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• W2076494274 abstract "In skeletal muscle the dihydropyridine receptor is the voltage sensor for excitation-contraction coupling and an L-type Ca2+ channel. We cloned a dihydropyridine receptor (named Fgα1S) from frog skeletal muscle, where excitation-contraction coupling has been studied most extensively. Fgα1S contains 5600 base pairs coding for 1688 amino acids. It is highly homologous with, and of the same length as, the C-truncated form predominant in rabbit muscle. The primary sequence has every feature needed to be an L-type Ca2+ channel and a skeletal-type voltage sensor. Currents expressed in tsA201 cells had rapid activation (5–10 ms half-time) and Ca2+-dependent inactivation. Although functional expression of the full Fgα1S was difficult, the chimera consisting of Fgα1S domain I in the rabbit cardiac Ca channel had high expression and a rapidly activating current. The slow native activation is therefore not determined solely by the α1 subunit sequence. Its Ca2+-dependent inactivation strengthens the notion that in rabbit skeletal muscle this capability is inhibited by a C-terminal stretch (Adams, B., and Tanabe, T. (1997)J. Gen. Physiol. 110, 379–389). This molecule constitutes a new tool for studies of excitation-contraction coupling, gating, modulation, and gene expression. In skeletal muscle the dihydropyridine receptor is the voltage sensor for excitation-contraction coupling and an L-type Ca2+ channel. We cloned a dihydropyridine receptor (named Fgα1S) from frog skeletal muscle, where excitation-contraction coupling has been studied most extensively. Fgα1S contains 5600 base pairs coding for 1688 amino acids. It is highly homologous with, and of the same length as, the C-truncated form predominant in rabbit muscle. The primary sequence has every feature needed to be an L-type Ca2+ channel and a skeletal-type voltage sensor. Currents expressed in tsA201 cells had rapid activation (5–10 ms half-time) and Ca2+-dependent inactivation. Although functional expression of the full Fgα1S was difficult, the chimera consisting of Fgα1S domain I in the rabbit cardiac Ca channel had high expression and a rapidly activating current. The slow native activation is therefore not determined solely by the α1 subunit sequence. Its Ca2+-dependent inactivation strengthens the notion that in rabbit skeletal muscle this capability is inhibited by a C-terminal stretch (Adams, B., and Tanabe, T. (1997)J. Gen. Physiol. 110, 379–389). This molecule constitutes a new tool for studies of excitation-contraction coupling, gating, modulation, and gene expression. excitation-contraction dihydropyridine dihydropyridine receptor cAMP-dependent protein kinase. In EC1 coupling an action potential activates DHP receptors in the transverse tubular membrane, which somehow open ryanodine receptors in the sarcoplasmic reticulum to release Ca2+ and initiate muscle contraction. The skeletal muscle DHPr is a slowly activating L-type Ca2+channel (2Beam K.G. Adams B.A. Niidome T. Numa S. Tanabe T. Nature. 1992; 360: 169-171Crossref PubMed Scopus (76) Google Scholar) that also controls Ca2+ release, working as a voltage sensor (3Rios E. Brum G. Nature. 1987; 325: 717-720Crossref PubMed Scopus (651) Google Scholar, 4Tanabe T. Beam K.G. Powell J.A. Numa S. Nature. 1988; 336: 134-139Crossref PubMed Scopus (582) Google Scholar). How DHPrs interact with ryanodine receptors during EC coupling is still not clear. Most experiments on EC coupling have been done in frog skeletal muscle fibers, although the cDNAs of skeletal muscle DHPr have been cloned from rabbit (5Tanabe T. Takeshima H. Mikami A. Flockerzi V. Takahashi H. Kangawa K. Kojima M. Matsuo H. Hirose T. Numa S. Nature. 