily detectable mRNA expression for each subunit (Fig 1). As shown, nontransfected BHK cells do not express detectable mRNA for these subunits. Expression and function of Ca²⁺ channels in BHKC112 cells was further assessed by radioligand binding using the DHP antagonist [³H]PN 200-110 and by patch-clamp techniques as described below. Saturation binding studies revealed a K_d of 0.81±0.23 nmol/L (n=3, Fig 2), in good general agreement with binding affinities observed in endogenous myocardial channels.³¹ The density of L-type Ca²⁺ channels in membranes prepared from transfected BHK cells was 1227±199 fmol/mg protein (n=7). Nontransfected BHK cells did not display any specific [³H]PN 200-110 binding sites (data not shown).

View larger version (45K): [in this window] [in a new window]   Figure 1. Northern analysis of baby hamster kidney (BHK) cells (lanes 1, 2, 5, 6, 9, and 10) and transfected recombinant cardiac Ca2+ channel BHK cells (lanes 3, 4, 7, 8, 11, and 12). Total RNA (5 µg) was electrophoresed, transferred to nylon membranes, and individually hybridized with double-stranded cDNA probes for the 1, 2, and ß Ca2+ channel subunits as described in "Materials and Methods." Autoradiograms were exposed at -70°C for 72 hours before developing. Lanes are as follows: 1 through 4, 1 probe; 5 through 8, 2 probe; and 9 through 12, ß probe. Equivalent loading of RNA was confirmed by using a double-stranded actin probe, which gave a strong signal in all lanes (not shown).

View larger version (17K): [in this window] [in a new window]   Figure 2. Graph showing saturation binding of membranes from baby hamster kidney cells expressing L-type Ca2+ channel. Membranes were prepared as described in "Materials and Methods" and incubated with [3H]PN200-110 in the presence () or absence () of 1 µmol/L nitrendipine for 2 hours at room temperature. Reactions were stopped by dilution and rapid filtration over glass fiber filters before being counted in a scintillation counter. Nonlinear regression of specific binding () yielded a Kd of 0.49 nmol/L. Data shown are representative of three experiments, each performed in duplicate.

Voltage-activated Ca²⁺ channel currents were measured in the presence of 2 mmol/L Ba²⁺ (Fig 3). Nontransfected BHK cells did not demonstrate voltage-activated inward Ba²⁺ current (I_Ba) (Fig 3A), in agreement with radioligand binding data. In contrast, BHKC112 cells expressed large inward I_Ba, which inactivated slowly during 300-millisecond depolarizing pulses (Fig 3B). The size of peak inward I_Ba was 861±117 pA (n=60; range, 110 to 3100 pA). The whole-cell current properties were further analyzed by using cells expressing current densities <1000 pA in order to avoid series resistance artifacts associated with large current amplitude (Table).

View larger version (27K): [in this window] [in a new window]   Figure 3. Typical whole-cell currents recorded in baby hamster kidney (BHK) cells (A) in the presence of 20 mmol/L Ba2+ and in recombinant cardiac Ca2+ channel BHK (BHKC112) cells (B) in the presence of 2 mmol/L Ba2+ as the charge carrier. Tracings show currents recorded from a holding potential of -80 mV to indicated test potentials. The normalized conductance (G/Gmax)–voltage curve for the BHKC112 cell shown in panel B was plotted in panel C. The solid line is fit to a Boltzmann equation. The midpoint potential (V0.5) and the slope factor k are -10.9 and 3.6 mV, respectively. Panel D shows voltage-dependent inactivation of the Ba2+ current in the BHKC112 cell. The current inactivation was examined by a double-pulse protocol (inset). The cells were held at -80 mV and stepped to different membrane potentials between -100 and +20 mV in 20-mV increments for 5 seconds and then stepped to test pulse of 0 mV. Gaps between two pulses were 50 milliseconds, and double pulses were applied every 30 seconds. Panel E shows the peak current during the test pulse normalized to the maximal peak current (Ip/Ipmax). The solid line is fit to a Boltzmann equation. V0.5 and k are -35 and 9.0 mV, respectively.

