The II-III Loop of the Skeletal Muscle Dihydropyridine Receptor Is Responsible for the Bi-directional Coupling with the Ryanodine Receptor

Abstract

The dihydropyridine receptor (DHPR) in the skeletal muscle plasmalemma functions as both voltage-gated Ca2+ channel and voltage sensor for excitation-contraction (EC) coupling. As voltage sensor, the DHPR regulates intracellular Ca2+ release via the skeletal isoform of the ryanodine receptor (RyR-1). Interaction with RyR-1 also feeds back to increase the Ca2+ current mediated by the DHPR. To identify regions of the DHPR important for receiving this signal from RyR-1, we expressed in dysgenic myotubes a chimera (SkLC) having skeletal (Sk) DHPR sequence except for a cardiac (C) II-III loop (L). Tagging with green fluorescent protein (GFP) enabled identification of expressing myotubes. Dysgenic myotubes expressing GFP-SkLC or SkLC lacked EC coupling and had very small Ca2+currents. Introducing a short skeletal segment (α1Sresidues 720–765) into the cardiac II-III loop (replacing α1C residues 851–896) of GFP-SkLC restored both EC coupling and Ca2+ current densities like those of the wild type skeletal DHPR. This 46-amino acid stretch of skeletal sequence was recently shown to be capable of transferring strong, skeletal-type EC coupling to an otherwise cardiac DHPR (Nakai, J., Tanabe, T., Konno, T., Adams, B., and Beam, K.G. (1998) J. Biol. Chem.273, 24983–24986). Thus, this segment of the skeletal II-III loop contains a motif required for both skeletal-type EC coupling and RyR-1-mediated enhancement of Ca2+ current. The dihydropyridine receptor (DHPR) in the skeletal muscle plasmalemma functions as both voltage-gated Ca2+ channel and voltage sensor for excitation-contraction (EC) coupling. As voltage sensor, the DHPR regulates intracellular Ca2+ release via the skeletal isoform of the ryanodine receptor (RyR-1). Interaction with RyR-1 also feeds back to increase the Ca2+ current mediated by the DHPR. To identify regions of the DHPR important for receiving this signal from RyR-1, we expressed in dysgenic myotubes a chimera (SkLC) having skeletal (Sk) DHPR sequence except for a cardiac (C) II-III loop (L). Tagging with green fluorescent protein (GFP) enabled identification of expressing myotubes. Dysgenic myotubes expressing GFP-SkLC or SkLC lacked EC coupling and had very small Ca2+currents. Introducing a short skeletal segment (α1Sresidues 720–765) into the cardiac II-III loop (replacing α1C residues 851–896) of GFP-SkLC restored both EC coupling and Ca2+ current densities like those of the wild type skeletal DHPR. This 46-amino acid stretch of skeletal sequence was recently shown to be capable of transferring strong, skeletal-type EC coupling to an otherwise cardiac DHPR (Nakai, J., Tanabe, T., Konno, T., Adams, B., and Beam, K.G. (1998) J. Biol. Chem.273, 24983–24986). Thus, this segment of the skeletal II-III loop contains a motif required for both skeletal-type EC coupling and RyR-1-mediated enhancement of Ca2+ current. Excitation-contraction (EC) 1The abbreviations used are: The abbreviations used are: EC, excitation-contraction; DHPR, dihydropyridine receptor; RyR-1, skeletal ryanodine receptor; GFP, green fluorescent protein; SR, sarcoplasmic reticulum; nt, nucleotide number; V, ohms; F, farads; S, siemens; C, coulombs.. coupling in skeletal muscle depends upon a functional interaction between dihydropyridine receptors (DHPRs) in the plasmalemma and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR). In skeletal muscle, the DHPR functions both as an L-type Ca2+ channel and as the voltage sensor, which in response to plasmalemmal depolarization, transmits a signal that causes RyR-1 (the skeletal RyR isoform) to release Ca2+ from the SR (1Rios E. Brum G. Nature. 1987; 325: 717-720Crossref PubMed Scopus (654) Google Scholar, 2Tanabe T. Beam K.G. Powell J.A. Numa S. Nature. 1988; 336: 134-139Crossref PubMed Scopus (584) Google Scholar, 3Takeshima H. Iino M. Takekura H. Nishi M. Kuno J. Minowa O. Takano H. Noda T. Nature. 