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Mice with cardiac-specific sequestration of the β -subunit of the L-type calcium channel
BBRC Biochemical and Biophysical Research Communications 293 (2002) 1405–1411 www.academicpress.com
Mice with cardiac-specific sequestration of the b-subunit of the L-type calcium channelq Vladimir Serikov,1,2 Ilona Bodi,2 Sheryl E. Koch,2 James N. Muth, Gabor Mikala,3 Sergey G. Martinov,4 Hannelore Haase,5 and Arnold Schwartz* Department of Surgery, Institute of Molecular Pharmacology and Biophysics, University of Cincinnati, College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0828, USA Received 16 April 2002
Abstract The b subunit of the L-type voltage-dependent calcium channel modifies the properties of the channel complex by both allosteric modulation of the a1 subunit function and by chaperoning the translocation of the a1 subunit to the plasma membrane. The goal of this study was to investigate the functional effect of changing the in vivo stoichiometry between the a1 and b subunits by creating a dominant negative expression system in a transgenic mouse model. The high affinity b subunit-binding domain of the a1 subunit was overexpressed in a cardiac-specific manner to act as a b subunit trap. We found that the predominant b isoform was located primarily in the membrane bound fraction of heart protein, whereas the b1 and b3 were mostly cytosolic. There was a significant diminution of the amount of b2 in the membrane fraction of the transgenic animals, resulting in a decrease in contractility of the heart and a decrease in L-type calcium current density in the myocyte. However, there were no distinguishable differences in b1 and b3 protein expression levels in the membrane bound fraction between transgenic and non-transgenic animals. Since the b1 and b3 isoforms only make up a small portion of the total b subunit in the heart, slight changes in this fraction are not detectable using Western analysis. In contrast, b1 and b3 in skeletal muscle and brain, the predominant isoforms in these tissues, respectively, are membrane bound. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Sequestration of b subunit in mouse heart
Voltage-dependent calcium channels (VDCCs) [1] play a major role in vital physiological processes, such as excitation–contraction coupling and excitation–
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Abbreviations: VDCC, voltage-dependent calcium channel; aMHC, alpha-myosin heavy chain promoter; Tg, transgenic; Ntg, non-transgenic; AID, a1 interactive domain. * Corresponding author. Fax: +1-513-558-1778. E-mail address: [email protected] (A. Schwartz). 1 Present address: Department of Human Physiology, University of California, Davis, CA 95616, USA. 2 These authors contributed equally to this work. 3 Present address: Department of Internal Medicine and Geriatrics, Faculty of Health Sciences, Semmelweis Medical University, Budapest, Hungary H-1135. 4 Present address: Department of Environmental Health, University of Cincinnati, Cincinnati, OH 45267, USA. 5 Present address: Franz Volhard Clinic at the Max Delbr€ uck Center for Molecular Medicine, Humboldt University, Berlin, Germany.
secretion coupling [1–3]. These channels are heteromultimeric proteins, built of four known subunits (a1 ; a2 =d, b, and c). The a1 subunit serves as the channel pore and voltage sensor and also possesses the binding sites for various drugs and channel modulators [2,4–7]. The functional role of the different subunits was initially studied by coexpressing the cDNAs that coded for these subunits in heterologous expression systems. These studies provided insight into the important role of the b subunit for the overall functioning of L-VDCC [8–11]. Coexpression of the a1 and b subunits of LVDCC, e.g., demonstrated a multifold increase in peak calcium current density compared to the expression of a1 alone [7,8]. Other characteristics include an acceleration of activation and inactivation kinetics, a leftward shift in the current/voltage (I–V) relationship and an increase in dihydropyridine binding [9–12]. It is now accepted that the presence of the b subunit and its
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 3 9 6 - 0
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interaction with the a1 subunit are rate-limiting steps in L-VDCC functioning in a variety of isolated cell systems [1,8–14]. There are four known genes encoding the b subunit [15,16]. The splice variant b1A is predominantly expressed in skeletal muscle, while b1B is expressed in brain [16]. b2 protein is preferentially expressed in the adult hearts of mouse, rat, rabbit, and human [17], as well as in the aorta, brain, lung, kidney, and pancreas ([15] and references cited therein, [18]). b3 is expressed in the heart, lung, aorta, and brain and b4 is expressed in the brain [15,16]. Knockout studies on animals demonstrated that heterozygous mice with an inactivated b1 gene developed normally; however, homozygous mice died of asphyxia at birth, due to a lack of excitation–contraction coupling [19]. Mice deficient in the b3 gene lived longer than a year, but showed a decreased high-voltage-activated calcium current and a reduction in the number of calcium channel-binding sites [20]. Mutation in the b4 gene, which is expressed in the brain, but was not detected in heart and liver [21]. This lethargic mutant can generate a spectrum of disease phenotypes in the mammalian central nervous system. The role of the b subunits in the heart, with cardiacspecific expression, has not been investigated. The goal of our study was to determine the physiological significance of a change in stoichiometry between the a1 and b subunits of the cardiac L-VDCC in vivo. Interaction between the a1 and b subunits occurs through a specific and highly conserved domain, found in the I–II loop of the a1 subunit [22–24]. The high affinity a1 interactive domain (AID) was overexpressed in a cardiac-specific manner in transgenic animals. Our hypothesis was that this interaction domain would act as a ‘‘trap’’ for the b subunits and thus changes the stoichiometry of available b subunits of the L-VDCC in myocytes. In this animal model, we observed a slight but significant decrease in basal heart contractility and peak calcium current
density, which demonstrates an important role of the b subunit in myocardial performance.
