Modified Cardiovascular L-type Channels in Mice Lacking the Voltage-dependent Ca2+ Channel β3 Subunit

Abstract

The β subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming α1 subunits. Of the four β subunits reported to date, the β3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the β3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in β3-null mice. Western blot analysis indicated a significant reduction in expression of the α1 subunit in the plasma membrane of β3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in β3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, β3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the β3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the β3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells. The β subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming α1 subunits. Of the four β subunits reported to date, the β3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the β3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in β3-null mice. Western blot analysis indicated a significant reduction in expression of the α1 subunit in the plasma membrane of β3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in β3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, β3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the β3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the β3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells. Voltage-dependent calcium channels (VDCCs) 1The abbreviations used are: VDCCs, Voltage-dependent calcium channels; DHP, dihydropyridine; RyR, ryanodine-sensitive calcium release; CHO, Chinese hamster ovary; RT, reverse transcriptase; GFP, green fluorescent protein. are the main mediators of calcium entry in many cell types and play pivotal roles in the control of calcium-dependent cellular functions, such as smooth muscle contraction. These channels are classified electrophysiologically and pharmacologically into five groups (L, N, T, R, and P/Q). L-type calcium channels are crucial for excitation-contraction coupling as voltage sensors in skeletal muscle and as the main mediators of calcium entry in heart and smooth muscle tissues. VDCCs are composed of four subunits, α1, α2, δ, β, and γ (1Hofmann F. Biel M. Flockerzi F. Annu. Rev. Neurosci. 1994; 17: 399-418Crossref PubMed Scopus (452) Google Scholar, 2Catterall W.A. Annu. Rev. Cell. Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1956) Google Scholar). The α1 subunits are the pore-forming subunits and are targets of calcium channel blockers such as dihydropyridines (DHPs), phenylalkylamines, and benzothiazepines, which are widely used for the treatment of hypertension, angina pectoris, and cardiac arrhythmias. Ten genes encoding pore-forming α1 subunits have been reported to date (3Perez-Reyes E. Cribbs L.L. Daud A. Lacerda A.E. Barclay J. Williamson M.P. Fox M. Rees M. Lee J.-H. Nature. 1998; 391: 896-900Crossref PubMed Scopus (643) Google Scholar). The β subunits accelerate activation and inactivation of calcium channel currents, increase the channel population, and influence the coupling of gating charge movement; in addition, the βγ dimer (Gβγ) is thought to be involved in G protein-mediated channel inhibition (4Pragnell M. De Waard M. Mori Y. Tanabe T. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Crossref PubMed Scopus (553) Google Scholar, 5De Waard M. Witcher D.R. Pragnell M. Liu H. Campbell K.P. J. Biol. Chem. 1995; 270: 12056-12064Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 6Singer D. Biel M. Lotan I. Flokerzi V. Hofman F. Dascal N. Science. 1991; 253: 1553-1557Crossref PubMed Scopus (441) Google Scholar, 7Tareilus E. Roux M. Qin N. Olcese R. Zhou J. Stefani E. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1703-1708Crossref PubMed Scopus (154) Google Scholar, 8Varadi G. Lory P. Schultz D. Varadi M. Schwartz A. Nature. 1991; 352: 159-162Crossref PubMed Scopus (232) Google Scholar). The β subunits are non-glycosylated hydrophilic proteins located on the intracellular side of the membrane; four variants are known (β1, β2, β3, and β4). Due to its wide expression in the aorta, trachea, and lung, β3 is considered the β subunit of smooth muscle calcium channels; it is also thought to couple with α1C subunits and comprise L-type calcium channels in these cells (9Hullin R. Singer-Lahat D. Freichel M. Biel M. Dascal N. Hofmann F. Flockerzi V. EMBO J. 1992; 11: 885-890Crossref PubMed Scopus (280) Google Scholar, 10Collin T. Lory P. Taviaux S. Courtieu C. Guilbault P. Berta P. Nargeot J. Eur. J. Biochem. 