1994; 369: 556-559Crossref PubMed Scopus (326) Google Scholar). The nature of the signal transmitted from the skeletal DHPR to RyR-1 is not yet understood, although there is strong evidence that skeletal-type EC coupling does not rely upon the entry of external Ca2+ (4Armstrong C.M. Bezanilla F.M. Horowicz P. Biochim. Biophys. Acta. 1972; 267: 605-608Crossref PubMed Scopus (301) Google Scholar). An approach to identifying regions of the skeletal DHPR that are important for EC coupling has been to express cDNAs encoding chimeric DHPRs in dysgenic myotubes, which lack endogenous skeletal DHPRs. This work has shown that a purely cardiac DHPR expressed in dysgenic myotubes results in EC coupling which is cardiac type (dependent on entry of Ca2+) (5Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 344: 451-453Crossref PubMed Scopus (194) Google Scholar), whereas skeletal-type EC coupling results from expression of a chimeric DHPR having cardiac sequence except for a skeletal II-III loop (6Tanabe T. Beam K.G. Adams B.A. Niidome T. Numa S. Nature. 1990; 346: 567-569Crossref PubMed Scopus (493) Google Scholar). More recently, it was shown that strong skeletal-type coupling could be produced by a chimeric DHPR that contained only a 46-amino acid skeletal segment within the II-III loop and weak skeletal-type coupling by a chimera containing only an 18-amino acid skeletal segment (7Nakai J. Tanabe T. Konno T. Adams B. Beam K.G. J. Biol. Chem. 1998; 273: 24893-24896Google Scholar). Analysis of myotubes from dyspedic mice, which lack RyR-1, has revealed that in addition to the orthograde (EC coupling) signal transmitted from the skeletal DHPR to RyR-1, there also appears to be a retrograde signal whereby RyR-1 increases the magnitude of the L-type Ca2+ current mediated by the DHPR. In particular, Ca2+ current density is very low in dyspedic myotubes even though the surface density of DHPRs appears to be essentially normal (8Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-75Crossref PubMed Scopus (401) Google Scholar, 9Fleig A. Takeshima H. Penner R. J. Physiol. (Lond.). 1996; 496: 339-345Crossref Scopus (29) Google Scholar). Expression of cDNA encoding RyR-1 causes the density of L-type current in dyspedic myotubes to increase toward normal (8Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-75Crossref PubMed Scopus (401) Google Scholar). However, these experiments did not reveal whether the region of the skeletal DHPR that is crucial for orthograde coupling is also important for the RyR-1-mediated enhancement of DHPR Ca2+current. Here we describe experiments to identify regions of the skeletal DHPR that are critical for the ability of the DHPR to receive the retrograde (current-enhancing) signal from RyR-1. The results demonstrate that the II-III loop is critical for both orthograde and retrograde signaling. Within the II-III loop, the 46-amino acid segment found to be important for skeletal-type EC coupling is also important for transducing the retrograde signal from RyR-1. Chimeras between the α1 subunits of the skeletal muscle DHPR (Sk (10Tanabe 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 (970) Google Scholar)) and the cardiac muscle DHPR (C (11Mikami A. Imoto K. Tanabe T. Niidome T. Mori Y. Takeshima H. Narumiya S. Numa S. Nature. 1989; 340: 230-233Crossref PubMed Scopus (770) Google Scholar)) had amino acid composition (numbers in parentheses) as follows. SkLC: Sk (1–654), C (777–927), Sk (797–1873). SkLCS46: Sk (1–654), C (777–850), Sk (720–765), C (897–927), Sk (797–1873). SkLCS18: Sk (1–654), C (777–855), Sk (725–742), C (874–927), Sk (797–1873). The chimeras were constructed and inserted into mammalian expression vectors as described below (nucleotide numbers (nt) indicated in parentheses): The EcoRI-XmnI fragment of Sk (nt 1007–1964) was coligated with the ligation product from theXmnI-HincII fragment of C (nt 2330–2782) plus the HincII-XhoI fragment of Sk (nt 2389–2654) into the corresponding EcoRI/XhoI restriction sites of a SacII-XhoI subclone of Sk (nt 86–2654) in pBluescript SK+ (Stratagene). Finally, theSacII-XhoI insert of the modified subclone (now carrying the cardiac II-III loop sequence) was ligated into the corresponding SacII/XhoI restriction sites of plasmid pCAC6, which contains the complete skeletal DHPR coding region in the mammalian expression vector pKCRH2 (6Tanabe T. Beam K.G. Adams B.A. Niidome T. Numa S. Nature. 