Methods Generation of transgenic mice. The b-trap construct consists of two main components, the S-fragment of RNase A (S-tag) and the I–II loop of the L-VDCC a1 subunit (bp 2551–2714) (Fig. 1A). The Sfragment of RNase A serves as a tag for detection and isolation of the trap construct. The I–II loop is known to bind to all known b subunit genes and isoforms [23–25]. Southern blots. Genomic DNA was extracted from the liver, digested with EcoRI, and separated on a 0.7% agarose gel. The a-MHCL-VDCC construct (50 pg, 100 pg, 250 pg, 500 pg, and 1 ng) was also cleaved with EcoRI and loaded onto the gel for quantitation. Following transfer to a supported nitrocellulose (Hybond-C extra, Amersham Pharmacia Biotech), the DNA was probed with a 1350 bp EcoRI fragment from the full-length a1C cDNA (bases 2098–3448). The fragment contains the I–II linker, from which the b-trap construct was created. Radioactive bands were quantified using the Image software (Scion). Northern analysis. Total RNA was isolated from frozen heart samples of Tg and Ntg littermate controls using Trizol reagent (Life Technologies) according to manufacturer’s recommendations. Forty lg of total RNA from each sample heart was loaded onto a 1% formaldehyde agarose gel. Following transfer to supported nitrocellulose, the RNA was probed with a 1350 bp EcoRI fragment from the full-length HHT a1C cDNA (bases 2098–3448). The fragment contains the I–II linker, from which the b-trap construct was created. All probes were 32 P-labeled by the random priming technique. Isolation of particulate and cytosolic fractions and Western analysis of the b-subunit. Separation of particulate and cytosolic fractions was performed, as previously described [26]. Production and purification of the polyclonal b2 antibody. The antibody was affinity purified on an antigen column and has been characterized previously [17,18,25]. Physiological studies. The heart was perfused in a retrograde manner at constant coronary perfusion pressure (50 mm Hg) with oxygenated Krebs–Henseleit solution at 37.7 °C, as described previously [27]. Electrophysiological measurements. Cardiomyocytes were dissociated from the ventricles of 18–20-month-old Ntg and Tg mice. The whole-cell patch-clamp technique and data analysis, which was used in this study are identical to those previously described [28].
Fig. 1. (A) A schematic representation of the a-MHC-b-trap transgene. Included are the a-MHC promoter and a portion of the I–II loop of the human cardiac calcium channel a1 subunit. (B) A Southern blot analysis comparing six Ntg and six Tg animals. Twenty lg of genomic DNA and the indicated amount of standard transgene were cleaved, blotted, and hybridized. From this blot, the copy number was determined to be 10.
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Results Molecular studies Southern blotting and hybridization with a I–II loop specific probe provided evidence for the presence of the b trap construct in the genome of transgenic animals. A quantitative comparison of the hybridization signal between the Tg animal DNA and known amounts of plasmid construct indicated that the Tg mice had approximately 10 copies of the transgene (Fig. 1A and B). Examination of total RNA isolated from Tg and Ntg hearts indicated the expression of the b-trap construct mRNA in the Tg mice (Fig. 2). According to our hypothesis, selective overexpression of this region, the so-called AID, in the mouse heart, using the a-MHC promoter [29], should decrease the amount of all b subunit isoforms available to interact with the a1 subunit (Fig. 1A). Western analysis revealed that in Ntg animals, the majority of the b2 subunits, the predominant b subunit in the adult mammalian heart, was localized in the membrane fraction of total protein (Fig. 3A). On the other hand, the b2 subunit in the Tg animals is equally distributed between the cytosolic and membrane fractions (Fig. 3A) (particulate to cytosol ratio: Tg 1:22 0:19 versus Ntg 4:43 1:4, n ¼ 7; P < 0:05). Surprisingly, we found that the majority of the b1 (cytosol to particulate ratio: Tg 4:99 1:0 versus Ntg 4:21 1:38, n ¼ 6 and 4, respectively; NS) and the b3 subunits (cytosol to particulate ratio: 4:97 1, n ¼ 3) were isolated from the cytosolic fraction of the cell (Fig. 3B and C). In skeletal muscle (particulate to cytosol ratio: 7:3 1:8, n ¼ 4) (Fig. 3D) and brain (particulate to cytosol ratio: 3:8 0:9, n ¼ 4) (Fig. 3E), the predominant b subunits, b1 and b3 , respectively, were found to be more abundant in the membrane fraction. Western analysis did not reveal significant quantitative changes in the amount of the
Fig. 2. Northern blot analysis of total RNA isolated from four Ntg and four Tg mouse hearts. Forty lg of total RNA, isolated using the Trizol reagent (Life Technologies), was blotted and hybridized. Lanes 1–4 are the Ntg RNA and lanes 5–8 are the Tg RNA. A fragment of the a1 subunit (1350 bp), containing I–II loop, was used as the probe for this blot. The construct mRNA is only present in the Tg total RNA samples (arrow).
Fig. 3. Western blot analysis of different b subunit isoforms in the heart, skeletal muscle, and brain. The b1 antibody recognizes both isoforms of the b1 subunit, the b1a (57.8 kD), and the b1b (65.5 kD). (A) In the mouse heart, the majority of the b2a subunits are present in the membrane fraction (M) for the Ntg animals and are evenly distributed between the two fractions, membrane and cytosolic (C) for the Tg animals. Fifty lg of total protein was loaded into each lane. Lanes 1–4 are the Ntg and lanes 5–8 are the Tg. In the mouse heart, the majority of the b1 (B) and b3 (C) subunits are present in the cytosolic fraction and not in the membrane fraction of total protein isolated. Twenty-five and 150 lg of total protein for (B) and (C) were loaded into each lane, respectively. Lanes 1–4 are the Ntg and lanes 5–8 are the Tg. However, in skeletal muscle (D) and brain (E), the majority of the b1 and b3 subunits, respectively, are found in the membrane fraction. One hundred lg of total protein was loaded into each lane (all samples are from Ntg animals).
b1 or the b3 subunit between Tg and their Ntg littermates. The b2 subunit, however, which was predominately in the membrane fraction, was lower in the transgenic heart (Fig. 3). There was an increase in the amount of RNase A activity in the Tg (n ¼ 17) heart protein, versus the Ntg (n ¼ 20) heart protein samples (0:001 0:00027 pmol=ll versus 0:0019 0:00040 pmol=ll, p ¼ 0:024, Ntg and Tg, respectively), as detected by the S-Tag Rapid Assay (Novagen). We did not observe any obvious differences in appearance, body weight, or life span of the Tg animals or any pathological changes at autopsy. The heart-to-body
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weight ratio was 5:97 0:18 in Tg animals and 6:05 0:15 in wild-type littermates. Cardiovascular and contractile parameters of isolated work-performing mouse heart preparation Heart performance of the Tg and Ntg animals was studied under similar hemodynamic conditions. The mean aortic pressure, the left atrial pressure, and the cardiac output did not change in Tg animals compared with Ntg littermates. We found that the dP =dt max in the Ntg mice (control, n ¼ 9) was 3760 170 mm Hg/s and in Tg animals (n ¼ 9) it was 3420 190 mm Hg/s, which is a 12% decrease (p ¼ 0:044) (Fig. 4A) with no change in relaxation. Pacing was performed with a stimulating frequency range from 325 to 550 min1 . Comparison of the contractility response to stimulation in Ntg and Tg animals is displayed in Fig. 4B. Despite an overall tendency for lower contractility in Tg animals, the response to pacing was similar in both groups with rather moderate increases in þdP =dt to pacing.
