Volume 308. number 1, 7-13 FEBS 11384 lq92 Federation of European Biochemical Socie|ie~ 0014S?93/92#$S.00
August 1992
Molecular cloning and characterization of a novel calcium channel from rabbit brain T e t s u h i r o N i i d o m e ' , M a n - S u k K i m , T h o m a s F r i c d r i c h " and Y a s u o M o r t Deparnnents of Medical Chcmi~lo' and Molecular G¢netirs. lf).o~a Onirersio, Faculty of M~dichw. A~'ota 606.01. Japan Received 22 3uric L992 The comptct¢amino acid aglucn~ of a novelcalciumchannel (dcsillnatcd BIL}from rabbit brain has been deduced by cloning and sequm¢inll the eDNA. The BII calcium channel is structurally afore ¢[or~ly related to the B| calciuqn channel than to the cardiac and skeletal mu~l~ k-typm
calciumchannels. Blot hybridizationanalysisof RNA from different tissues and from different regionsof the brain shows that the BII calcium channel is distributed predominantly in the brain, l~in~ abundant in the cxrcbr'.zl cortex, hippocampus and corpus striaturn. Calcium channel: eDNA doninlp Nucleolid¢ ~qtaen~; RNA blot hybridization; Rabbit brai,~
l, I N T R O D U C T I O N
button o f B I t calcium channel m R N A studied by blot hybridization analysis,
Voltage-dependent calcium channels arc essential for the regulation of a variety of cellular functions, including membrane excitability, muscle contraction, synaptic transmission and other forms o f secretion, At leastfour types of calcium channel (designated T-. L-. N- and P-type calcium channels) have been aistinguished by their electrophysiological and pharmacological properties [L-3]. Recently, attempts have been made to understand the molecular basis o f the functional heterogeneity of the calcium channel, c D N A cloning studies have revealed the existence of multiple calcium channel gone products.
Expression o f the c D N A s
for the d i h y -
dropyridine ( D H P ) receptor from skeletal muscle, heart, smooth muscle and brain yields DHP-sensitive L-typ= calcium channe!s [4-10]. On the other hand, the brain calcium channel BI exhibits calcium channel activity that is insensitive both to nifedipin¢ and to caconotoxin (oJ.CgTx), resembling P-type calcium channels in neurons [I I]. Here wc report the complete nuclcotid¢ sequence and deduced amino-acid sequence of a novel calcium channel from rabbit brain (designated BII). The tissue distriA/d~rr~'iraio.#: DHP. dihydropyridin¢; a~.CBTx, co-¢onotoxin. " Pre,~nl address: Tsukuba Research Laboratories, Eisai Co.. I-3
Tok0dai S-chom~, Tsukuba.shi, lbaraki 300.26, Japan, "" Presenl tzddre#s: BASF AG. ZHB/P
A30, D-6700 Ludwi~hafen,
Germany, Corr~spaJ~de~:cr a.tdress: Y, Marl. Departmentsof Medical Chemistry and Molr.cular (3¢neties, Kyoto University Faculty of Medicine, Yoshida. Sakyo.ku, Kyoto 606.01, Japan, Fax: (81) (75) 753-43~8,
Puhl{d~¢dby Ei~'cv/¢r$ci#nc~ Puhtidwr# fl. V.