1990; 346: 567-569Crossref PubMed Scopus (493) Google Scholar). The EcoRI-XmnI fragment of Sk (nt 1007–1964) was coligated with theXmnI-AflII* fragment (nt C2330-Sk2297) (partial cut) of clone CSk53 (7Nakai J. Tanabe T. Konno T. Adams B. Beam K.G. J. Biol. Chem. 1998; 273: 24893-24896Google Scholar) which is C (1–850), Sk (720–765), C (897–2171) into the corresponding EcoRI/AflII restriction sites (nt Sk1007/C2690) of the modified (the cardiac II-III loop carrying) SacII-XhoI subclone of Sk (nt 86–2654) in pBluescript SK+. To yield SkLCS46, theSacII-XhoI insert was ligated into the corresponding SacII/XhoI restriction sites of plasmid pCAC6 (see above). The asterisks here and below indicate restriction sites generated by polymerase chain reaction. The EcoRI-XmnI fragment of Sk (nt 1007–1964) was coligated with theXmnI-AflII fragment (nt C2330-C2690) of clone CSk58 (7Nakai J. Tanabe T. Konno T. Adams B. Beam K.G. J. Biol. Chem. 1998; 273: 24893-24896Google Scholar), which is C (1–855), Sk (725–742), C (874–2171), into the corresponding EcoRI/AflII restriction sites (nt Sk1007/C2690) of the modified (with the cardiac II-III loop inserted)SacII-XhoI subclone of Sk (nt 86–2654) in pBluescript SK+. Finally, the SacII-XhoI insert was ligated into the corresponding SacII/XhoI restriction sites of plasmid pCAC6 (see above) to yield SkLCS18. The coding sequence of the α1 subunit of the skeletal muscle DHPR (10Tanabe 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 (970) Google Scholar) was inserted in-frame and downstream of the coding region of a modified green fluorescence protein (GFP), cloned in a proprietary mammalian expression vector (kindly provided by P. Seeburg) as described in detail elsewhere (12Grabner M. Dirksen R.T. Beam K.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1903-1908Crossref PubMed Scopus (136) Google Scholar). The SalI*-EcoRI fragment of GFP-α1S (nt 5′ polylinker-Sk1007) was coligated with theEcoRI-BglII of SkLCS46, and (nt into the corresponding restriction sites of plasmid The Ca2+ channel subunit cDNA (kindly provided by K. was cloned as a fragment and into the sites of the mammalian expression vector The of the chimeric DHPRs was by sequence an of myotubes from dysgenic were as described B.A. Beam K.G. J. Physiol. 1989; PubMed Scopus Google Scholar). myotubes were T. Beam K.G. Powell J.A. Numa S. Nature. 1988; 336: 134-139Crossref PubMed Scopus (584) Google Scholar) into a with of expression carrying cDNAs for or myotubes were for the of green were for T. Beam K.G. Powell J.A. Numa S. Nature. 1988; 336: 134-139Crossref PubMed Scopus (584) Google Scholar) in response to V, Ca2+ B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google Scholar), and channel for In a of experiments the of the subunit for Ca2+ channel dysgenic myotubes were with GFP-SkLC cDNA and mammalian expression dyspedic myotubes were in as described for dysgenic myotubes B.A. Beam K.G. J. Physiol. 1989; PubMed Scopus Google Scholar) and T. Beam K.G. Powell J.A. Numa S. Nature. 1988; 336: 134-139Crossref PubMed Scopus (584) Google Scholar) with mammalian expression plasmid with vector to the identification of expressing Ca2+ were the A. E. B. J. Physiol. PubMed Scopus Google Scholar). The had of with an containing and with The composition of the external was and with were by a to to endogenous B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google Scholar). were for of and by and the of by voltage from the of Ca2+ were by in the of Ca2+ and were to the external to the of The for and densities and the used was described in detail elsewhere B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google Scholar, B.A. Mori Y. Tanabe T. Beam K.G. J. Physiol. 1994; PubMed Scopus Google Scholar). To the of on Ca2+ and Ca2+ were J. Beam K.G. J. Physiol. 1994; PubMed Scopus Google Scholar) in normal myotubes with the external described for Ca2+ and containing an of with or with the of Ca2+ in dysgenic myotubes expressing chimeric the contained with and the external was with the of Ca2+ it was not to the that had fluorescence and to those of Thus, cDNAs coding for SkLCS46, and were inserted into the expression plasmid pKCRH2 M. T. T. Y. Noda M. T. M. J. Takahashi T. Kuno M. Numa S. Nature. PubMed Scopus Google Scholar) and were with cDNA encoding the subunit of the surface Y. G. 1994; Google Scholar). expressing the were with as described J. J. Imoto K. Beam K.G. Biophys. J. PubMed Scopus Google Scholar). in fluorescence were by the fluorescence The of of was by a segment