Electrophysiological studies The mean capacitance of the myocytes from Tg animals was 270:4 10:98 pF (n ¼ 75), which was not significantly different from Ntg control animals (252:8 10:6 pF, n ¼ 70). However, the ICa density was significantly decreased (25%) in the myocytes isolated from Tg mice (5:85 0:24 pA/pF, n ¼ 75) compared to control Ntg cells (7:81 0:28 pA/pF, n ¼ 70, p < 0:05) (Fig. 5A). The voltage dependence of the peak ICa (halfmaximal voltage for activation: V1=2 ) was slightly shifted towards more positive potentials, from 6:3 0:64 to 3:5 0:75 mV, p < 0:05 for Ntg and Tg myocytes, respectively (Fig. 5B).
A
A
B
Effects of the b-adrenoreceptor agonist isoproterenol on contractility for the Ntg and Tg animals are illustrated in Fig. 4C. The response to isoproterenol for both Ntg and Tg animals for these variables was similar. For the contractility measurement, the estimated ED50 was 11 nM for Ntg and 11.5 nM for Tg animals.
C B
Fig. 4. (A) Maximal rate of contraction (dP =dt, mm Hg/s) in perfused working hearts of wild type (closed bar) and Tg animals (open bar). (B) Effect of pacing on maximal rate of contraction (þdP =dt, mm Hg/s). Data for Ntg animals shown as closed circles, data for Tg animals are shown as open circles. Differences between Ntg and Tg animals were not significant. (C) Dose–response curves to isoproterenol (IP). The maximal rate of contraction (þdP =dt, mm Hg/s).
Fig. 5. (A) Comparison of ICa density values for Tg and Ntg littermates. (B) Average current–voltage (I–V) relationship of b-trap transgenic myocytes and Ntg myocytes. ICa was normalized to the cell capacitance (pA/pF). Data are means SEM of Ntg (n ¼ 27) and Tg (n ¼ 64) cells from five Tg mice and six Ntg littermates. Smooth lines represent the best fit to a Boltzmann equation ðy ¼ ðA2 þ ðA1 A2 ÞÞ= ð1 þ expððx x0 Þ=dxÞÞGðx ErÞÞ.
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B
A
C
Fig. 6. Steady-state inactivation of ICa caused a slight, but significant shift to more positive potentials. A standard two-pulse protocol was used to assess the voltage dependence of steady-state channel availability of the L-type Ca2þ current. Data were normalized to peak current amplitude by using a 1 s prepulse of )80 mV followed by a test pulse to +10 mV. Representative steady-state current traces from Ntg myocyte (A) and from b-trap transgenic myocytes (B). Steady-state inactivation curves of calcium currents were plotted as I=Imax against the voltages of the conditioning pulse (C). Solid lines represent the inactivation curves calculated from mean values for half-inactivation voltage and slope obtained by fitting a Boltzmann distribution to the data in each individual cell of the respective cell population (n ¼ 7, V1=2 ¼ 19:8 mV, k ¼ 5:4 mV in Ntg versus n ¼ 13, V1=2 ¼ 13:0 mV, k ¼ 5:5 mV in b-trap transgenic myocytes).
In the Tg cardiomyocytes, we observed a significant shift to more positive potentials of the half-maximal voltage for inactivation (V1=2 ) from 19:84 1:67 (Ntg, n ¼ 7) to 13:0 1:83 mV (Tg, n ¼ 13) without altering the slope factor (5:5 0:04, control versus 5:4 0:08, Tg), Fig. 6A–C. When compared at the test potential of +10 mV, it is obvious that the steady-state inactivation of the L-type channels in myocytes from Tg animals occurs at more depolarized potentials compared with the Ntg animals.
Discussion We used a dominant negative overexpression system to generate mice that express a synthetic peptide (AID) corresponding to a partial sequence of the a1 subunit as a tool to disrupt the a1 –b interaction. The AID structure
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involved in the a1 –b interaction is located in the cytoplasmic loop between domains I–II of the a1 subunit [23,24,30–32]. Previous immunohistochemical observations have shown that the b subunit contributes to cell surface redistribution of a1 subunits of L-VDCC [23,33]. The b2 subunit of L-VDCCs facilitates the trafficking and incorporation of the a1 subunit into the plasma membrane and strongly modulates the a1 function [8,13,14]. In addition, studies by Bichet et al. [23] revealed an endoplasmic reticulum retention signal in the I–II loop of the a1 subunit. This signal appears to prevent the a1 subunit from reaching the plasma membrane, unless a b subunit is attached to this interaction domain. Prior to this study, it was believed that the majority of the b subunit population would be found in the membrane fraction of total protein in the mouse heart cell. This assumption was due in part to the co-precipitation of the a1 and b subunits in early studies of the calcium channel and its accessory subunits [34,35]. Based on this belief, it was assumed that the amount of b subunit in the cytosolic fraction would increase in animals expressing the ‘‘trap’’ protein, since it would prevent the b subunit from associating with the a1 subunit thereby trapping the b subunit in the cytosol. However, preliminary experiments demonstrated a large concentration of b1 and b3 already in the cytosol of the wild-type protein preparation as compared to the membrane fraction. Further investigation, examining the tendency of the predominant isoform, the b2 subunit, found that the majority of this isoform was associated with the membrane. The relative amounts of b2a subunit isoform were difficult to determine because the immunoreactivity was about 30%. In contrast, Haase et al. [personal communication] found about 65% immunoreactivity with the affinity-purified b2a antibody from 50 lg total mouse heart proteins. The discrepancy between our results and those of Haase et al. [18] is