1994; 220: 257-262Crossref PubMed Scopus (46) Google Scholar). Although the role of the β subunits has been investigated by heterologous co-expression studies and antisense experiments with cultured cells, little is known about the physiological importance of the β3 subunit in situ. The purpose of the present study was to clarify the importance of the voltage-dependent calcium channel β3 subunit in the cardiovascular system by using gene-targeted mutant mice. We examined channel populations in the aorta, calcium channel currents in aortic smooth muscle cells, calcium sparks in response to high KCl, and expression patterns of calcium channel subunits in the cardiovascular system of β3-null mice. We also measured blood pressure and heart rate in β3-null mice and compared them with wild-type controls. β3-Null mice showed high blood pressure, hypertrophic changes in the aortic smooth muscle layer, and cardiac enlargement in response to a high salt diet. The wild-type (β3+/+) and β3-deficient (β3–/–) mice were 12–16-week-old experimentally naive mice (11Murakami M. Yamamura H. Murakami A. Okamura T. Nunoki K. Mistui-Saito M. Muraki K. Hano K. Imaizumi Y. Flockerzi V. Yanagisawa T. J. Cardiovasc. Pharmacol. 2002; 36: S69-S73Crossref Scopus (17) Google Scholar, 12Murakami M. Fleischmann B. De Felipe C. Freichel M. Trost C. Ludwig A. Wissenbach U. Schwegler H. Hofmann F. Hescheler J. Flockerzi V. Cavalie A. J. Biol. Chem. 2002; 277: 40342-40351Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Animals were maintained at 22 ± 0.5 °C under a 12-h light-dark cycle. All experiments were conducted during the light phase of the cycle. RNA Isolation and RT-PCR Analysis—Total RNA was isolated from the mouse aorta using an RNeasy extraction kit (Qiagen Inc., Valencia, CA). Reverse transcription reaction was performed in a solution of 10 pmol of oligo(dT) primer, 1 μg of RNA, 1× first strand cDNA buffer (Invitrogen), 10 mm dithiothreitol, 0.4 mm dNTPs, 40 units of RNAsin, and 200 units of Superscript II (Invitrogen), in a volume of 25 μl, at 42 °C for 45 min. β3 and β2 subunit-specific sequences were amplified by PCR with the following primers: MB3S (5′-CTC AAA CAG GAA CAG AAG GCC-3′) and MB3A (5′-CAT AGC CTT TCA GAG AGG GTC-3′), corresponding to the sequences of the murine β3 subunit 128LKQEQKAR136 and 184PSLKGYE191; MB2S (5′-CTA GAG AAC ATG AGG CTA CAG-3′) and MB2A (5′-ACT GTT TGC ACT GGG CTT AGG-3′), corresponding to the sequences of the murine β2 subunit 130LENMRLQ137 and 197PKPSANS204. RT-PCR amplification of the α1C subunit was performed using the specific primers MA1C3 (5′-TTG GCC ATT GCG GTG GAC AAC CTG-3′) and MA1C4 (5′-CTG GAG TGC ATC CAT GTG TAT CTT G-3′), which generated a PCR product of 237 bp. Western Blot Analysis—For analysis of α1, β2a, and β3, the aorta was dissected from each mouse and homogenized in homogenization buffer containing 20 mm Na4O7P2, 20 mm NaH2PO4·H2O, 1 mm MgCl2, 0.3 mm sucrose, 0.5 mm EDTA, and a mixture of protease inhibitors. Aliquots of 100 μg of the homogenate from each mouse were resolved by 10% gradient SDS-PAGE and subjected to Western blot analysis as described previously R. Mori Y. Campbell K.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and specific for the calcium channel subunits β2a, β3, and have been described previously D.R. M. J. C. Pragnell M. Campbell K.P. Science. PubMed Scopus Google Scholar). aortic smooth muscle were and in mm buffer as described previously J. A. K. H. PubMed Scopus Google Scholar). The were in a volume of 0.5 in the for at 25 in the of The was and binding was using was by on and by the with of The was by were as described previously Y. Muraki K. M. J. Scopus Google Scholar). cells from the aorta were in using a The were with a solution containing mm mm MgCl2, mm mm and 10 mm The was to with 1 The solution mm mm KCl, mm mm MgCl2, mm and 10 mm The was to with 10 currents were by voltage from to from the of currents were and membrane have been for to the of E. 1992; PubMed Scopus Google Scholar). was measured by the charge during the by 10 from using the and analysis were on a using at the of are as the ± The of and mice was using first in mm in were and into solution containing mm mm KCl, 25 mm mm mm mm MgCl2, mm EDTA, and mm The of the was The was with solution containing 10 and 1 a light to the of smooth muscle cells at ± 1 for min. the was in the using a and was with solution at a rate of solution has an of mm mm KCl, mm mm MgCl2, mm and 10 mm The of the was to with were using a