1987; 328: 313-318Crossref PubMed Scopus (967) Google Scholar), fish (carp) (6Grabner M. Friedrich K. Knaus H.G. Striessnig J. Scheffauer F. Staudinger R. Koch W.J. Schwartz A. Glossmann H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 727-731Crossref PubMed Scopus (51) Google Scholar), and human (7Hogan K. Powers P.A. Gregg R.G. Genomics. 1994; 24: 608-609Crossref PubMed Scopus (20) Google Scholar). Functional differences exist between frog and mammalian skeletal muscle DHPrs. Seeking additional insights into the molecular basis of skeletal muscle EC coupling, we cloned and expressed a skeletal muscle DHPr from a frog. The frog DHPr (Fgα1S) was cloned from mRNA of skeletal muscle of Rana catesbeiana by combining RT-PCR with construction and screening of a cDNA library. The molecule has high homology with other DHPrs at transmembrane domains, voltage sensors (5Tanabe T. Takeshima H. Mikami A. Flockerzi V. Takahashi H. Kangawa K. Kojima M. Matsuo H. Hirose T. Numa S. Nature. 1987; 328: 313-318Crossref PubMed Scopus (967) Google Scholar), pore regions (8Tang S. Mikala G. Bahinski A. Yatani A. Varadi G. Schwartz A. J. Biol. Chem. 1993; 268: 13026-13029Abstract Full Text PDF PubMed Google Scholar), and DHP binding sites (9Tang S. Yatani A. Bahinski A. Mori Y. Schwartz A. Neuron. 1993; 11: 1013-1021Abstract Full Text PDF PubMed Scopus (93) Google Scholar). Despite those conserved features, Fgα1S has also several differences with known DHPrs. The cloned rabbit skeletal muscle L-type Ca channel has been expressed in dysgenic myotubes (4Tanabe T. Beam K.G. Powell J.A. Numa S. Nature. 1988; 336: 134-139Crossref PubMed Scopus (582) Google Scholar) and in Xenopus oocytes (10Ren D. Hall L.M. J. Biol. Chem. 1997; 272: 22393-22396Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). However, functional expression of this channel in mammalian cell lines has been difficult (11Perez-Reyes E. Kim H.S. Lacerda A.E. Horne W. Wei X.Y. Rampe D. Campbell K.P. Brown A.M. Birnbaumer L. Nature. 1989; 340: 233-236Crossref PubMed Scopus (232) Google Scholar, 12Lacerda A.E. Kim H.S. Ruth P. Perez-Reyes E. Flockerzi V. Hofmann F. Birnbaumer L. Brown A.M. Nature. 1991; 352: 527-530Crossref PubMed Scopus (243) Google Scholar, 13Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-75Crossref PubMed Scopus (398) Google Scholar, 14Johnson B.D. Brousal J.P. Peterson B.Z. Gallombardo P.A. Hockerman G.H. Lai Y. Scheuer T. Catterall W.A. J. Neurosci. 1997; 17: 1243-1255Crossref PubMed Google Scholar). In our hands, the functional expression of Fgα1S also occurred infrequently. In contrast, a chimera (named α1C-FSDI) of domain I of Fgα1S in a rabbit cardiac background was expressed easily and at high levels. Total RNA samples were isolated from the liver, cardiac, and skeletal muscle of a frog by the acid guanidinium-phenol-chloroform method (15Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63228) Google Scholar). Poly(A)+ RNA was purified from the total RNA by using the Mini-oligo(dT) cellulose spin kit (5 Prime → 3 Prime, Inc., Boulder, CO). Total RNA harvested from frog skeletal muscle was reverse transcribed using random primers. Based on the conserved amino acid sequences of known DHP receptors, two pairs of degenerate primers (C5/C6 and C12/J3) were synthesized for amplifying a cDNA segment (segment 7) between IIIS6 and IVS6 and a segment (segment 6) between IS1 and IIS3 of the channel molecule. Primer C5 (5′-CGCGGAATTC TT(T,C)ATGATGAA(C,T)AT(T,C,A)TT(T,C)GT-3′, with base degeneracy represented in parentheses) had an EcoRI restriction site at the 5′ end (bold) fused to the DNA sequence of FMMNIFV in IIIS6. Primer C6 (5′-CGG CGG ATCC