partially due to the solubilization and the procedure we used to purify the membranous fraction. Apparently, TritonX-100 is rather insufficient, especially for b2a extraction. We used TritonX to solubilize the membranous fraction before the last centrifugation. However, we did not have any problems detecting the b1a -subunit ([18] and references cited therein). The stoichiometry between the a1 and b subunits in the membrane has been assumed to be 1:1; however, there may be an abundance of the b subunit in the cell to efficiently facilitate the membrane translocation of the a1 subunit. The significant excess of b1 and b3 in the cytosolic fraction of cardiomyocytes may reflect a safety mechanism: viz., there is always present a large excess of the b subunit to quickly assist the translocation of nascent a1 polypeptide chains into the plasma membrane. Upon comparison of protein isolated from heart, brain, and skeletal muscle (leg) of wild-type mice, we found that only the b1 and b3 isoforms in heart are more
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abundant in the cytosol versus the membrane. The predominant isoform, the b3 in the brain, and b1 in skeletal muscle, were membrane bound, as reported [15]. The increase in the cytosolic fraction of the b2 isoform in Tg mice indicates that the AID ‘‘trap’’ might have been effective in preventing this particular isoform of the b subunit from interacting with the a1 subunit. In our model, the alteration of the stoichiometry between the a1 and b subunits was moderate in that only the b2 subunit was lower in the membrane fraction in the Tg hearts. This modest effect on the totality of the b subunits was enough to effect a concomitant decrease in the contractility and the peak calcium current density. Western analysis did not illustrate a marked decrease in the concentration of b1 and b3 subunits in the Tg heart protein fractions, as compared to the Ntg littermates. The AID ‘‘trap,’’ if effective, should prevent all b subunit isoforms from interacting with the membranebound a1 subunit. Our data show, however, that only the b2 was prevented from reaching the membrane therefore one can conclude that the b1 and b3 subunits in the mammalian heart do not play a significant role in calcium channel assembly. Furthermore, no changes were seen between the membrane and cytosolic fractions isolated from the Tg and Ntg animals for the b1 and b3 subunits because these subunits are already localized to the cytosol, where the AID ‘‘trap’’ would sequester them. Due to the abundance of these subunits in the cytosol, it is impossible, using Western analysis, to distinguish between trapped b1 and b3 and non-trapped b1 and b3 . The only difference is seen with the b2 subunit, since the majority is found in the membrane fraction of the Ntg animals and is more evenly distributed between cytosol and membrane fractions of the Tg animals. Previous studies on cell cultures demonstrated that post-translational modification and phosphorylation of b subunits affect the interaction between the channel subunits and channel function [36]. A mutation in the I– II loop interaction domain was shown to slow the inactivation rate of the calcium channel and decreased the use dependency by phenylalkylamines [23]. Furthermore, the calcium channel b subunits may compete with the G-protein bc complex for the binding site on the a1 subunit [33]. Palmitoylated b2a subunits anchored in the membrane, like the b subunit of the G-protein, may have an advantage over cytoplasmically localized protein. The fact that we did not observe substantial differences in Ntg and Tg animals to various stimuli, that include G-protein-related mechanisms, points to a possibility of the development of compensatory mechanisms in the intact animal. Another possibility is that an alternative interaction domain, not necessarily part of the I–II loop, may exist in the a1 subunit. This second hypothetical domain may interact with b subunits with low affinity. This may be consistent with the proposed existence of several isoform-specific interaction domains
in both the a1 and b subunits [33]. Several laboratories have investigated the consequences of removing the b subunit from binding to the a1 interactive domain. In stably transfected HEK293 cells, there was a decrease in the open probability of the calcium channel when the interactive domain peptide was transiently transfected into the cells [25]. In oocytes coinjected with the a1A (Cav 2.1), and b2a subunits and the interactive domain peptide, the slow inactivating a1A channel was found to be fast inactivating [32]. The results from these in vitro studies are consistent with what we found regarding the b subunits in vivo. The AID ‘‘trap’’ peptide was able to disrupt the interaction between the a1 subunit and the b2 subunit in the heart, as indicated by a decrease in calcium channel current density and contractility of the heart. It would appear that the b1 and b3 subunits in the heart are not as involved in the trafficking of the a1 subunit to membrane or calcium channel assembly, so their role in the heart is still undefined.
Acknowledgments This work was supported in part by NIH Grant HL22619 (A.S.) and NIH Training Grant HL07382 (V.S., S.E.K., J.N.M., S.G.M., and A.S., Program Director). We acknowledge with thanks Dr. Gyula Varadi for his critical comments and assistance. The costs of publication of this paper were defrayed in part by the payment of page charges. This paper must therefore be thereby marked ‘‘advertisement’’ in accordance with 18 USC Section 1734 solely to indicate this fact.