View this table: [in this window] [in a new window]   Table 1. Properties of Whole-Cell Ba2+ Currents Expressed in Recombinant Cardiac Ca2+ Channel Baby Hamster Kidney Cells

The voltage dependence of current activation was determined by measuring the peak current elicited by depolarizing pulses to various potentials. Typically, the inward current activated at a threshold potential between -30 and -20 mV, peaked between -10 and 0 mV, and reversed around +50 mV. Conductance was then calculated by dividing the peak current by the driving force (Fig 3C and Table). It was difficult to estimate the true reversal potential for the Ca²⁺ channels, because a small outward current in nontransfected cells overlapped at positive potentials (Fig 3A). Therefore, the apparent reversal potential was obtained by extrapolating the peak current-voltage (I-V) plot through the zero current axis. The voltage-dependent channel inactivation was determined by applying 5-second depolarizing pulses to a series of different potentials (Fig 3D). V_0.5 and k values were determined from a Boltzmann equation (Fig 3E and Table). These data demonstrate that the activation and inactivation kinetics of expressed currents in BHKC112 cells are comparable to the L-type Ca²⁺ channel seen in native cardiac myocytes.^{4 36} As previously reported in various cell types, we found that the Ca²⁺ channel currents run down after the whole-cell recording configuration has been established.³ In control experiments (n=10), we generally observed that I_Ba initially increased in amplitude immediately after puncturing the membrane and then exhibited rundown. During rundown, peak inward current amplitude decreased by 50% at a steady rate over 20 to 30 minutes.

The pharmacological properties of the channels expressed in BHKC112 cells are shown in Fig 4. The DHP-agonist Bay K 8644 increased I_Ba in a concentration-dependent manner with an average EC₅₀ of 80 nmol/L (n=4, Fig 4A). As in the case of native cardiac cells, Bay K 8644 (1 µmol/L) shifted the I-V curve to more negative potentials by 11.5±1.3 mV. The benzothiazepine Ca²⁺ channel blocker diltiazem reversibly reduced I_Ba with an average IC₅₀ of 5 µmol/L (n=4, Fig 4B), without changing the I-V relations. Diltiazem at 5 µmol/L shifted the voltage-dependent inactivation curves to more negative potentials by 14.8±1.4 mV.

View larger version (11K): [in this window] [in a new window]   Figure 4. Effects of 1 µmol/L Bay K 8644 (A) and 10 µmol/L diltiazem (B) on Ba2+ current (IBa) in the recombinant cardiac Ca2+ channel baby hamster kidney cell. Current tracings show superimposed IBa before and after the application of the agents. Depolarizing pulses to -10 mV (A) or 0 mV (B) were applied from a holding potential of -80 mV. Bars=100 milliseconds and 200 pA.

To examine the effects of cAMP-dependent phosphorylation on these recombinant Ca²⁺ channel currents, a membrane-permeable cAMP analogue, dibutyryl cAMP (dB-cAMP), was applied to the cells. In the presence of dB-cAMP (50 µmol/L), the amplitude of I_Ba gradually increased and reached a new steady state level 3 minutes after application (Fig 5A). Neither the voltage-dependent activation nor inactivation was significantly affected by dB-cAMP exposure (Fig 5B). An average increase in I_Ba of 22.9±5.6% was observed in seven cells of the 14 studied. The reasons for the failures of 50% of the experiments are not known. As noted above, in most of the cells, I_Ba declines gradually in control conditions. Thus, the varied effects after dB-cAMP treatment are probably due to competition between the slow increase in I_Ba due to dB-cAMP and the steady decrease of control current. The membrane currents in nontransfected BHK cells were not affected by the addition of dB-cAMP (n=10). Together, these results indicate that increased levels of intracellular cAMP potentiate Ca²⁺ channel currents expressed in BHKC112 cells.