ANAGCAT(A,G)TA(A,G)AA(A,G)CT(A,G,T)AT(A,G)AA-3′) had a BamHI site at the beginning fused to the cDNA sequence of FISFYML in IVS6. Primers C12 (5′-AT(A,C,T)GTNGA(A,G)TGGAA(G,A)CCNTT-3′) and J3 (5′-AA(A,G)CA(A,G)TC(A,G)AANC(G,T)(A,G)TT(A,G)AA-3′) were derived from the conserved amino acid sequences IVEWKPF at the beginning of domain IS1 and FNRFDCF in domain IIS3, respectively. Based on the cDNA sequences of cloned frog cDNA segments 6 and 7, nondegenerate primers (J4 and J5) were designed to amplify the cDNA (segment 9) between domains II and IV. Primer J4 (5′-GCC CTT GGT TTC CAG TCA TAT TTC-3′) corresponds to the amino acid sequence ALGFQSYF in IIS2, and J5 (5′-CAT ACC AAG GCT GAT GGT GTT CAG-3′) presents the cDNA sequence of LNTISLGM in IVS1. Control reactions were performed in the absence of reverse transcriptase to rule out possible contamination by genomic DNA. First-strand cDNA synthesis kits were from either Boehringer Mannheim or Pharmacia Biotech, Inc. PCR products were subcloned into pGEM-T vectors (Promega, Madison, WI). Because there are no conserved sequences at both C and N termini of known Ca channels, no gene-specific primers could be designed for RT-PCR. A frog skeletal muscle cDNA library was constructed instead, and the probes derived from RT-PCR clones (segments 6 and 7) were used to screen it and to obtain both 5′ and 3′ cDNAs. Double-stranded cDNA was synthesized using the Superscript Choice system for cDNA synthesis (Life Technologies, Inc.). The size-fractionated cDNA (>1 kb) was ligated into the λZAP Express vector (Stratagene, La Jolla, CA). The recombinant phage vectors packaged with a protein coat (Gigapack II Gold, Stratagene) were used to infect XL1-Blue-MRF′ bacteria (Stratagene) to construct the library. Positive clones pBK/5-13 (covering the 5′ untranslated region), pBK/5-8, pBK/5-10 (covering the 3′ untranslated region), and pBK/5-5 were obtained by screening the library. All cDNA clones were sequenced using the Sequenase 2.0 DNA sequencing kit (Amersham Corp.) and the ABI PRISM dye terminator cycle sequencing ready reaction kit (Perkin-Elmer). Sequence similarity searches of GenBank™ were performed with PCGENE (IntelliGenetics, Mountain View, CA) and DNASIS (Hitachi-Software, South San Francisco, CA). Based on suitable restriction enzyme sites (Fig. 1), RT-PCR clones (segments 6 and 9) and the library clones (5-13, 5-5, and 5-8) were ligated to obtain the full-length molecule (Fgα1S) in the mammalian expression vector pCR3.1 (Invitrogen, San Diego, CA). Total RNA (20 μg) isolated from various frog tissues was separated on a 1% agarose-formaldehyde gel, transferred to Nytron BA-S-supported nitrocellulose membranes (Schleicher & Schuell), and cross-linked by baking for 2 h at 80 °C. The cDNA probe, including Fgα1S nucleotides 4859–5600, was cut out from clone pBK/5-8 by NotI and XmnI digestion. The probe was radiolabeled with [α-32P]dCTP using random primers (Ready to Go DNA labeling beads, Pharmacia). The membrane was hybridized with the probe for 24 h at 42 °C in 6 × SSC, 50% formamide, 0.5% SDS, 100 μg/ml sheared salmon sperm DNA, and 5 × Denhardt's solution. The high-stringency wash was performed in 0.5 × SSC and 0.1% SDS at 60 °C. X-ray film (X-Omat AR, Eastman Kodak Co.) was exposed to the membrane at −80 °C. Glyceraldehyde-3-P dehydrogenase probes were also used for hybridization to estimate relative total RNA for each tissue. cDNA coding for domain I of rabbit cardiac channel (α1C) at amino acid positions 165–422 was replaced by the corresponding cDNA segment from either Fgα1S or rabbit skeletal muscle α1S to obtain chimeras. 