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[22] M. Pragnell, M. DeWaard, Y. Mori, T. Tanabe, T.P. Snuch, K.P. Campbell, Calcium channel b-subunit binds to a conserved motif in the I–II cytoplasmic linker of the a1 -subunit, Nature 368 (1994) 67–69. [23] D. Bichet, V. Cornet, S. Geib, E. Carlier, S. Volsen, T. Hoshi, Y. Mori, M. DeWaard, The I–II loop of the Ca2þ channel a1 subunit contains an endoplasmic reticulum retention signal antagonized by the b subunit, Neuron 25 (2000) 177–190. [24] S. Sokolov, R.G. Weiss, B. Kurka, F. Gapp, S. Hering, Inactivation derminants in the loop of the Ca2þ channel a1 -subunit and b-subunit interaction affect sensitivity for the phenylalkylamine ())gallopamil, J. Physiol. (London) 519 (1999) 315–322. [25] A. Hohaus, M. Poteser, C. Romanin, N. Klugbauer, F. Hofmann, I. Morano, H. Haase, K. Groschner, Modulation of the smooth muscle L-type Ca2þ channel a1 subunit (a1Cb ) by the b2 subunit: a peptide which inhibits binding of b to I–II linker of a1 induces functional uncoupling, Biochem. J. 348 (2000) 657–665. [26] Y. Takeishi, T. Jalili, N.A. Ball, R.A. Walsh, Responses of cardiac protein kinase C isoforms to distinct pathological stimuli are differentially regulated, Circ. Res. 85 (1999) 264–271. [27] I.L. Grupp, G. Grupp, in: A. Schwartz (Ed.), Methods in Pharmacology, vol. 5, Plenum Press, New York, NY, 1984. [28] J.N. Muth, I. Bodi, W. Lewis, G. Varadi, A.A. Schwartz, Ca2þ dependent transgenic model of cardiac hypertrophy. A role for protein kinase Ca , Circulation 103 (2001) 140–147. [29] A. Subramaniam, W.K. Jones, J. Gulick, S. Wert, J. Neumann, J. Robbins, Tissue-specific regulation of the alpha-myosin heavy chain gene promoter in transgenic mice, J. Biol. Chem. 266 (1991) 24613–24620. [30] A. Marquart, V. Flockerzi, a1 –b interaction in voltage-gated cardiac L-type calcium channels, FEBS Lett. 407 (1997) 137–140. [31] M. DeWaard, V.E. Scott, M. Pragnell, K.P. Campbell, Identification of critical amino acids involved in a1 –b interaction in voltage-dependent Ca2þ channels, FEBS Lett. 380 (1996) 272–276. [32] T. Cens, S. Restituito, S. Galas, P. Charnet, Voltage and calcium use the same molecular determinants to inactivate calcium channels, J. Biol. Chem. 274 (1999) 5483–15490. [33] B. Neuhuber, U. Gerster, J. Mitterdorfer, H. Glossmann, B.E. Flucher, Differential effects of Ca2þ channel b1a and b2a subunits on complex formation with a1S and on current expression in tsA201 cells, J. Biol. Chem. 273 (1998) 9110–9118. [34] B.M. Curtis, W.A. Catterall, Purification of the calcium antagonist receptor of the voltage-sensitive calcium channel from skeletal muscle transverse tubules, Biochemistry 23 (1984) 2113–2118. [35] V. Flockerzi, H.J. Oeken, F. Hofmann, Purification of functional receptor for calcium-channel blockers from rabbit skeletal-muscle microsomes, Eur. J. Biochem. 161 (1986) 217–224. [36] A.J. Chien, M.M. Hosey, Post-translational modifications of b subunits of voltage-dependent calcium channels, J. Bioenerg. Biomembr. 30 (1998) 377–385.
Mice with cardiac-specific sequestration of the b-subunit of the L-type calcium channelq Vladimir Serikov,1,2 Ilona Bodi,2 Sheryl E. Koch,2 James N. Muth, Gabor Mikala,3 Sergey G. Martinov,4 Hannelore Haase,5 and Arnold Schwartz* Department of Surgery, Institute of Molecular Pharmacology and Biophysics, University of Cincinnati, College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0828, USA Received 16 April 2002
Abstract The b subunit of the L-type voltage-dependent calcium channel modifies the properties of the channel complex by both allosteric modulation of the a1 subunit function and by chaperoning the translocation of the a1 subunit to the plasma membrane. The goal of this study was to investigate the functional effect of changing the in vivo stoichiometry between the a1 and b subunits by creating a dominant negative expression system in a transgenic mouse model. The high affinity b subunit-binding domain of the a1 subunit was overexpressed in a cardiac-specific manner to act as a b subunit trap. We found that the predominant b isoform was located primarily in the membrane bound fraction of heart protein, whereas the b1 and b3 were mostly cytosolic. There was a significant diminution of the amount of b2 in the membrane fraction of the transgenic animals, resulting in a decrease in contractility of the heart and a decrease in L-type calcium current density in the myocyte. However, there were no distinguishable differences in b1 and b3 protein expression levels in the membrane bound fraction between transgenic and non-transgenic animals. Since the b1 and b3 isoforms only make up a small portion of the total b subunit in the heart, slight changes in this fraction are not detectable using Western analysis. In contrast, b1 and b3 in skeletal muscle and brain, the predominant isoforms in these tissues, respectively, are membrane bound. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Sequestration of b subunit in mouse heart
Voltage-dependent calcium channels (VDCCs) [1] play a major role in vital physiological processes, such as excitation–contraction coupling and excitation–
q
Abbreviations: VDCC, voltage-dependent calcium channel; aMHC, alpha-myosin heavy chain promoter; Tg, transgenic; Ntg, non-transgenic; AID, a1 interactive domain. * Corresponding author. Fax: +1-513-558-1778. E-mail address: [email protected] (A. Schwartz). 1 Present address: Department of Human Physiology, University of California, Davis, CA 95616, USA. 2 These authors contributed equally to this work. 3 Present address: Department of Internal Medicine and Geriatrics, Faculty of Health Sciences, Semmelweis Medical University, Budapest, Hungary H-1135. 4 Present address: Department of Environmental Health, University of Cincinnati, Cincinnati, OH 45267, USA. 5 Present address: Franz Volhard Clinic at the Max Delbr€ uck Center for Molecular Medicine, Humboldt University, Berlin, Germany.
secretion coupling [1–3]. These channels are heteromultimeric proteins, built of four known subunits (a1 ; a2 =d, b, and c). The a1 subunit serves as the channel pore and voltage sensor and also possesses the binding sites for various drugs and channel modulators [2,4–7]. The functional role of the different subunits was initially studied by coexpressing the cDNAs that coded for these subunits in heterologous expression systems. These studies provided insight into the important role of the b subunit for the overall functioning of L-VDCC [8–11]. Coexpression of the a1 and b subunits of LVDCC, e.g., demonstrated a multifold increase in peak calcium current density compared to the expression of a1 alone [7,8]. Other characteristics include an acceleration of activation and inactivation kinetics, a leftward shift in the current/voltage (I–V) relationship and an increase in dihydropyridine binding [9–12]. It is now accepted that the presence of the b subunit and its
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interaction with the a1 subunit are rate-limiting steps in L-VDCC functioning in a variety of isolated cell systems [1,8–14]. There are four known genes encoding the b subunit [15,16]. The splice variant b1A is predominantly expressed in skeletal muscle, while b1B is expressed in brain [16]. b2 protein is preferentially expressed in the adult hearts of mouse, rat, rabbit, and human [17], as well as in the aorta, brain, lung, kidney, and pancreas ([15] and references cited therein, [18]). b3 is expressed in the heart, lung, aorta, and brain and b4 is expressed in the brain [15,16]. Knockout studies on animals demonstrated that heterozygous mice with an inactivated b1 gene developed normally; however, homozygous mice died of asphyxia at birth, due to a lack of excitation–contraction coupling [19]. Mice deficient in the b3 gene lived longer than a year, but showed a decreased high-voltage-activated calcium current and a reduction in the number of calcium channel-binding sites [20]. Mutation in the b4 gene, which is expressed in the brain, but was not detected in heart and liver [21]. This lethargic mutant can generate a spectrum of disease phenotypes in the mammalian central nervous system. The role of the b subunits in the heart, with cardiacspecific expression, has not been investigated. The goal of our study was to determine the physiological significance of a change in stoichiometry between the a1 and b subunits of the cardiac L-VDCC in vivo. Interaction between the a1 and b subunits occurs through a specific and highly conserved domain, found in the I–II loop of the a1 subunit [22–24]. The high affinity a1 interactive domain (AID) was overexpressed in a cardiac-specific manner in transgenic animals. Our hypothesis was that this interaction domain would act as a ‘‘trap’’ for the b subunits and thus changes the stoichiometry of available b subunits of the L-VDCC in myocytes. In this animal model, we observed a slight but significant decrease in basal heart contractility and peak calcium current
density, which demonstrates an important role of the b subunit in myocardial performance.