2. M A T E R I A L S
AND
has also been
METHODS
An olillo(dT}-primcd, sizc-r,etected ( > - 2 kiloba..~ pair tkb))eDNA library was constructed [4] in phaje Ag| 10 using poly(AT RNA prepared [7] from adult rabbit bruin, it was ~¢r~ned with a Bl calcium channel eDNA protm, that is, the EcoRl(3,72?yEroRl(5,050) £raj. meat from clone ACB:I [| I}; restriction endonudea..~ ~1.¢~are identified by numbers (in parenthe~) indicating the $'.tcrminml n~lcotid¢ i|enerutcd b), ¢leaval|c. Thu~ done ACDA240 (¢arryins nucl~otides 3.6t~9,-G,293x was isolated;numbers in parenthese~indicate the nudeotidc residuesof the eDNA ~rried by the clone. Restriction fragments from ,,1,CBA240 were u~d as pro~s to clone adjaent cDNAs and similar clonin~ proce..durcswere repeated. The synthetic primer complemen|ary to nucleofide residues 4.242-4,258 was ¢lonpted and cloned into A,IPl0 by the pro¢,edures dczcril>'.d previously [4)(siz~ selection > - I kb). A,CBP201 (I 32.-4,253x wai isolated from the synth¢|ic prlmer-derived cDNAs. A,CB204 (-598 to 1,648) and ;.CB215 (-59~ to 1,0a3) was isolated from randomly primed, size.loitered (>-. l kb) eDNA llbraw, and ~,CB236 (6,1t16-~,010). A,CB244 (6,0236,i1(f4) and 3,CB264 (5,446-tl.010 with a deletion of 6,300-7,115) from another ru.domly primed eDNA library, Appropriate restriction frug. ments from the isolated clones were subcloned into pBlucr,crlpt KS(-). Ml3mpl~ or Ml3mpl9. Nested deletions were made (4l and DNA sequencing was carried out on both str.,nds by |he didcoxy chaintermination n'lethr~t [12]. RNA blot hybridimtion analysis was pc~r. formed as in [13],
3. RESULTS A N D DISCUSSION Fig. 1 shows the primary structure o f the rabbit BII calcium channel deduced from the nucl¢otid¢ sequence of the cloned eDNA (for cloning procedures see section 2); the open reading frame corresponding to the amino acid sequences of the skeletal muscle, cardiac and BI calcium channels [4, I I, 14] was adopted and the translation initiation si:¢ was assigned to the first ATfi triplet 7
V o l u m e 308, n u m b e r I
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August L992
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Fis, I, Nu¢IcotLdc sequence of the c D N A encoding the BI[ calcium channel ;Ll'id|he dcduc,~d mnino acid sequence, (A) The nu¢IcotLd¢ ~qum'~c~ of BII.I was detcrmincd usLn$ clone ACB204 (carrying nucleotidcs -sgi~ to 1.64t~),ACBP201 (132-4,2~,3},A C B A 4 0 (3.689-6,293) and ACB236 (6,i 86-~.0 L0), Nucl¢otidc residues arc nurnbercd in the 5" to 3' dirccLion from the first rcsidu~ of th~ A T G initiation triplet and the pr¢ccdin~ residues arc indicated by neBaLivc numbeTs, The Y-termL.~aL nucleotid¢ residue. R,010. Lsnot l'ol]nwcd by a poly(dA) tracL, Numbers oi'th¢ nucl¢otide residues -L Lh¢ rig{'~t-handcnd of the individual {incs urc sivcn, Amino acid rcshJucsarc nmllbePcd I'rom I,hc i~itiating meLhioninc. The puLativ¢ tPans,ncnl.,~ ;tssign~d,The tcnt;tti~eboundaries brnn¢ segmcnLs SI-$6 in each or the Tep~s~tS I-IV are overlined; Lhe termini orcaci~ segment ha v ¢ b('¢ntenLatiV"', or" th~ inscr~cd sequence of 818 .ucl¢otid¢ residues arc shown. (U) Thc 3'-t¢~'minal rcsion o1' the c D N A cncodin~ BIi-2, Thc nuclcotidc sequence oT BIL-2 w~s detennine..d using clones ~.CB204, ACBP201, ~ C B A 4 0 and ~CB2~4 CS.~6=8,010 with ~ d~i0~ion of 6,300-'/,115), Th~ arrowhead indicates the position whcrc the t;i6 nucLcotid¢ sc~iucncc is in.fred in BII.I,
Volumc 30~, numl~r I
FEBL~ LF./I'TERS
August 1992
Jttsl l||t fell |~ll II*l lOl l ll+l+l fill ll4YI 114J l l I 114111A I 6 t4v I | S l | l I l l'/~l l l ~ + | IA I l ~ I I~ l yAl| LllMI I I l l + I d I VIyI Jl I ~4~4||VL¥1~I &| | ~ l l l & | I ~ I l [ 11 I pIIA ~ l l I | I I I I • | • LII I l l b l l ~ I f l l l ~ I k I ¢CCI~IPg?('I+~ItI;I~&I~IgPIT?lPIIilflC?II?4;II(IIIa'it'II"(al jJ64'~4~4AI~J~8~&~i`~lI~A~P.4~T~P~'k4+~k4t
August 1992
Molecular cloning and characterization of a novel calcium channel from rabbit brain T e t s u h i r o N i i d o m e ' , M a n - S u k K i m , T h o m a s F r i c d r i c h " and Y a s u o M o r t Deparnnents of Medical Chcmi~lo' and Molecular G¢netirs. lf).o~a Onirersio, Faculty of M~dichw. A~'ota 606.01. Japan Received 22 3uric L992 The comptct¢amino acid aglucn~ of a novelcalciumchannel (dcsillnatcd BIL}from rabbit brain has been deduced by cloning and sequm¢inll the eDNA. The BII calcium channel is structurally afore ¢[or~ly related to the B| calciuqn channel than to the cardiac and skeletal mu~l~ k-typm
calciumchannels. Blot hybridizationanalysisof RNA from different tissues and from different regionsof the brain shows that the BII calcium channel is distributed predominantly in the brain, l~in~ abundant in the cxrcbr'.zl cortex, hippocampus and corpus striaturn. Calcium channel: eDNA doninlp Nucleolid¢ ~qtaen~; RNA blot hybridization; Rabbit brai,~
l, I N T R O D U C T I O N
button o f B I t calcium channel m R N A studied by blot hybridization analysis,
Voltage-dependent calcium channels arc essential for the regulation of a variety of cellular functions, including membrane excitability, muscle contraction, synaptic transmission and other forms o f secretion, At leastfour types of calcium channel (designated T-. L-. N- and P-type calcium channels) have been aistinguished by their electrophysiological and pharmacological properties [L-3]. Recently, attempts have been made to understand the molecular basis o f the functional heterogeneity of the calcium channel, c D N A cloning studies have revealed the existence of multiple calcium channel gone products.