2The replaced segment starts in the middle of IS1 and ends in the middle of IS6. The replacement can be termed a full domain swap, however, because IS1 and IS6 are almost identical in the frog protein and rabbit α1C. The cDNA of α1C has unique restriction sites, MfeI at nucleotide 689 of IS1 and BamHI at nucleotide 1456 of IS6. These unique sites also appear at the corresponding positions of Fgα1S (491 and 1258, respectively). The cDNA (491–1258) was cut out from Fgα1S and then ligated into MfeI and BamHI predigested pCR3/α1C to produce the frog chimeric channel α1C-FSDI.MfeI and BamHI sites are not present at the corresponding positions of domain I of rabbit α1S, and the sites were introduced by PCR. The PCR product, which covers at 415–1185 of rabbit α1S, was cut by MfeI and BamHI and then ligated into pCR3/α1C to produce the rabbit chimeric channel α1C-RSDI. Large T-antigen-transformed human embryonic kidney cells (tsA201, a gift from Dr. M. M. Hosey, Northwestern University, Chicago, IL) were maintained in DMEM (Sigma) containing 10% fetal bovine serum (Biowhittaker, Walkersville, MD), 100 units/ml penicillin/streptomycin (Sigma) at 37 °C in 5% CO2. Rabbit skeletal muscle α2/δ (in pMT2) was a gift from Dr. T. Tanabe (Tokyo Medical/Dental University, Tokyo, Japan). Rabbit cardiac α1C was carried in pCR3 and rat brain β2a in pCMV. Seven micrograms of each cDNA (total <25 μg) were used for transfection by calcium phosphate precipitation (16Ferreira G. Yi J. Rios E. Shirokov R. J. Gen. Physiol. 1997; 109: 449-461Crossref PubMed Scopus (107) Google Scholar). Expression efficiency was defined as the fraction of cells that had Ca2+ currents among those selected and patched. Ionic currents were recorded by whole-cell patch clamp. Voltage clamping, pulse generation, and data acquisition were carried out with an Axopatch 200 A (Axon Instruments, Foster City, CA) and a 16-bit digital conversion card in a PC-compatible computer. The analog-to-digital and digital-to-analog routines were written by Ivan Stavrovsky in our laboratory. Pipettes were from borosilicate glass (Corning 7052; Garner Glass, Claremont, CA) heat polished to a tip outside diameter of 2–4 μm for a resistance of 2–4 MΩ. Ion currents were recorded with a pipette solution containing (in mm) 150 Cs+, 125 Asp−, 15 Cl−, 5 MgATP, 10 HEPES, and 10 EGTA, pH 7.6, adjusted by CsOH, and an external solution containing (in mm) 130 NaCl, 10 HEPES, and 10 Ca2+ or Ba2+, pH 7.3. Experiments were performed at 20–23 °C. Single, nonclustered cells were chosen for recording. Cells were pulsed from a holding potential of −90 mV. Asymmetric currents were obtained by subtraction of scaled control currents elicited with pulses from −130 to −100 mV. The composite cDNA sequence of Fgα1S was obtained by sequencing multiple overlapping clones. The sequence map of all clones is shown in Fig. 1. The 5′-end sequence, including an untranslated region, is present in the library clone 5-13. The 3′-end, including an untranslated region and a portion of the poly(A) tail, is covered by library clones 5-8 and 5-10. The cDNA sequences of the library clones (5-13, 5-5, 5-8, and 5-10) are equal to those of RT-PCR clones (segments 6, 7, and 9), with several single-base differences: 1) clone 5-13 has CGT780, whereas segment 6 and the full-length molecule have CGG780; both CGT and CGG encode arginine; 2) clones 5-13 and 5-5 have G876TA encoding valine, whereas segment 6 and the full-length cDNA have A876TA, encoding isoleucine; 3) clone 5-5 has T2151CT (serine), whereas segment 9 and the full-length cDNA have G2151CT (alanine) at this position; and 4) segment 7, clone 5-8, and the full-length cDNA have CTC4274, whereas clone 5-10 has CTT4274. Both CTC and CTT code for leucine. These single-base differences could be explained by polymorphism, also observed in α1 cDNA clones from rabbit skeletal muscle (5Tanabe T. Takeshima H. Mikami A. Flockerzi V. Takahashi H. Kangawa K. Kojima M. Matsuo H. Hirose T. Numa S. Nature. 