to the of the were and are as dysgenic myotubes on were with a normal and with and a The was on the of a were a with a and the was with a for were with a of between was were the work that the cardiac DHPR expressed in dysgenic myotubes was to orthograde skeletal EC However, a chimera with cardiac sequence except for a skeletal II-III loop (5Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 344: 451-453Crossref PubMed Scopus (194) Google Scholar)) could orthograde signaling. More recently, it was found that the expression of RyR-1 in dyspedic myotubes the of L-type Ca2+ current produced by the skeletal DHPR (8Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-75Crossref PubMed Scopus (401) Google Scholar). To whether the II-III loop an important in this signal from RyR-1, we constructed a chimeric DHPR (SkLC) that was the of a skeletal DHPR except for a cardiac II-III were in dysgenic myotubes that had been with with the that the chimera was or that a cardiac II-III loop orthograde signaling. could not be used to identify myotubes expressing we constructed a cDNA that to the amino of SkLC to identification by of in of this were shown not to the of muscle or Ca2+ (12Grabner M. Dirksen R.T. Beam K.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1903-1908Crossref PubMed Scopus (136) Google Scholar). expressing GFP-SkLC which were in those in myotubes To between were B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google Scholar) to yield a of The for GFP-SkLC was for This for GFP-SkLC did not to be a of a of DHPRs expressed in the surface for were for GFP-SkLC and GFP-α1S and The of to the endogenous in dysgenic B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google Scholar) for GFP-SkLC was that for GFP-α1S Thus, it appears that the of a cardiac II-III loop GFP-SkLC from receiving the signal from RyR-1. the for GFP-SkLC was very to the found for dyspedic myotubes (8Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-75Crossref PubMed Scopus (401) Google Scholar), which lack from dysgenic myotubes expressing GFP-α1S GFP-SkLC and Ca2+ were by from a of to the indicated on the were by and are expressed as were in response to a to a B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google Scholar). of were from the myotubes as shown Ca2+ with a containing and The (C) for was as a and C and C and C and in dyspedic myotubes and in dysgenic myotubes expressing and the GFP-SkLC clone are as with the numbers in the of myotubes the Ca2+ were by the to the B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google current for of of were as described B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google Scholar) and were V, which the has is the and the endogenous found in dysgenic myotubes B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google the the was indicate by an indicate whereas for dyspedic myotubes and for dysgenic myotubes were for and were (8Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-75Crossref PubMed Scopus (401) Google Scholar, B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google Scholar). in a are as with the numbers in the of myotubes the Ca2+ were by the to the B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google current for of of were as described B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google Scholar) and were V, which the has is the and the endogenous found in dysgenic myotubes B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google the the was indicate by an indicate whereas for dyspedic myotubes and for dysgenic myotubes were for and were (8Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-75Crossref PubMed Scopus (401) Google Scholar, B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google Scholar). (7Nakai J. Tanabe T. Konno T. Adams B. Beam K.G. J. Biol. Chem. 1998; 273: 24893-24896Google Scholar) that of a 46-amino acid segment of skeletal sequence into the II-III loop of an otherwise cardiac DHPR produced a chimera residues 720–765) capable of strong, skeletal-type EC coupling upon expression in dysgenic myotubes. To whether this motif is also to of the Ca2+ signal from RyR-1, we this segment into the cardiac II-III loop of the otherwise skeletal chimera The not only mediated skeletal-type EC coupling of of the fluorescent in not also produced Ca2+ current