Methods Generation of transgenic mice. The b-trap construct consists of two main components, the S-fragment of RNase A (S-tag) and the I–II loop of the L-VDCC a1 subunit (bp 2551–2714) (Fig. 1A). The Sfragment of RNase A serves as a tag for detection and isolation of the trap construct. The I–II loop is known to bind to all known b subunit genes and isoforms [23–25]. Southern blots. Genomic DNA was extracted from the liver, digested with EcoRI, and separated on a 0.7% agarose gel. The a-MHCL-VDCC construct (50 pg, 100 pg, 250 pg, 500 pg, and 1 ng) was also cleaved with EcoRI and loaded onto the gel for quantitation. Following transfer to a supported nitrocellulose (Hybond-C extra, Amersham Pharmacia Biotech), the DNA was probed with a 1350 bp EcoRI fragment from the full-length a1C cDNA (bases 2098–3448). The fragment contains the I–II linker, from which the b-trap construct was created. Radioactive bands were quantified using the Image software (Scion). Northern analysis. Total RNA was isolated from frozen heart samples of Tg and Ntg littermate controls using Trizol reagent (Life Technologies) according to manufacturer’s recommendations. Forty lg of total RNA from each sample heart was loaded onto a 1% formaldehyde agarose gel. Following transfer to supported nitrocellulose, the RNA was probed with a 1350 bp EcoRI fragment from the full-length HHT a1C cDNA (bases 2098–3448). The fragment contains the I–II linker, from which the b-trap construct was created. All probes were 32 P-labeled by the random priming technique. Isolation of particulate and cytosolic fractions and Western analysis of the b-subunit. Separation of particulate and cytosolic fractions was performed, as previously described [26]. Production and purification of the polyclonal b2 antibody. The antibody was affinity purified on an antigen column and has been characterized previously [17,18,25]. Physiological studies. The heart was perfused in a retrograde manner at constant coronary perfusion pressure (50 mm Hg) with oxygenated Krebs–Henseleit solution at 37.7 °C, as described previously [27]. Electrophysiological measurements. Cardiomyocytes were dissociated from the ventricles of 18–20-month-old Ntg and Tg mice. The whole-cell patch-clamp technique and data analysis, which was used in this study are identical to those previously described [28].
Fig. 1. (A) A schematic representation of the a-MHC-b-trap transgene. Included are the a-MHC promoter and a portion of the I–II loop of the human cardiac calcium channel a1 subunit. (B) A Southern blot analysis comparing six Ntg and six Tg animals. Twenty lg of genomic DNA and the indicated amount of standard transgene were cleaved, blotted, and hybridized. From this blot, the copy number was determined to be 10.
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Results Molecular studies Southern blotting and hybridization with a I–II loop specific probe provided evidence for the presence of the b trap construct in the genome of transgenic animals. A quantitative comparison of the hybridization signal between the Tg animal DNA and known amounts of plasmid construct indicated that the Tg mice had approximately 10 copies of the transgene (Fig. 1A and B). Examination of total RNA isolated from Tg and Ntg hearts indicated the expression of the b-trap construct mRNA in the Tg mice (Fig. 2). According to our hypothesis, selective overexpression of this region, the so-called AID, in the mouse heart, using the a-MHC promoter [29], should decrease the amount of all b subunit isoforms available to interact with the a1 subunit (Fig. 1A). Western analysis revealed that in Ntg animals, the majority of the b2 subunits, the predominant b subunit in the adult mammalian heart, was localized in the membrane fraction of total protein (Fig. 3A). On the other hand, the b2 subunit in the Tg animals is equally distributed between the cytosolic and membrane fractions (Fig. 3A) (particulate to cytosol ratio: Tg 1:22 0:19 versus Ntg 4:43 1:4, n ¼ 7; P < 0:05). Surprisingly, we found that the majority of the b1 (cytosol to particulate ratio: Tg 4:99 1:0 versus Ntg 4:21 1:38, n ¼ 6 and 4, respectively; NS) and the b3 subunits (cytosol to particulate ratio: 4:97 1, n ¼ 3) were isolated from the cytosolic fraction of the cell (Fig. 3B and C). In skeletal muscle (particulate to cytosol ratio: 7:3 1:8, n ¼ 4) (Fig. 3D) and brain (particulate to cytosol ratio: 3:8 0:9, n ¼ 4) (Fig. 3E), the predominant b subunits, b1 and b3 , respectively, were found to be more abundant in the membrane fraction. Western analysis did not reveal significant quantitative changes in the amount of the
Fig. 2. Northern blot analysis of total RNA isolated from four Ntg and four Tg mouse hearts. Forty lg of total RNA, isolated using the Trizol reagent (Life Technologies), was blotted and hybridized. Lanes 1–4 are the Ntg RNA and lanes 5–8 are the Tg RNA. A fragment of the a1 subunit (1350 bp), containing I–II loop, was used as the probe for this blot. The construct mRNA is only present in the Tg total RNA samples (arrow).