1987; 328: 313-318Crossref PubMed Scopus (967) Google Scholar). The 5600-nucleotide cDNA sequence of Fgα1S and its amino acid sequence are available (GenBank™ accession number AF037625). The polyadenylation signal sequence, AATAAA (17Sambrook J Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), is found between nucleotides 5554 and 5559, 16 nucleotides upstream of the poly(A) tail. Translation of the deduced channel protein (1688 amino acids) starts at nucleotide 303 with the first methionine and ends at nucleotide 5367, which is followed by the stop codon TGA. Transmembrane domains and pore regions were deduced from an alignment of Fgα1S with rabbit cardiac (Rbα1C) (18Mikami A. Imoto K. Tanabe T. Niidome T. Mori Y. Takeshima H. Narumiya S. Numa S. Nature. 1989; 340: 230-233Crossref PubMed Scopus (768) Google Scholar), skeletal (Rbα1S) (5Tanabe T. Takeshima H. Mikami A. Flockerzi V. Takahashi H. Kangawa K. Kojima M. Matsuo H. Hirose T. Numa S. Nature. 1987; 328: 313-318Crossref PubMed Scopus (967) Google Scholar), and carp skeletal muscle (Cpα1S) DHP receptors (6Grabner M. Friedrich K. Knaus H.G. Striessnig J. Scheffauer F. Staudinger R. Koch W.J. Schwartz A. Glossmann H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 727-731Crossref PubMed Scopus (51) Google Scholar). Like other L-type Ca2+channels, Fgα1S has four putative transmembrane domains, but its C terminus is shorter than that of Rbα1S and Cpα1S. Table I lists the homology between different portions of Fgα1S and rabbit and carp L-type Ca2+channels. The homology is high with all L-type channels, but in every domain the highest homology is with Rbα1S.Table IDegrees of homology of Fgα1S with other L-type Ca channelsChannelFgα1S domainFgα1S loopFgα1S C terminusIIIIIIIVI-IIII-IIIIII-IV166 aa downstream from IVS6After downstream IVS6 160 aa%%%Rbα1S7778818066869096Very diverseRbα1C6875777744529092Very diverseCpα1S6874786854708592Very diverseRbα1C, rabbit cardiac channel; Rbα1S, rabbit skeletal muscle channel; Cpα1S, Carp skeletal muscle channel. Note the greater homology with the skeletal muscle channels and the high degree of homology with Rbα1S in the critical II-III linker. Open table in a new tab Rbα1C, rabbit cardiac channel; Rbα1S, rabbit skeletal muscle channel; Cpα1S, Carp skeletal muscle channel. Note the greater homology with the skeletal muscle channels and the high degree of homology with Rbα1S in the critical II-III linker. The structural basis of the voltage sensitivity has been located at transmembrane segments S4 for voltage-gated K+, Na+, and Ca2+ channels (19Papazian D.M. Timpe L.C. Jan Y.N. Jan L.Y. Nature. 1991; 349: 305-310Crossref PubMed Scopus (431) Google Scholar, 20Stuhmer W. Conti F. Suzuki H. Wang X.D. Noda M. Yahagi N. Kubo H. Numa S. Nature. 1989; 339: 597-603Crossref PubMed Scopus (950) Google Scholar, 21Garcia J. Nakai J. Imoto K. Beam K.G. Biophys. J. 1997; 72: 2515-2523Abstract Full Text PDF PubMed Scopus (54) Google Scholar). An alignment of the S4 segments of Fgα1S with rabbit and carp channels (Fig. 2 A) shows identical charges and almost perfect homology, consistent with the requirements for the voltage sensor of EC coupling and a voltage-operated ion channel. The pore regions of ion channels have been localized to the linkers between segments S5 and S6 (22Tomaselli G.F. Backx P.H. Marban E. Circ. Res. 1993; 72: 491-496Crossref PubMed Google Scholar). Four glutamic acid residues at equivalent positions in pore regions have been demonstrated to determine the high-affinity Ca2+ binding in L-type channels (23Yang J. Ellinor P.T. Sather W.A. Zhang J.F. Tsien R.W. Nature. 