densities with like those of GFP-α1S or (7Nakai J. Tanabe T. Konno T. Adams B. Beam K.G. J. Biol. Chem. 1998; 273: 24893-24896Google Scholar) also that an otherwise cardiac chimera containing an even skeletal segment residues was to skeletal-type EC this coupling was weak (7Nakai J. Tanabe T. Konno T. Adams B. Beam K.G. J. Biol. Chem. 1998; 273: 24893-24896Google Scholar). To this 18-amino acid segment of the signal from RyR-1, we constructed chimera a and The for was not from that of GFP-SkLC for was to that found for GFP-SkLC expressed in dysgenic myotubes or that of endogenous in dyspedic myotubes that lack RyR-1 the DHPR sequence that strong enhancement of Ca2+ current by RyR-1 is or the skeletal segment in the II-III loop of However, this sequence is contained within the skeletal segment of the The a of the of retrograde coupling enhancement from To a of the of orthograde (EC) coupling to RyR-1, we Ca2+ Ca2+ were with SkLC of were for of The the that skeletal-type EC coupling were of magnitude for to Ca2+ current is and Ca2+ current is small as a of the was also to skeletal-type coupling the Ca2+ were and However, the of increase of the signal was only for which is the of for Thus, skeletal-type coupling is for for is to the enhancement of Ca2+ current that been for to that for the that there is a between the of retrograde and orthograde signaling. The enhancement of current for be as and are the and the from the enhancement of current for was the enhancement of current for like orthograde for (see it yield a enhancement of only a small to been As described the chimeric DHPR to the Ca2+ signal from RyR-1 were the as those to the EC coupling signal to RyR-1. Thus, it that Ca2+ from RyR-1 response to the EC coupling the the enhancement of Ca2+ current. As a of this we of Ca2+ and Ca2+ in normal myotubes of these contained the we used for Ca2+ plus to Ca2+ into the The contained Ca2+ and lacked as to Ca2+ into the To we only small myotubes with and used low to the the low entry into Ca2+ and Ca2+ by the indicated into a normal with or results were for a of with and with in the Ca2+ release was to a increase in the fluorescence of the that both and the fluorescence between and with that this was into the both and it that was for between the and the in the to a increase in and the fluorescence very Ca2+ was that of the the in the The of fluorescence increases with that there was of Ca2+ from the SR Ca2+ had been the with an of the of Ca2+ in myotubes Ca2+ release sites were As is in a and Ca2+ current as a of into the with or In the in these experiments (with small and low was that the we used for of Ca2+ to the with the the of Ca2+ and the of However, Ca2+ were Ca2+ were it that Ca2+ release the critical signal whereby RyR-1 Ca2+current. of skeletal DHPRs in normal myotubes were shown to in that with RyR S. A. Powell J.A. J. Biol. PubMed Scopus Google Scholar). also in dysgenic myotubes expressing DHPRs (12Grabner M. Dirksen R.T. Beam K.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1903-1908Crossref PubMed Scopus (136) Google Scholar). To whether the of orthograde or retrograde by GFP-SkLC a and was a of to with we used to whether or not were in dysgenic myotubes expressing this chimeric As shown in were in dysgenic myotubes. The of of these does not from that of DHPRs (12Grabner M. Dirksen R.T. Beam K.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1903-1908Crossref PubMed Scopus (136) Google Scholar), which are capable of with the of the found that the II-III loop of the skeletal DHPR with the corresponding region of the cardiac DHPR causes the of This skeletal DHPR with a cardiac II-III loop (SkLC) the orthograde (EC coupling) signal to the skeletal ryanodine receptor receive the retrograde from RyR-1. of a 46-amino acid segment of skeletal sequence into the cardiac loop of SkLC both orthograde and retrograde signaling. Ca2+ are of small in skeletal muscle RyR-1, we in an that the small Ca2+ expression of skeletal DHPRs in be a of the of RyR-1 in these (8Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-75Crossref PubMed Scopus (401) Google Scholar). it was shown that Ca2+ could be produced with skeletal DHPRs expressed in the was used of J. Biol. Chem. PubMed Scopus Google Scholar), the isoform in skeletal muscle M. Flockerzi V. 1994; PubMed