Expression o f the c D N A s
for the d i h y -
dropyridine ( D H P ) receptor from skeletal muscle, heart, smooth muscle and brain yields DHP-sensitive L-typ= calcium channe!s [4-10]. On the other hand, the brain calcium channel BI exhibits calcium channel activity that is insensitive both to nifedipin¢ and to caconotoxin (oJ.CgTx), resembling P-type calcium channels in neurons [I I]. Here wc report the complete nuclcotid¢ sequence and deduced amino-acid sequence of a novel calcium channel from rabbit brain (designated BII). The tissue distriA/d~rr~'iraio.#: DHP. dihydropyridin¢; a~.CBTx, co-¢onotoxin. " Pre,~nl address: Tsukuba Research Laboratories, Eisai Co.. I-3
Tok0dai S-chom~, Tsukuba.shi, lbaraki 300.26, Japan, "" Presenl tzddre#s: BASF AG. ZHB/P
A30, D-6700 Ludwi~hafen,
Germany, Corr~spaJ~de~:cr a.tdress: Y, Marl. Departmentsof Medical Chemistry and Molr.cular (3¢neties, Kyoto University Faculty of Medicine, Yoshida. Sakyo.ku, Kyoto 606.01, Japan, Fax: (81) (75) 753-43~8,
Puhl{d~¢dby Ei~'cv/¢r$ci#nc~ Puhtidwr# fl. V.
2. M A T E R I A L S
AND
has also been
METHODS
An olillo(dT}-primcd, sizc-r,etected ( > - 2 kiloba..~ pair tkb))eDNA library was constructed [4] in phaje Ag| 10 using poly(AT RNA prepared [7] from adult rabbit bruin, it was ~¢r~ned with a Bl calcium channel eDNA protm, that is, the EcoRl(3,72?yEroRl(5,050) £raj. meat from clone ACB:I [| I}; restriction endonudea..~ ~1.¢~are identified by numbers (in parenthe~) indicating the $'.tcrminml n~lcotid¢ i|enerutcd b), ¢leaval|c. Thu~ done ACDA240 (¢arryins nucl~otides 3.6t~9,-G,293x was isolated;numbers in parenthese~indicate the nudeotidc residuesof the eDNA ~rried by the clone. Restriction fragments from ,,1,CBA240 were u~d as pro~s to clone adjaent cDNAs and similar clonin~ proce..durcswere repeated. The synthetic primer complemen|ary to nucleofide residues 4.242-4,258 was ¢lonpted and cloned into A,IPl0 by the pro¢,edures dczcril>'.d previously [4)(siz~ selection > - I kb). A,CBP201 (I 32.-4,253x wai isolated from the synth¢|ic prlmer-derived cDNAs. A,CB204 (-598 to 1,648) and ;.CB215 (-59~ to 1,0a3) was isolated from randomly primed, size.loitered (>-. l kb) eDNA llbraw, and ~,CB236 (6,1t16-~,010). A,CB244 (6,0236,i1(f4) and 3,CB264 (5,446-tl.010 with a deletion of 6,300-7,115) from another ru.domly primed eDNA library, Appropriate restriction frug. ments from the isolated clones were subcloned into pBlucr,crlpt KS(-). Ml3mpl~ or Ml3mpl9. Nested deletions were made (4l and DNA sequencing was carried out on both str.,nds by |he didcoxy chaintermination n'lethr~t [12]. RNA blot hybridimtion analysis was pc~r. formed as in [13],
3. RESULTS A N D DISCUSSION Fig. 1 shows the primary structure o f the rabbit BII calcium channel deduced from the nucl¢otid¢ sequence of the cloned eDNA (for cloning procedures see section 2); the open reading frame corresponding to the amino acid sequences of the skeletal muscle, cardiac and BI calcium channels [4, I I, 14] was adopted and the translation initiation si:¢ was assigned to the first ATfi triplet 7
V o l u m e 308, n u m b e r I
FEB$
LETTERS
August L992
A
b'.*.