1993; 366: 158-161Crossref PubMed Scopus (529) Google Scholar), and other cloned high-voltage-activated Ca2+channels also have these conserved glutamic acid residues at equivalent positions. The alignment of pore regions of Fgα1S, rabbit, and carp channels is shown in Fig. 2 B. Fgα1S has conserved sequences in those regions, as well as the critical glutamic acid residues. DHP binding affinity is an established criterion to distinguish L- and non-L-type Ca channels (24Tsien R.W. Lipscombe D. Madison D.V. Bley K.R. Fox A.P. Trends Neurosci. 1988; 11: 431-438Abstract Full Text PDF PubMed Scopus (1132) Google Scholar). The DHP binding site of L-type Ca channels appears to involve regions between transmembrane segments IIIS6 and IVS6, where residues Tyr1048, Ile1049, and Ile1052 in IIIS6 and Tyr1365, Met1366, Ile1372, and Ile1373 in IVS6 are critical (25Peterson B.Z. Tanada T.N. Catterall W.A. J. Biol. Chem. 1996; 271: 5293-5296Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Fgα1S has a sequence almost identical to other DHP receptors in the DHP binding regions, including all required residues at corresponding sites (Tyr1046, Ile1047, Ile1050, Tyr1352, Met1353, Ile1359, and Ile1360). The alignment of DHP binding regions of Fgα1S with rabbit and carp DHP receptors is shown in Fig. 2 C. Unlike cardiac cells, in skeletal muscle Ca2+ release is triggered by DHPrs without requirement for entry of extracellular Ca2+ (26Armstrong C.M. Bezanilla F.M. Horowicz P. Biochim. Biophys. Acta. 1972; 267: 605-608Crossref PubMed Scopus (299) Google Scholar, 27Endo M. Physiol. Rev. 1977; 57: 71-108Crossref PubMed Scopus (1140) Google Scholar). A crucial molecular determinant of skeletal muscle type EC coupling is at the intracellular loop between domains II and III of rabbit α1S (28Tanabe T. Beam K.G. Adams B.A. Niidome T. Numa S. Nature. 1990; 346: 567-569Crossref PubMed Scopus (492) Google Scholar). The II-III loop of Fgα1S has high (86%) homology with Rbα1S (Table I), suggesting that Fgα1S is a skeletal muscle-type voltage sensor. Skeletal muscle L-type Ca2+ channels are regulated by phosphorylation. In rat skeletal muscle fibers, both Ca2+ current and intramembranous charge movement are increased by PKA (29Garcia J. Gamboa-Aldeco R. Stefani E. Pflugers Arch. 1990; 417: 114-116Crossref PubMed Scopus (26) Google Scholar), whereas in frog fibers, only Ca2+ currents are increased by PKA (29Garcia J. Gamboa-Aldeco R. Stefani E. Pflugers Arch. 1990; 417: 114-116Crossref PubMed Scopus (26) Google Scholar) or cAMP (30Arreola J. Calvo J. Garcia M.C. Sanchez J.A. J. Physiol. 1987; 393: 307-330Crossref PubMed Scopus (92) Google Scholar). Serine 687, in the intracellular II-III loop of rabbit α1S, is a major PKA phosphorylation site in vitro (31Rohrkasten A. Meyer H.E. Nastainczyk W. Sieber M. Hofmann F. J. Biol. Chem. 1988; 263: 15325-15329Abstract Full Text PDF PubMed Google Scholar). Fgα1S has no Ser or Thr at the corresponding position. In intact rabbit skeletal muscle myotubes other putative PKA sites have been located at Ser1757 and Ser1854 at the C terminus of the full-length channel (32Rotman E.I. Murphy B.J. Catterall W.A. J. Biol. Chem. 1995; 270: 16371-16377Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Fgα1S, with a shorter C terminus, has no corresponding sites. By searching the derived amino acid sequence, we located a PKA phosphorylation site motif, RRXS (33Kemp B.E. Pearson R.B. Trends Biochem. Sci. 1990; 15: 342-346Abstract Full Text PDF PubMed Scopus (807) Google Scholar), at the intracellular I-II loop of Fgα1S (Ser424). To determine the tissue specificity of the cloned molecule, we carried out Northern blot analysis. Total