View larger version (16K): [in this window] [in a new window]   Figure 5. Modulation of Ba2+ current in the recombinant cardiac Ca2+ channel baby hamster kidney cell by dibutyryl cAMP (dB-cAMP, 50 µmol/L). Current tracings (A) are shown before (left) and 5 minutes after (right) dB-cAMP in response to the test potential indicated. The holding potential was -80 mV. The corresponding peak current-voltage relations are plotted in panel B. Bars=100 milliseconds and 100 pA.

Fig 6 shows single-channel recordings of BHKC112 cells in the presence of 1 µmol/L Bay K 8644. Single-channel data obtained from cell-attached patches also demonstrated the features of native cardiac L-type Ca²⁺ channels. For example, the open-state probability of the channel increased with depolarization (Fig 6A). From the I-V relations of unitary Ba²⁺ currents, a single-channel conductance of 24 pS was estimated (Fig 6B). This value correlates well with the value of 24 to 25 pS observed in cardiac myocytes that was determined under identical conditions.^{37 38} From whole-cell and single-channel measurements (Figs 4A and 6B), we estimated the Ca²⁺ channel density in BHKC112 cells, assuming a probability of opening of 0.2 at 0 mV and a unitary current amplitude of 1.1 pA, to be 2700 channels per cell, or about three channels per square micrometer.

View larger version (16K): [in this window] [in a new window]   Figure 6. Single-channel currents recorded from the cell-attached patch on the recombinant cardiac Ca2+ channel baby hamster kidney cell. A, Typical single-channel current evoked by step pulses to indicated voltages from a holding potential of -80 mV in the presence of Bay K 8644 (1 µmol/L). B, Current-voltage relations of single-channel currents. The solid line is linear regression with a slope conductance of 24 pS. Each data point is the mean from four to six experiments with 100 to 1000 events.

Functional Coupling of Ca²⁺ Channel Currents With Coexpressed ß₁ARs
We transiently transfected the ß₁AR cDNA into BHKC112 cells and determined receptor expression by radioligand binding. Nontransfected BHKC112 cells expressing only the DHP-sensitive Ca²⁺ channel demonstrated no detectable ICYP binding (data not shown). In contrast, membranes from BHKC112 cells transfected with pcNeoß₁, as described in "Materials and Methods," expressed 200 fmol ICYP binding sites per milligram protein (range, 150 to 300 fmol/mg). It should be noted that because of the transient nature of these transfections, only a portion of BHKC112 cells treated in this manner actually take up and express the ß₁AR; as a result, the receptor density obtained by radioligand binding represents a mean value for all cells treated, including those cells that failed to take up exogenous DNA. Our previous experience with these assays has suggested that the actual efficiency of transient expression (ie, the proportion of total cells that express the recombinant protein of interest) is between 30% and 60% (S.A. Green and S.B. Liggett, unpublished data, 1994). As described in detail below, we noted electrophysiological evidence of ß₁AR activity in 32 of 78 cells studied, a figure that is consistent with this phenomenon.

The identity and functional nature of the expressed ßAR was confirmed as the ß₁AR subtype by competition binding and cAMP determinations. As depicted in Fig 7A, membranes prepared from BHKC112 cells transfected with pcNeoß₁ demonstrated rank-order antagonist profiles typical of ß₁AR receptors.²⁸ In addition, transfection of pcNeoß₁ conferred Iso responsiveness for cAMP accumulation in whole cells, which was not observed in nontransfected BHKC112 cells (Fig 7B). This response confirms the presence of ß₁AR functionally coupled to the G_s/adenylyl cyclase pathway.

View larger version (24K): [in this window] [in a new window]   Figure 7. Graphs showing pharmacological characteristics of recombinant cardiac Ca2+ channel baby hamster kidney (BHKC112) cells transfected with pcNeoß1. A, Antagonist competition binding with [125I]cyanopindolol ([125I]CYP) performed in membranes prepared from transfected cells as described in "Materials and Methods." Nonlinear regression of binding data yielded Ki values of 1.91 and 114 nmol/L for CGP 20712 (ß1-specific antagonist) and ICI 118 551 (ß2-specific antagonist), respectively. B, Accumulation of cAMP in response to isoproterenol. cAMP content in intact BHKC112 cells was determined by radioimmunoassay along with that observed in cells transfected with pcNeoß1, as described in "Materials and Methods." Nonlinear regression yielded an EC50 value for isoproterenol-promoted cAMP accumulation of 2.9 nmol/L. Data shown are representative of two experiments, with each point determined in duplicate. ß1AR indicates ß2-adrenoceptor.