Scopus Google Scholar). This work did not whether the subunit expression of DHPRs in the plasmalemma or the current the of plasmalemmal DHPRs we shown is the for enhancement of current by experiments that for DHPRs in normal expression of does not the of interaction with RyR-1. In particular, expression of in dyspedic myotubes of with GFP-SkLC in dysgenic myotubes that were from the corresponding of and The of orthograde and retrograde between the skeletal DHPR and RyR-1 to be of retrograde is that the Ca2+ EC coupling feeds back the DHPR to current. This is with the that those chimeras that did not enhancement of current were those that also lacked (SkLC) or had only weak EC coupling. B. P. J. Physiol. PubMed Scopus Google Scholar) evidence that Ca2+ release the Ca2+ current in from skeletal muscle, the that of the to or the complete of current. the the of Ca2+ current and of B. P. J. Physiol. PubMed Scopus Google Scholar) to a of the that there been of the or a of the ability of the DHPR to the of these of not which that functional coupling of the DHPR to RyR-1 is with the of Ca2+ to in to the results on skeletal muscle B. P. J. Physiol. PubMed Scopus Google Scholar), experiments that Ca2+ were in myotubes in which Ca2+ release sites been by with on Ca2+ also been for of myotubes with or J. Physiol. PubMed Google Scholar). from work on DHPR chimeras B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google Scholar) also an of Ca2+ release in enhancement of current. In that it was found for (the cardiac and for (the cardiac DHPR with a skeletal II-III Thus, it appears that the of a skeletal II-III loop the current via a not on Ca2+ both and Ca2+ release the used for of Ca2+ (7Nakai J. Tanabe T. Konno T. Adams B. Beam K.G. J. Biol. Chem. 1998; 273: 24893-24896Google Scholar). from chimeras that Ca2+ via skeletal-type EC coupling is not required for enhancement of current. In particular, expression in dyspedic myotubes of the chimera produced enhancement of Ca2+ current not of skeletal-type EC coupling J. Allen P.D. Beam K.G. J. Biol. Chem. 1998; 273: PubMed Scopus Google Scholar). Finally, experiments that are in dyspedic myotubes expression of a ryanodine which response to Allen P.D. Beam K.G. Biophys. J. Scholar). An to the that the release of Ca2+ from RyR-1 causes enhancement of current is to that are a in which EC coupling of a signal from the skeletal DHPR to RyR-1 via the II-III loop, and enhancement of current of a retrograde signal from RyR-1 to the DHPR, via the II-III loop protein coupling between the II-III loop and RyR-1 is The nature of both the orthograde and retrograde the retrograde signal to a of the However, in the interaction of RyR-1 with the II-III loop the DHPR in a which increases channel current with the of the DHPR found in the of this interaction cardiac II-III skeletal-type EC coupling and the enhancement of current are restored by of a small segment of the skeletal II-III loop The of the II-III loop is by and of EC coupling) was shown with in which the skeletal II-III loop was into the cardiac DHPR (5Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 344: 451-453Crossref PubMed Scopus (194) Google Scholar). As these experiments also a of of for for B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: PubMed Scopus Google Scholar). The experiments here demonstrate a of both functions EC enhancement of the cardiac II-III loop is into the skeletal DHPR and a of both functions with a like the in the that orthograde and retrograde coupling are weak for is that the of DHPRs and are not in of the required However, it very that into sites the plasmalemma with regions of the SR, even SkLC into a of of DHPRs and the does not evidence for between DHPRs and in skeletal muscle has been provided by This has shown that skeletal DHPRs to be in to be of which is in with of the subunits of a B.A. A. J. Biol. 1988; PubMed Scopus Google Scholar). cardiac DHPRs to be not in are not in cardiac muscle Takahashi M. Takeshima H. J. Biol. PubMed Scopus Google Scholar). Thus, it be important to to whether or not are upon expression of SkLCS46, and SkLCS18. very are for it that the weak coupling for this was a of of with RyR-1. is for SkLCS46, it that of a channel of a RyR-1 of P. for the of an of the expression K. for the P. Allen for dyspedic mice, and for