g t t ~ i * "o¢ Pll.oql| ~ I oL I 6& | i& | i ~ 1TArI P#1hl | i l l I (: | IPG|ldl l le~* pie l'i,i 141114;| y~ ! r&o ll~4.1~llle4 ITfll# P'I I v * | N I~ | 6 I * 1'4| y I oI &| 6 L I *t, I 6'1"1.'1#8¢ I IS11 W IL | ,I 11~,,~L#I& | | • ;I ~'l lit Ill I |t Tt il tf l|l lff i | i b 4 1 ~ / f i l i P l o | t IIIIV6IAI J G | A l J ~ l l / ~ S r l l l V l | & S i i ! | | l l ~ d ~ i | J e l h ¢ |y ~|e ~. F J ~i l i ~ l v i | J ~ ! w i g y l i ~ i ~ , r l & y | m | l l l | p f l p h o P l l / 1 4 1 | l ~ l ~ I I il~,4u~iiVb~ I | • i | l [41| J ~ l J | I¥11 k I l l i i LMI| | vG | ~ | l IAi P lO~ | v i i l i 4 p ~yithl l # l ~ l $1 f ~ l ~ | ~4K | wL11til~ J l t # ¢ ~ l t ~ | | I ~ | y | JI I bl~/I ~kl$| v&| i t J t | J I ~ i | i o¥4 | i i i b4 K | ~/~4 | |0 P ~ I ~ i ~ 1 ¢ | | h i t ~ l b4I ~ l i i l ~
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Fis, I, Nu¢IcotLdc sequence of the c D N A encoding the BI[ calcium channel ;Ll'id|he dcduc,~d mnino acid sequence, (A) The nu¢IcotLd¢ ~qum'~c~ of BII.I was detcrmincd usLn$ clone ACB204 (carrying nucleotidcs -sgi~ to 1.64t~),ACBP201 (132-4,2~,3},A C B A 4 0 (3.689-6,293) and ACB236 (6,i 86-~.0 L0), Nucl¢otidc residues arc nurnbercd in the 5" to 3' dirccLion from the first rcsidu~ of th~ A T G initiation triplet and the pr¢ccdin~ residues arc indicated by neBaLivc numbeTs, The Y-termL.~aL nucleotid¢ residue. R,010. Lsnot l'ol]nwcd by a poly(dA) tracL, Numbers oi'th¢ nucl¢otide residues -L Lh¢ rig{'~t-handcnd of the individual {incs urc sivcn, Amino acid rcshJucsarc nmllbePcd I'rom I,hc i~itiating meLhioninc. The puLativ¢ tPans,ncnl.,~ ;tssign~d,The tcnt;tti~eboundaries brnn¢ segmcnLs SI-$6 in each or the Tep~s~tS I-IV are overlined; Lhe termini orcaci~ segment ha v ¢ b('¢ntenLatiV"', or" th~ inscr~cd sequence of 818 .ucl¢otid¢ residues arc shown. (U) Thc 3'-t¢~'minal rcsion o1' the c D N A cncodin~ BIi-2, Thc nuclcotidc sequence oT BIL-2 w~s detennine..d using clones ~.CB204, ACBP201, ~ C B A 4 0 and ~CB2~4 CS.~6=8,010 with ~ d~i0~ion of 6,300-'/,115), Th~ arrowhead indicates the position whcrc the t;i6 nucLcotid¢ sc~iucncc is in.fred in BII.I,
Volumc 30~, numl~r I
FEBL~ LF./I'TERS
August 1992
Jttsl l||t fell |~ll II*l lOl l ll+l+l fill ll4YI 114J l l I 114111A I 6 t4v I | S l | l I l l'/~l l l ~ + | IA I l ~ I I~ l yAl| LllMI I I l l + I d I VIyI Jl I ~4~4||VL¥1~I &| | ~ l l l & | I ~ I l [ 11 I pIIA ~ l l I | I I I I • | • LII I l l b l l ~ I f l l l ~ I k I ¢CCI~IPg?('I+~ItI;I~&I~IgPIT?lPIIilflC?II?4;II(IIIa'it'II"(al jJ64'~4~4AI~J~8~&~i`~lI~A~P.4~T~P~'k4+~k4t