Potentiation of I_Ba in BHKC112 cells transfected with the ß₁AR was examined with various concentrations of Iso. An increase in I_Ba was detectable with 10 nmol/L Iso and reached a maximum at 10 µmol/L. As noted above, an effect of Iso on I_Ba was observed in 32 of 78 cells transfected with the pcNeoß₁. An increase in the Ca²⁺ channel currents by Iso was not observed in BHKC112 cells not transfected with the ß₁AR (n=24), consistent with the lack of ßAR measured by the radioligand binding.

During the steady state exposure to Iso at a concentration of 10 µmol/L, the mean relative increase of I_Ba by Iso (10 µmol/L) was 2.39±0.35-fold (n=12, Fig 8A and 8B). Several studies have reported that the ß-adrenergic stimulation of Ca²⁺ channel currents causes a shift in the I-V curve to more negative potentials.^{39 40} Therefore, we examined the effects of Iso on the I-V relation of I_Ba. The peak I-V relations before and after Iso exposure are illustrated in Fig 8D. Iso increased I_Ba at all potentials measured without changing the peak I-V relation. In four experiments, the conductance-voltage relation was analyzed in the same way as described in Fig 3C. In the absence of Iso, the mean values of V_0.5 and k were -6.8±3.5 and 4.5±0.6 mV. With 10 µmol/L Iso, the values were -8.5±3.9 and 4.4±0.7 mV for V_0.5 and k, respectively. These results indicate that ß₁-adrenoceptor stimulation did not affect the voltage-dependent activation kinetics. In most experiments, such as that depicted in Fig 8C, the current waveform appeared to be scaled, suggesting that the time course of I_Ba was not altered by Iso. Fig 8E depicts the dose-response relations for Iso on I_Ba. Analysis of data by nonlinear techniques using a one-to-one drug-receptor relation revealed a mean EC₅₀ of 231 nmol/L (n=5). Iso-mediated increases in I_Ba were completely inhibited by 50 µmol/L propranolol (data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 8. Effects of isoproterenol (Iso) on Ba2+ current (IBa) in recombinant cardiac Ca2+ channel baby hamster kidney cells expressing the ß1-adrenoceptor (ß1AR). Currents recorded before (A) and 2 minutes after (B) the applications of Iso (10 µmol/L) are shown. Holding potential was -80 mV. The control current at 0 mV was scaled for comparison with the waveform in panel C. Current-voltage relations (D) are shown in the absence and presence of Iso (same data as shown in panels A and B). Concentration-response curve (E) is shown for the effect of Iso on IBa. The relative increase of peak current amplitude at different Iso concentrations was obtained by normalizing to the value produced by the drug (10 µmol/L). Solid line is fit to one-to-one binding model with an EC50 of 231 nmol/L. Data are the mean from four experiments.

