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• W2054539671 abstract "Cav1.4 channels are unique among the high voltage-activated Ca2+ channel family because they completely lack Ca2+-dependent inactivation and display very slow voltage-dependent inactivation. Both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained Ca2+ influx through Cav1.4 channels is required to couple slow graded changes of the membrane potential with tonic glutamate release. Loss of Cav1.4 function causes severe impairment of retinal circuitry function and has been linked to night blindness in humans and mice. Recently, an inhibitory domain (ICDI: inhibitor of Ca2+-dependent inactivation) in the C-terminal tail of Cav1.4 has been discovered that eliminates Ca2+-dependent inactivation by binding to upstream regulatory motifs within the proximal C terminus. The mechanism underlying the action of ICDI is unclear. It was proposed that ICDI competitively displaces the Ca2+ sensor calmodulin. Alternatively, the ICDI domain and calmodulin may bind to different portions of the C terminus and act independently of each other. In the present study, we used fluorescence resonance energy transfer experiments with genetically engineered cyan fluorescent protein variants to address this issue. Our data indicate that calmodulin is preassociated with the C terminus of Cav1.4 but may be tethered in a different steric orientation as compared with other Ca2+ channels. We also find that calmodulin is important for Cav1.4 function because it increases current density and slows down voltage-dependent inactivation. Our data show that the ICDI domain selectively abolishes Ca2+-dependent inactivation, whereas it does not interfere with other calmodulin effects. Cav1.4 channels are unique among the high voltage-activated Ca2+ channel family because they completely lack Ca2+-dependent inactivation and display very slow voltage-dependent inactivation. Both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained Ca2+ influx through Cav1.4 channels is required to couple slow graded changes of the membrane potential with tonic glutamate release. Loss of Cav1.4 function causes severe impairment of retinal circuitry function and has been linked to night blindness in humans and mice. Recently, an inhibitory domain (ICDI: inhibitor of Ca2+-dependent inactivation) in the C-terminal tail of Cav1.4 has been discovered that eliminates Ca2+-dependent inactivation by binding to upstream regulatory motifs within the proximal C terminus. The mechanism underlying the action of ICDI is unclear. It was proposed that ICDI competitively displaces the Ca2+ sensor calmodulin. Alternatively, the ICDI domain and calmodulin may bind to different portions of the C terminus and act independently of each other. In the present study, we used fluorescence resonance energy transfer experiments with genetically engineered cyan fluorescent protein variants to address this issue. Our data indicate that calmodulin is preassociated with the C terminus of Cav1.4 but may be tethered in a different steric orientation as compared with other Ca2+ channels. We also find that calmodulin is important for Cav1.4 function because it increases current density and slows down voltage-dependent inactivation. Our data show that the ICDI domain selectively abolishes Ca2+-dependent inactivation, whereas it does not interfere with other calmodulin effects. Retinal photoreceptors and bipolar cells contain a highly specialized type of synapse designated ribbon synapses. Glutamate release in these synapses is controlled via graded and sustained changes in membrane potential that are maintained throughout the duration of a light stimulus (1Thoreson W.B. Mol. Neurobiol. 2007; 36: 205-223Crossref PubMed Scopus (55) Google Scholar, 2Juusola M. French A.S. Uusitalo R.O. Weckström M. Trends Neurosci. 1996; 19: 292-297Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In recent years, it became clear that Cav1.4 L-type Ca2+ channels are the main channel subtype converting these analog input signals into corresponding permanent glutamate release (1Thoreson W.B. Mol. Neurobiol. 2007; 36: 205-223Crossref PubMed Scopus (55) Google Scholar, 3Mansergh F. Orton N.C. Vessey J.P. Lalonde M.R. Stell W.K. Tremblay F. Barnes S. Rancourt D.E. Bech-Hansen N.T. Hum. Mol. Genet. 2005; 14: 3035-3046Crossref PubMed Scopus (207) Google Scholar, 4Haeseleer F. Imanishi Y. Maeda T. Possin D.E. Maeda A. Lee A. Rieke F. Palczewski K. Nat. Neurosci. 2004; 7: 1079-1087Crossref PubMed Scopus (228) Google Scholar, 5Barnes S. Kelly M.E. Adv. Exp. Med. Biol. 2002; 514: 465-476Crossref PubMed Scopus (75) Google Scholar). In support of this mechanism, mutations in the Cav1.4 gene have been identified in patients suffering from congenital stationary night blindness type 2 and X-linked cone rod dystrophy (6Bech-Hansen N.T. Naylor M.J. Maybaum T.A. Pearce W.G. Koop B. Fishman G.A. Mets M. Musarella M.A. Boycott K.M. Nat. Genet. 