RNA from frog heart, liver, and skeletal muscle tissues was hybridized with a cDNA probe derived from Fgα1S, covering the 3′-untranslated region and the diverse sequence of the C terminus. Despite the differences in glyceraldehyde-3-P dehydrogenase signal between lanes, the ethidium bromide staining gel (data not shown) indicated that approximately the same amount of total RNA was loaded on each lane. The blot revealed a single RNA band in skeletal muscle (Fig. 3), consistent with the result found in mammalian skeletal muscle (34Ellis S.B. Williams M.E. Ways N.R. Brenner R. Sharp A.H. Leung A.T. Campbell K.P. McKenna E. Koch W.J. Hui A. Schwartz A. Harpold M.M. Science. 1988; 241: 1661-1664Crossref PubMed Scopus (442) Google Scholar). There were no detectable transcripts in any of the other tissues, even using longer exposure times (data not shown). tsA201 cells were chosen as a functional expression system for the cloned frog channel because HEK cells have no endogenous L-type Ca2+ currents (35Berjukow S. Doring F. Froschmayr M. Grabner M. Glossmann H. Hering S. Br. J. Pharmacol. 1996; 118: 748-754Crossref PubMed Scopus (100) Google Scholar), and large T antigen-transformed HEK cells (tsA201) express rabbit cardiac calcium channels efficiently (16Ferreira G. Yi J. Rios E. Shirokov R. J. Gen. Physiol. 1997; 109: 449-461Crossref PubMed Scopus (107) Google Scholar). In general, the functional expression of the wild-type clone was poor. Only in a few cells we obtained large, clearly extrinsic L-type Ca2+ currents. Nontransfected cells were patch clamped first as a control, and most had no detectable inward currents during depolarization. Three of 20 cells had small Ba2+ currents of 0.3 pA/pF. These currents were not sensitive to 5 μmextracellular Bay K (Table II), indicating the absence of endogenous L-type channels.Table IIExpression efficiency and current properties in cells transfected with Fgα1SDNA transfectionNo. of cellsCells with currentsIon carrierτ½I PmspA/pFNone203Ba2+0.3 ± 0.1Ba2+ + Bay K0.3 ± 0.1β2A405Ba2+0.4 ± 0.1Ba2+ + Bay K6.6 ± 0.81.1 ± 0.4β2A302Ba2+1.1 ± 1.0+α2/δBa2+ + Bay K8.0 ± 0.02.0 ± 1.3Fgα1S202Ba2+1.9 ± 1.2+β2ABa2+ + Bay K6.0 ± 1.53.0 ± 1.5Fgα1S303Ca2+3.9 ± 0.710.1 ± 4.7+β2A+ α2/δBa2+4.0 ± 0.520.2 ± 1.8One hundred forty cells were stable after whole-cell patching. The currents recorded in 15 of those were studied and included, either because they were >0.3 pA/pF (the native nontransfected cells) or because they had L-type characteristics (all others).I P is the normalized peak current, and τ½ is the time to half-activation at the maximum of the current-voltage dependence. Open table in a new tab One hundred forty cells were stable after whole-cell patching. The currents recorded in 15 of those were studied and included, either because they were >0.3 pA/pF (the native nontransfected cells) or because they had L-type characteristics (all others).I P is the normalized peak current, and τ½ is the time to half-activation at the maximum of the current-voltage dependence. Cells transfected with β2a or β2a plus α2/δ were studied in search of L-type Ca2+ channel currents induced by transfection of auxiliary subunits. Five of 40 β2a-transfected cells patched had Bay K-sensitive currents. Fig. 4 A shows Ba2+currents from one of those. Peak and tail currents were increased three times by 5 μm Bay K (Table II). Two of 30 cells cotransfected with β2a plus α2/δ had Ba2+ currents that were increased by Bay K. On average, these currents were somewhat greater than those of the β2a-transfected cells (Table II). In conclusion, extrinsic auxiliary subunits induce the expression of DHP-sensitive endogenous current of very low amplitude. Fgα1S was cotransfected