with an 40 The from an was and the was The of the was and in the The Ca2+ to a and to 100 in each at were by with solution containing mm are as the ± The of groups was using analysis of of were the groups and were The heart rate and and blood pressure were measured using an system with the The were the high salt diet for the were by measured analysis of are as ± and of and mice were under The were in in buffer at °C and in of with was analysis was performed using the amplification were with The was as a with solution mm mm buffer and and was performed with was to the for 20 at by the at a of for at the α1C subunit were were with to the of isolated smooth muscle cells was with at a of as the and with at a of with hamster ovary cells were cultured in with 10% cells at were cells were with an expression the smooth muscle α1C subunit in the in the β3 subunit in with a green fluorescent expression by E. R. T. Hescheler J. A. Hofmann F. Flockerzi V. EMBO J. 1992; 11: PubMed Scopus (46) Google Scholar). of the channel subunits was by the of green light using an was currents were by voltage from the at to are expressed as the ± were using The of was β3 and β2 are in the from and mice were using the MB3S and and MB2S and RT-PCR analysis of the wild-type aorta showed and of and the expression of subunits in this We also performed the with and which PCR The expression of the α1C subunit was in wild-type and mutant mice. We also the expression of β2, β3, and α1C subunit genes with we also performed the with primers for α1 subunits thought to in the of high calcium such as PCR with these Western Blot the of the β3 subunit and the expression of the β2 subunit in the aortic at the we performed Western blot analysis using and subunit A corresponding to β3 was in whereas corresponding was in mice a of in β2 subunit expression and this subunit is a of voltage-dependent calcium channels in the vascular was significant in the of β2 subunit expression in the of as compared with was increase in the of β2 expression in this Western blot a with a of was in the of and mice with the subunit We also performed Western blot analysis with α1 with the the of calcium channels are by a α1C subunit VDCCs in the cardiovascular mice showed a reduced of the α1C subunit in the aortic plasma membrane reduction as by which a decrease in the channel population in the smooth muscle cell membrane by the of the β3 subunit the decrease in the channel population, we performed binding analyses using the dihydropyridine which to a of binding in aortic smooth muscle from a The were ± and ± and the were ± (β3+/+) and ± (β3–/–) a decrease in the of in the cell membrane of the mutant with changes in the channel reduction in the channel population with a reduction in the of expression of α1 subunits by Western blot The binding were high which analysis in to skeletal muscle membrane binding was about 10% and of Voltage-dependent was used to the of β3 expression on voltage-dependent Ca2+ channel currents in aortic smooth muscle cells. The cell was ± in cells and ± in cells cells from mice for of were in the of mm as a charge of voltage-dependent Ca2+ channel currents were in and cells In the density of the peak current was the The density of the voltage-dependent current at of β3 A reduction of about 30% in the current density was in cells ± ± in which the peak current was at was examined with In the currents were by 100 a significant decrease was in the in cells ± ± inactivation of the voltage-dependent Ca2+ channel current was significantly slower in cells. The of the voltage-dependent Ca2+ channel current at was by an the was ± and ± in cells and cells is with the the inactivation was by β subunits (9Hullin R. Singer-Lahat D. Freichel M. Biel M. Dascal N. Hofmann F. Flockerzi V. EMBO J. 1992; 11: 885-890Crossref PubMed Scopus (280) Google Scholar). to in and in has been VDCCs play an role in coupling sparks and currents, and this coupling a for regulating Ca2+ sparks in were of Ca2+ changes in the first of a wild-type in response to mm of in and mice of and in and The decrease in the in mice on of an L-type calcium channel was significantly in mice and decreased responsiveness to in the were significant in the of in and cells from mice each in high in the of The of Ca2+ sparks was also significantly by β3 ± and ± in and cells, in The of Ca2+ were also in these The and were ± and ± in cells cells from and were significantly from in cells ± and ± cells from of the in the and the decrease in channel population by Western blot