Fig. 3. Western blot analysis of different b subunit isoforms in the heart, skeletal muscle, and brain. The b1 antibody recognizes both isoforms of the b1 subunit, the b1a (57.8 kD), and the b1b (65.5 kD). (A) In the mouse heart, the majority of the b2a subunits are present in the membrane fraction (M) for the Ntg animals and are evenly distributed between the two fractions, membrane and cytosolic (C) for the Tg animals. Fifty lg of total protein was loaded into each lane. Lanes 1–4 are the Ntg and lanes 5–8 are the Tg. In the mouse heart, the majority of the b1 (B) and b3 (C) subunits are present in the cytosolic fraction and not in the membrane fraction of total protein isolated. Twenty-five and 150 lg of total protein for (B) and (C) were loaded into each lane, respectively. Lanes 1–4 are the Ntg and lanes 5–8 are the Tg. However, in skeletal muscle (D) and brain (E), the majority of the b1 and b3 subunits, respectively, are found in the membrane fraction. One hundred lg of total protein was loaded into each lane (all samples are from Ntg animals).
b1 or the b3 subunit between Tg and their Ntg littermates. The b2 subunit, however, which was predominately in the membrane fraction, was lower in the transgenic heart (Fig. 3). There was an increase in the amount of RNase A activity in the Tg (n ¼ 17) heart protein, versus the Ntg (n ¼ 20) heart protein samples (0:001 0:00027 pmol=ll versus 0:0019 0:00040 pmol=ll, p ¼ 0:024, Ntg and Tg, respectively), as detected by the S-Tag Rapid Assay (Novagen). We did not observe any obvious differences in appearance, body weight, or life span of the Tg animals or any pathological changes at autopsy. The heart-to-body
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weight ratio was 5:97 0:18 in Tg animals and 6:05 0:15 in wild-type littermates. Cardiovascular and contractile parameters of isolated work-performing mouse heart preparation Heart performance of the Tg and Ntg animals was studied under similar hemodynamic conditions. The mean aortic pressure, the left atrial pressure, and the cardiac output did not change in Tg animals compared with Ntg littermates. We found that the dP =dt max in the Ntg mice (control, n ¼ 9) was 3760 170 mm Hg/s and in Tg animals (n ¼ 9) it was 3420 190 mm Hg/s, which is a 12% decrease (p ¼ 0:044) (Fig. 4A) with no change in relaxation. Pacing was performed with a stimulating frequency range from 325 to 550 min1 . Comparison of the contractility response to stimulation in Ntg and Tg animals is displayed in Fig. 4B. Despite an overall tendency for lower contractility in Tg animals, the response to pacing was similar in both groups with rather moderate increases in þdP =dt to pacing.
Electrophysiological studies The mean capacitance of the myocytes from Tg animals was 270:4 10:98 pF (n ¼ 75), which was not significantly different from Ntg control animals (252:8 10:6 pF, n ¼ 70). However, the ICa density was significantly decreased (25%) in the myocytes isolated from Tg mice (5:85 0:24 pA/pF, n ¼ 75) compared to control Ntg cells (7:81 0:28 pA/pF, n ¼ 70, p < 0:05) (Fig. 5A). The voltage dependence of the peak ICa (halfmaximal voltage for activation: V1=2 ) was slightly shifted towards more positive potentials, from 6:3 0:64 to 3:5 0:75 mV, p < 0:05 for Ntg and Tg myocytes, respectively (Fig. 5B).
A
A
B
Effects of the b-adrenoreceptor agonist isoproterenol on contractility for the Ntg and Tg animals are illustrated in Fig. 4C. The response to isoproterenol for both Ntg and Tg animals for these variables was similar. For the contractility measurement, the estimated ED50 was 11 nM for Ntg and 11.5 nM for Tg animals.
C B
Fig. 4. (A) Maximal rate of contraction (dP =dt, mm Hg/s) in perfused working hearts of wild type (closed bar) and Tg animals (open bar). (B) Effect of pacing on maximal rate of contraction (þdP =dt, mm Hg/s). Data for Ntg animals shown as closed circles, data for Tg animals are shown as open circles. Differences between Ntg and Tg animals were not significant. (C) Dose–response curves to isoproterenol (IP). The maximal rate of contraction (þdP =dt, mm Hg/s).
Fig. 5. (A) Comparison of ICa density values for Tg and Ntg littermates. (B) Average current–voltage (I–V) relationship of b-trap transgenic myocytes and Ntg myocytes. ICa was normalized to the cell capacitance (pA/pF). Data are means SEM of Ntg (n ¼ 27) and Tg (n ¼ 64) cells from five Tg mice and six Ntg littermates. Smooth lines represent the best fit to a Boltzmann equation ðy ¼ ðA2 þ ðA1 A2 ÞÞ= ð1 þ expððx x0 Þ=dxÞÞGðx ErÞÞ.
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B
A
C
Fig. 6. Steady-state inactivation of ICa caused a slight, but significant shift to more positive potentials. A standard two-pulse protocol was used to assess the voltage dependence of steady-state channel availability of the L-type Ca2þ current. Data were normalized to peak current amplitude by using a 1 s prepulse of )80 mV followed by a test pulse to +10 mV. Representative steady-state current traces from Ntg myocyte (A) and from b-trap transgenic myocytes (B). Steady-state inactivation curves of calcium currents were plotted as I=Imax against the voltages of the conditioning pulse (C). Solid lines represent the inactivation curves calculated from mean values for half-inactivation voltage and slope obtained by fitting a Boltzmann distribution to the data in each individual cell of the respective cell population (n ¼ 7, V1=2 ¼ 19:8 mV, k ¼ 5:4 mV in Ntg versus n ¼ 13, V1=2 ¼ 13:0 mV, k ¼ 5:5 mV in b-trap transgenic myocytes).
In the Tg cardiomyocytes, we observed a significant shift to more positive potentials of the half-maximal voltage for inactivation (V1=2 ) from 19:84 1:67 (Ntg, n ¼ 7) to 13:0 1:83 mV (Tg, n ¼ 13) without altering the slope factor (5:5 0:04, control versus 5:4 0:08, Tg), Fig. 6A–C. When compared at the test potential of +10 mV, it is obvious that the steady-state inactivation of the L-type channels in myocytes from Tg animals occurs at more depolarized potentials compared with the Ntg animals.