The degree of increase in I_Ba by Iso was dependent on the presence of guanine nucleotides; without GTP in the pipette solution, in the presence of 10 µmol/L Iso, the magnitude of enhancement was greatly reduced to 106±3% of control values (n=5). To further test the involvement of a G protein pathway in the potentiation of Ca²⁺ channel currents, cells were loaded with the nonhydrolyzable GDP analogue GDPßS instead of GTP in the patch pipette and were then depolarized from a holding potential of -80 to 0 mV every 10 seconds for 5 minutes. The relative current amplitudes of I_Ba during this time were reduced by 15% to 20% (n=4). No increase in I_Ba was observed after application of 10 µmol/L Iso, and the time course of the current amplitude was indistinguishable from that in the control experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
A number of neurotransmitters modulate cardiac L-type Ca²⁺ channel currents through G protein–coupled receptors, including various -adrenergic, ß-adrenergic, and muscarinic receptor subtypes.⁴¹ Because of the coexistent expression of different receptor subtypes with overlapping affinities for commonly used agonists and antagonists, separation of subtype-specific effects on native L-type Ca²⁺ channels is frequently problematic. To examine the relative functional coupling of a particular receptor subtype to Ca²⁺ channels, experiments should ideally be performed with tissue or cells expressing pure populations of the receptor of interest. To address this, we stably expressed cardiac L-type Ca²⁺ channels in a mammalian cell line and subsequently examined the effects of transiently expressed ß₁ARs on the Ca²⁺ channel regulation.

Our results indicate that this mammalian cell recombinant system can provide a useful tool that may be superior to the commonly used oocyte system for studying the regulation of Ca²⁺ channels. The expression of Ca²⁺ channel currents in this clonal cell line has been stable over extended periods with reproducible results. This may avoid some of the technical limitations associated with the oocyte expression system, including seasonal variability in oocyte quality and time constraints imposed by the transient expression system. Compared with oocytes, BHK cells have a smaller size and offer improved voltage-clamp quality for biophysical analysis of the channel. In addition, Ca²⁺ channels expressed in mammalian cells may undergo posttranslational modifications and regulation processes in a manner more akin to that occurring in native cells. For example, each ₁ subunit has multiple consensus sites for phosphorylation by PKA. In mammalian cells, phosphorylation of the cardiac Ca²⁺ channel ₁ subunit by cAMP increases Ca²⁺ channel currents,^{15 16} but Ca²⁺ channel currents expressed in oocytes are not modulated by cAMP.¹⁴

Previous studies have shown that expression of cardiac or smooth muscle Ca²⁺ channel ₁ subunit alone in CHO cells produces functional DHP-sensitive Ca²⁺ channel currents.^{15 42 43} The DHP sensitivity, high voltage–dependent activation, and single-channel conductance of these channel subunits are similar to those observed in native cells. However, whole-cell recordings revealed some differences in their biophysical properties. For example, expressed channels displayed slower rates of activation and inactivation than those observed in native cells. Coexpression of the ₁ subunit with Ca²⁺ channel ß and ₂/ subunits from skeletal muscle in CHO cells increased the current density and DHP-binding sites mediated by the ₁ subunit.⁴³ In addition, the coexpression produced rapid activation and inactivation rates and shifts in the steady state activation and inactivation to more negative potentials.

Therefore, in the present study, we expressed the cardiac Ca²⁺ channel ₁ subunit in combination with the skeletal ß and ₂/ subunits to produce high-level expression and native cell–like channel kinetics. BHK cells transfected with the cDNAs of the ₁, ß, and ₂/ subunits demonstrated specific high-affinity [³H]PN200-110 binding with a K_d of 0.81 nmol/L, a B_max of 1230 fmol/mg protein, and a current density measured in the presence of 2 mmol/L Ba²⁺ of 19.45 A/F. These values are comparable to those obtained in CHO cells expressing Ca²⁺ channel ₁, ß, and ₂ subunits.⁴³ Furthermore, the Ca²⁺ channels expressed in BHKC112 cells exhibit pharmacology, channel kinetics, and single-channel conductances characteristic of native cardiac L-type Ca²⁺ channels.