into tsA201 cells in different combinations with cDNAs of other auxiliary subunits (β2a from rat brain and α2δ from rabbit skeletal muscle). The frequency of cells with L-type currents was not increased compared with those transfected with auxiliary subunits only. However, the L-type current density was greater. Two of 20 Fgα1S- plus β2a-transfected cells patched had peak Ba2+ currents at least two times larger than the maximum observed in the 40 cells transfected with β2a alone. Bay K increased the amplitude of peak and tail currents in these cells (Fig. 4 B and Table II). Substitution of Ca2+ as current carrier reduced current amplitude substantially (data not shown). Again, most of the 30 Fgα1S plus β2a plus α2/δ cells patched had no evidence of L-type currents. However, in three of them Ba2+ current density was >5-fold greater than the largest currents recorded in Fgα1S plus β2a cells. As illustrated in Fig. 4, C1 and C2, and summarized in Table II, in those cells the current decreased ∼2-fold when Ca2+ was substituted for Ba2+ as current carrier. Half-times of activation of these currents (τ½) are listed in Table II. Against our expectations activation of current in the cells transfected with Fgα1S was much faster than in frog skeletal muscle, where τ½ at 20 °C is ∼70 ms at 0 mV (36Cota G. Nicola Siri L. Stefani E. J. Physiol. 1983; 338: 395-412Crossref PubMed Scopus (27) Google Scholar). Because functional expression of Fgα1S was so infrequent, we carried out additional expression tests with a chimera of Fgα1S and rabbit α1C. In rabbit channels domain I determines the slow activation kinetics (37Tanabe T. Adams B.A. Numa S. Beam K.G. Nature. 1991; 352: 800-803Crossref PubMed Scopus (107) Google Scholar). Because the currents induced by the full Fgα1S activated rapidly, neither domain I nor any other in the frog molecule was expected to make the activation of the chimeric construct slower. Still, we chose to splice domain I of Fgα1S to rabbit α1C (to make α1C-FSDI), because a similar chimera with DI from Rbα1S (named α1C-RSDI) should have slower kinetics, thus providing overall control of the procedures. In all cases, β2a cDNA was cotransfected with the α1 constructs. The expression efficiency of α1C was ∼80%. Ca2+ and Ba2+ currents are illustrated in Fig. 5 A. For IBa, the τ½ at +30 mV was ∼5 ms. Average τ½ values are plotted versus voltage in Fig. 6.Figure 6Kinetic properties of currents in cells transfected with α1C or its chimeras. (A) activation kinetics. τ½, determined in records such as those in Fig. 5as the time to one-half of peak amplitude, averaged in seven cells for each type of molecule. Bars, ±S.E. •, FSDI; ▾, a1C; ▪, RSDI. B, inactivation kinetics. Ratio of I Ca (open symbols) orI Ba (filled symbols) at 100 ms, over peak current in the same record, calculated in records such as those in Figs. 4 (for Fgα1S, triangles) and 5 (for α1C,circles, or its frog chimera FSDI, squares) and averaged (numbers of cells listed in Table II).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Ca2+ and Ba2+ currents of a cell transfected with rabbit chimera α1C-RSDI are shown in Fig. 5 B. Average τ½ values are plotted versus voltage in Fig. 6. τ½ at +30 mV was ∼13 ms, or two times that of α1C. The currents obtained by expression of frog chimera α1C-FSDI are shown in Fig. 5 C. The expression efficiency of α1C-FSDI was as high as that of rabbit α1C, and Ca2+ and Ba2+currents had a size similar to that of" @default.
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• W2076494274 title "Molecular Cloning and Functional Expression of a Skeletal Muscle Dihydropyridine Receptor from Rana catesbeiana" @default.
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