Discussion We used a dominant negative overexpression system to generate mice that express a synthetic peptide (AID) corresponding to a partial sequence of the a1 subunit as a tool to disrupt the a1 –b interaction. The AID structure
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involved in the a1 –b interaction is located in the cytoplasmic loop between domains I–II of the a1 subunit [23,24,30–32]. Previous immunohistochemical observations have shown that the b subunit contributes to cell surface redistribution of a1 subunits of L-VDCC [23,33]. The b2 subunit of L-VDCCs facilitates the trafficking and incorporation of the a1 subunit into the plasma membrane and strongly modulates the a1 function [8,13,14]. In addition, studies by Bichet et al. [23] revealed an endoplasmic reticulum retention signal in the I–II loop of the a1 subunit. This signal appears to prevent the a1 subunit from reaching the plasma membrane, unless a b subunit is attached to this interaction domain. Prior to this study, it was believed that the majority of the b subunit population would be found in the membrane fraction of total protein in the mouse heart cell. This assumption was due in part to the co-precipitation of the a1 and b subunits in early studies of the calcium channel and its accessory subunits [34,35]. Based on this belief, it was assumed that the amount of b subunit in the cytosolic fraction would increase in animals expressing the ‘‘trap’’ protein, since it would prevent the b subunit from associating with the a1 subunit thereby trapping the b subunit in the cytosol. However, preliminary experiments demonstrated a large concentration of b1 and b3 already in the cytosol of the wild-type protein preparation as compared to the membrane fraction. Further investigation, examining the tendency of the predominant isoform, the b2 subunit, found that the majority of this isoform was associated with the membrane. The relative amounts of b2a subunit isoform were difficult to determine because the immunoreactivity was about 30%. In contrast, Haase et al. [personal communication] found about 65% immunoreactivity with the affinity-purified b2a antibody from 50 lg total mouse heart proteins. The discrepancy between our results and those of Haase et al. [18] is partially due to the solubilization and the procedure we used to purify the membranous fraction. Apparently, TritonX-100 is rather insufficient, especially for b2a extraction. We used TritonX to solubilize the membranous fraction before the last centrifugation. However, we did not have any problems detecting the b1a -subunit ([18] and references cited therein). The stoichiometry between the a1 and b subunits in the membrane has been assumed to be 1:1; however, there may be an abundance of the b subunit in the cell to efficiently facilitate the membrane translocation of the a1 subunit. The significant excess of b1 and b3 in the cytosolic fraction of cardiomyocytes may reflect a safety mechanism: viz., there is always present a large excess of the b subunit to quickly assist the translocation of nascent a1 polypeptide chains into the plasma membrane. Upon comparison of protein isolated from heart, brain, and skeletal muscle (leg) of wild-type mice, we found that only the b1 and b3 isoforms in heart are more
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abundant in the cytosol versus the membrane. The predominant isoform, the b3 in the brain, and b1 in skeletal muscle, were membrane bound, as reported [15]. The increase in the cytosolic fraction of the b2 isoform in Tg mice indicates that the AID ‘‘trap’’ might have been effective in preventing this particular isoform of the b subunit from interacting with the a1 subunit. In our model, the alteration of the stoichiometry between the a1 and b subunits was moderate in that only the b2 subunit was lower in the membrane fraction in the Tg hearts. This modest effect on the totality of the b subunits was enough to effect a concomitant decrease in the contractility and the peak calcium current density. Western analysis did not illustrate a marked decrease in the concentration of b1 and b3 subunits in the Tg heart protein fractions, as compared to the Ntg littermates. The AID ‘‘trap,’’ if effective, should prevent all b subunit isoforms from interacting with the membranebound a1 subunit. Our data show, however, that only the b2 was prevented from reaching the membrane therefore one can conclude that the b1 and b3 subunits in the mammalian heart do not play a significant role in calcium channel assembly. Furthermore, no changes were seen between the membrane and cytosolic fractions isolated from the Tg and Ntg animals for the b1 and b3 subunits because these subunits are already localized to the cytosol, where the AID ‘‘trap’’ would sequester them. Due to the abundance of these subunits in the cytosol, it is impossible, using Western analysis, to distinguish between trapped b1 and b3 and non-trapped b1 and b3 . The only difference is seen with the b2 subunit, since the majority is found in the membrane fraction of the Ntg animals and is more evenly distributed between cytosol and membrane fractions of the Tg animals. Previous studies on cell cultures demonstrated that post-translational modification and phosphorylation of b subunits affect the interaction between the channel subunits and channel function [36]. A mutation in the I– II loop interaction domain was shown to slow the inactivation rate of the calcium channel and decreased the use dependency by phenylalkylamines [23]. Furthermore, the calcium channel b subunits may compete with the G-protein bc complex for the binding site on the a1 subunit [33]. Palmitoylated b2a subunits anchored in the membrane, like the b subunit of the G-protein, may have an advantage over cytoplasmically localized protein. The fact that we did not observe substantial differences in Ntg and Tg animals to various stimuli, that include G-protein-related mechanisms, points to a possibility of the development of compensatory mechanisms in the intact animal. Another possibility is that an alternative interaction domain, not necessarily part of the I–II loop, may exist in the a1 subunit. This second hypothetical domain may interact with b subunits with low affinity. This may be consistent with the proposed existence of several isoform-specific interaction domains
in both the a1 and b subunits [33]. Several laboratories have investigated the consequences of removing the b subunit from binding to the a1 interactive domain. In stably transfected HEK293 cells, there was a decrease in the open probability of the calcium channel when the interactive domain peptide was transiently transfected into the cells [25]. In oocytes coinjected with the a1A (Cav 2.1), and b2a subunits and the interactive domain peptide, the slow inactivating a1A channel was found to be fast inactivating [32]. The results from these in vitro studies are consistent with what we found regarding the b subunits in vivo. The AID ‘‘trap’’ peptide was able to disrupt the interaction between the a1 subunit and the b2 subunit in the heart, as indicated by a decrease in calcium channel current density and contractility of the heart. It would appear that the b1 and b3 subunits in the heart are not as involved in the trafficking of the a1 subunit to membrane or calcium channel assembly, so their role in the heart is still undefined.
Acknowledgments This work was supported in part by NIH Grant HL22619 (A.S.) and NIH Training Grant HL07382 (V.S., S.E.K., J.N.M., S.G.M., and A.S., Program Director). We acknowledge with thanks Dr. Gyula Varadi for his critical comments and assistance. The costs of publication of this paper were defrayed in part by the payment of page charges. This paper must therefore be thereby marked ‘‘advertisement’’ in accordance with 18 USC Section 1734 solely to indicate this fact.
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