Native cardiac Ca²⁺ channels are phosphorylated by PKA, although the primary target subunit(s) for phosphorylation is unclear.¹³ It has been previously shown that CHO cells expressing cardiac Ca²⁺ channel ₁ subunits alone are phosphorylated by membrane-permeable cAMP or intracellular PKA.^{15 16} We found that dB-cAMP increases I_Ba to an extent similar to that observed in CHO cells.¹⁵ Recently Perez-Reyes et al⁴⁴ have reported that I_Ba in human embryonic kidney (HEK) cells expressing cardiac L-type Ca²⁺ channel ₁ and ß subunits did not increase by phosphorylation unless endogenous kinases were previously inhibited. Our observation that phosphorylation of Ca²⁺ channels expressed in BHKC112 cells leads to stimulation of I_Ba comparable to the phosphorylation of CHO cells indicates that the basal phosphorylation of BHKC112 cells is rather low compared with HEK cells. Although this observation does not definitely establish the ₁ subunit as the sole target protein for phosphorylation under our experimental conditions, the presence of a cAMP-regulated component in the complete channels expressed in BHKC112 cells supports the use of this system for studying regulatory mechanisms controlling Ca²⁺ channel activity.

As noted in the present study, Ca²⁺ channel currents in BHKC112 cells not transfected with the human ß₁AR are not responsive to the external application of Iso. In agreement with these results, radioligand binding studies did not reveal measurable ßARs. Coexpression with ß₁AR rendered the cell responsive to Iso. The ß₁AR modulation of the recombinant cardiac Ca²⁺ channels by Iso is remarkably similar to that observed in native cardiac myocytes.^{1 3 4} The external application of Iso produced a concentration-dependent enhancement of Ca²⁺ channel current amplitudes. In contrast to the results of previous studies,^{1 3 4} several recent reports indicate that the Iso stimulation of Ca²⁺ channel currents causes changes in the voltage-dependent Ca²⁺ channel gating.^{39 40} In our experiments, potentiation of the current amplitude by Iso occurred at all membrane potentials, and the current waveform during depolarization was not affected by Iso, suggesting that the voltage-dependent parameters were not affected. The reasons for these differences are not clear. One possible explanation, however, is the contribution of other ßAR subtypes that may be present in native cells. For example, Xiao and Lakatta⁹ have noted that there are differences between ß₁AR and ß₂AR stimulation in their effects on Ca²⁺ channel currents; ß₂AR but not ß₁AR stimulation markedly prolonged the Ca²⁺ channel current inactivation time.

The maximum increase in the current amplitude by Iso in BHKC112 cells was close to that observed in mammalian cardiac myocytes.^{1 3 4 40} The maximum increase was a factor of 3 to 4; however, the EC₅₀ value (200 nmol/L) in BHKC112 cells was higher than that seen in native cells (38 nmol/L). The reason for this discrepancy is not clear. It is possible that an additional cofactor involved in ß₁AR/Ca²⁺ channel coupling is not present in BHK cells. However, other cellular components necessary for the signal transduction, such as G proteins and adenylyl cyclase, are clearly present, as indicated by our studies of cAMP accumulation. The differences may therefore reflect coupling characteristics specific to ß₁ARs. Recent pharmacological studies using mammalian cell lines that express ß₁AR or ß₂AR exclusively indicate that these subtypes mediate quantitatively different effects on G_s–adenylyl cyclase coupling.^{28 45} For example, Green et al²⁸ found that although both receptors mediated equivalent maximal increases in Iso-stimulated adenylyl cyclase activities, the EC₅₀ for the ß₂AR was significantly lower than that for the ß₁AR. Therefore, it is possible that the lower EC₅₀ observed for Iso increases in I_Ba in endogenous myocytes may in fact reflect a contribution from the ß₂AR that is not seen in our recombinant system. Further study will be necessary to clarify this relation.

In summary, our results show that heterologous expression in mammalian cells provides a useful system for studying functional properties of a Ca²⁺ channel protein. It is hoped that this system will facilitate the study of the receptor-mediated regulation of Ca²⁺ channel activities.

	Acknowledgments

 
This study was supported by American Heart Association Grant-in-Aid 93012860 (Dr Yatani). Dr Wakamori is a fellow supported by the International Human Frontier Science Foundation. The authors would like to thank Drs S. Liggett and A. Schwartz for encouragement and guidance throughout this work. The authors also thank Dr Piotr Chomczynski for performing the Northern analysis and Dr J. Heiny for critical review of the manuscript.

Received May 31, 1994; accepted November 9, 1994.

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