and binding we examined the of the α1C of which the of L-type channels in the heart and aorta is The α1C subunit is in the heart and aortic smooth muscle cell were in the patterns of and mutant mice with amplification to the of the β3 as the of were we examined expression of the β3 subunit by analysis in isolated aortic smooth muscle cells In the wild-type mice a of the β3 subunit was whereas only were in mice has high in high The corresponding phase are in the to and in of the L-type voltage-dependent calcium channels in the cardiovascular system is widely and its such as DHP, are widely used in the treatment of decreased blood pressure was significant were in blood pressure in mice on normal as compared with wild-type controls. We also measured blood pressure using were significant in the using the and we used the for of a significantly and blood pressure in whereas it significant in mice of of of of of of the β3 subunit on and blood pressure in mice on normal and high salt the ± of mice from to ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± in a high salt in is with high blood pressure Science. 1995; PubMed Scopus Google we examined the of a high salt containing on the blood pressure of and mice. with the high salt diet for and blood pressure in mice in mice with the high salt diet for hypertrophic changes in the aortic smooth muscle of mice whereas significant changes were in mice. mice on the high salt diet also showed an increase in heart these changes are in have been a of elevated blood Electrophysiological of the importance of β subunits in the voltage-dependent calcium channels in smooth muscle cells, we expression of the β2, and β3 subunits in cells. Ca2+ channel currents using as the charge A in was The cell was ± 0.4 To the Ca2+ channel we first cells with a on voltage-dependent Ca2+ channel currents The voltage-dependent Ca2+ channel current was from cells α1C with In the cells the current density from the peak current at 10 was ± 0.5 The voltage-dependent current during ± These are the as reported previously A. E. A. B. Ludwig A. Flockerzi V. Hofmann F. J. Scopus Google Scholar, M. Murakami M. T. Y. K. S. J. Full Text Full Text PDF PubMed Scopus Google Scholar). The currents were from cells in which the subunit the and β3 subunits β3 were expressed with the of β subunits on of β subunit significantly ± ± ± significant the of the β3 subunit significantly the rate of The for each is in cells, α1C with were Of the types of cells, the was with β3 ± and with β2 ± The is thought to aortic smooth muscle cells. The rate of inactivation for the expression of β2 significantly from the inactivation for the The expression of the β2 and β3 subunits an ± β3 the in this expression system as in the isolated smooth muscle cells of β3-deficient mice. In addition, to of β subunits expression of voltage-dependent Ca2+ we the β3 subunit in cells. the β3 an Ca2+ channel current the were on β2 of β3 The of these experiments indicated were the channel properties in cells β2a, and β3 subunits. the β3 subunit current as compared with the β2 the significant on the current in the smooth muscle cells was to the β3 isolated smooth muscle cells showed current which were to In the present we a significant reduction in the L-type Ca2+ channel population in the aorta of mice by Western blot and binding We also the resulted in reduced and Ca2+ channel currents in aortic smooth muscle cells and decreased responsiveness to in the whereas were changes in the Ca2+ sparks in In addition, of the voltage-dependent calcium channel β3 subunit resulted in a decrease in responsiveness to DHP, as by blood pressure and an increase in blood pressure in a high salt as as changes such as of the aortic smooth muscle layer and cardiac enlargement. We a reduction in the channel population at the cell membrane in the aorta in mice by and decreased peak currents and a reduction in the calcium channel The of studies the the β subunits have a and increase the channel population D. V. S. E. S. T. Mori Y. De Waard M. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, S. H. Hofmann F. Flockerzi V. K. PubMed Scopus Google Scholar). In A. Olcese R. L. Stefani E. Science. PubMed Scopus Google reported the β2 subunit influence gating currents, which is a charge of the voltage and to the channel with mice the at in the cardiovascular In this we a reduction of only 30% in the Ca2+ channel currents in cells, whereas experiments a of the β3 as compared with the with α1C cells we expression of the β2 subunit in the aorta, we compared the of co-expression of the β2 and α1C subunits with co-expression of the β2 and β3 subunits and the α1C The these were to and cells. The expression of the β2 as revealed in the RT-PCR the β2 subunit also with α1C subunits in the aortic smooth muscle cells in Of known β subunits, β3 subunit has the in the inactivation rate (9Hullin R. Singer-Lahat D. Freichel M. Biel M. Dascal N. Hofmann F. Flockerzi V. EMBO J. 1992; 11: 885-890Crossref PubMed Scopus (280) Google the inactivation rate in cells in the present study is also with these and calcium channels in vascular smooth muscle cells is at to the expression of the β subunits, β2 and is β2 is for the L-type calcium channel currents in vascular smooth muscle cells. a study revealed an increase in a of the β3 subunit in heart the importance of the β3 subunit in cardiovascular and the involvement of this subunit in R. S. P. Varadi G. Schwartz A. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We significant changes in Ca2+ such as sparks and in significant in responsiveness to were in by mm as compared with wild-type controls. intracellular calcium Ca2+ are and by the of ryanodine-sensitive calcium and Ca2+ in the J. 2000; PubMed Google Scholar). The physiological role of Ca2+ sparks in smooth muscle is the of membrane the activation of which a with and J. 2000; PubMed Google Scholar, Y. Yamamura H. N. S. Muraki K. M. Imaizumi Y. J. Scopus Google Scholar). A Ca2+ is of a increase in has little on the L-type calcium channel activation is known to increase the and of Ca2+ mouse in which the calcium channel current density is reduced by was used to Ca2+ Although we significant changes in Ca2+ sparks in L-type calcium channels play a significant role in the control of Ca2+ as showed a significant on and the Ca2+ through the β3 subunit during to mm significantly to the of Ca2+ Ca2+ to the rate of Ca2+ and the The mutant mice used in the present study have a 30% reduction in the calcium current density, and the of in smooth muscle cells be in a during in expression of RyR, Ca2+ and to be the coupling of channels with be as as of this is on the of α1C in and mice. In addition, the of its it to in to the 30% reduction in peak calcium channel We significant in blood pressure and mice on the normal diet. calcium channels are thought to play roles in the cardiovascular by regulating calcium into smooth muscle cells, be a for the decreased channel population in mice. the we decreased responsiveness to the in the of blood pressure we a significant reduction in the channel population, on the of and decreased in the decreased responsiveness to in the blood pressure with the decreased channel population and reduced responsiveness in the and was to the the reduced channel population in the smooth muscle cells. The increase in blood pressure in response to salt is to and in the we examined calcium channel subunit expression in the by in We significant and the of expression of the β3 calcium channel subunit in the were also changes in the of mice. we examined plasma changes were of the high of in the To date, genes to have been to the of the of β3 T. Lory P. Taviaux S. Courtieu C. Guilbault P. Berta P. Nargeot J. Eur. J. Biochem. 1994; 220: 257-262Crossref PubMed Scopus (46) Google as such genes have to be isolated and are this be a to for in The cardiac enlargement and hypertrophic changes in the aortic smooth muscle layer, are in and be a of the elevated blood blood pressure is through of and vascular the of expression of a voltage-dependent calcium channel β subunit is for by as under this is to normal under of salt studies are to determine the for the salt and the of changes in blood pressure in mice. of the calcium channels is to α1 which is the of many calcium channel The β subunit has been to the of the channels to calcium channel blockers A. L. K. Ludwig A. E. Flockerzi V. Hofmann F. 1995; PubMed Scopus Google Scholar, L. A. E. P. Flockerzi V. Hofmann F. J. Pharmacol. 1995; Google Scholar). we examined the of and on of the aortic smooth muscle cells, which be by reduced L-type we in the to these and mice the β2 and β3 genes are expressed in wild-type the calcium channels are composed of a mixture of β2, and β3 in aortic smooth muscle cells, and it be to significant by and mice. the of this study the β subunit the of calcium channels of vascular smooth muscle cells. We also the mouse voltage-dependent calcium channel β3 subunit product a role in the blood pressure increase is for a of We and for We also and for