1998; 19: 264-267Crossref PubMed Scopus (429) Google Scholar, 7Strom T.M. Nyakatura G. Apfelstedt-Sylla E. Hellebrand H. Lorenz B. Weber B.H. Wutz K. Gutwillinger N. Rüther K. Drescher B. Sauer C. Zrenner E. Meitinger T. Rosenthal A. Meindl A. Nat. Genet. 1998; 19: 260-263Crossref PubMed Scopus (395) Google Scholar, 8Doering C.J. Peloquin J.B. McRory J.E. Channels (Austin). 2007; 1: 3-10Crossref PubMed Scopus (23) Google Scholar). Individuals displaying congenital stationary night blindness type 2 as well as mice deficient in Cav1.4 typically have abnormal electroretinograms that indicate a loss of neurotransmission from the rods to second order bipolar cells, which is attributable to a loss of Cav1.4 (3Mansergh F. Orton N.C. Vessey J.P. Lalonde M.R. Stell W.K. Tremblay F. Barnes S. Rancourt D.E. Bech-Hansen N.T. Hum. Mol. Genet. 2005; 14: 3035-3046Crossref PubMed Scopus (207) Google Scholar). Retinal Cav1.4 channels are set apart from other high voltage-activated (HVA) 3The abbreviations used are: HVAhigh voltage-activatedCDICa2+-dependent inactivationVDIvoltage-dependent inactivationICDIinhibitor of CDICaMcalmodulinFRETfluorescence resonance energy transferFRFRET ratioYPFyellow fluorescent proteinCFPcyan fluorescent proteinpFpicofarads. Ca2+ channels by their total lack of Ca2+-dependent inactivation (CDI) and their very slow voltage-dependent inactivation (VDI). Recently, we and others discovered an inhibitory domain (ICDI: inhibitor of CDI) in the C-terminal tail of the Cav1.4 channel that eliminates Ca2+-dependent inactivation in this channel by binding to upstream regulatory motifs (9Wahl-Schott C. Baumann L. Cuny H. Eckert C. Griessmeier K. Biel M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 15657-15662Crossref PubMed Scopus (73) Google Scholar, 10Singh A. Hamedinger D. Hoda J.C. Gebhart M. Koschak A. Romanin C. Striessnig J. Nat. Neurosci. 2006; 9: 1108-1116Crossref PubMed Scopus (109) Google Scholar). Importantly, introducing the ICDI into the backbone of Cav1.2 or Cav1.3 almost completely abolishes the CDI of these channels. Contrasting with the clear cut function, the underlying mechanism by which ICDI abolishes CDI remains controversial. It was suggested that ICDI displaces the Ca2+ sensor calmodulin (CaM) from binding to the proximal C terminus (10Singh A. Hamedinger D. Hoda J.C. Gebhart M. Koschak A. Romanin C. Striessnig J. Nat. Neurosci. 2006; 9: 1108-1116Crossref PubMed Scopus (109) Google Scholar), suggesting that the binding sites of CaM and ICDI are largely overlapping or allosterically coupled to each other. Alternatively, our own data rather suggested that CaM and the ICDI domain bind to different portions of the proximal C terminus (9Wahl-Schott C. Baumann L. Cuny H. Eckert C. Griessmeier K. Biel M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 15657-15662Crossref PubMed Scopus (73) Google Scholar). We proposed that the interaction between the ICDI domain and the EF-hand, a motif with a central role for transducing CDI (11Mori M.X. Vander Kooi C.W. Leahy D.J. Yue D.T. Structure. 2008; 16: 607-620Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 12Zühlke R.D. Reuter H. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 3287-3294Crossref PubMed Scopus (160) Google Scholar, 13Pitt G.S. Zühlke R.D. Hudmon A. Schulman H. Reuter H. Tsien R.W. J. Biol. Chem. 2001; 276: 30794-30802Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 14Bernatchez G. Talwar D. Parent L. Biophys. J. 1998; 75: 1727-1739Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 15Peterson B.Z. Lee J.S. Mulle J.G. Wang Y. de Leon M. Yue D.T. Biophys. J. 2000; 78: 1906-1920Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 16Kim J. Ghosh S. Nunziato D.A. Pitt G.S. Neuron. 2004; 41: 745-754Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), switches off CDI without impairing binding of CaM to the channel. In this study, we designed experiments to differentiate between these two models. Here, using FRET in HEK293 cells, we provide evidence that in living cells, CaM is bound to the full-length C terminus of Cav1.4 (i.e. in the presence of ICDI). Furthermore, our data suggest that the steric orientation of the CaM/Cav channel complex differs between Cav1.2 and Cav1.4 channels. We show that CaM preassociation with Cav1.4 controls current density and also affects VDI. Thus, although CaM does not trigger CDI in Cav1.4 as it does in other HVA Ca2+ channels, it is still an important regulator of this channel. high voltage-activated Ca2+-dependent inactivation voltage-dependent inactivation inhibitor of CDI calmodulin fluorescence resonance energy transfer FRET ratio yellow fluorescent protein cyan fluorescent protein picofarads. For expression of murine Cav1.4 (17Baumann L. Gerstner A. Zong X. Biel M. Wahl-Schott C. Invest. Ophthalmol. Vis. Sci. 2004; 45: 708-713Crossref PubMed Scopus (100) Google Scholar) (accession number AJ579852), the bicistronic pIRES2-EGFP expression vector (Clontech) was used. For the construction of truncated Cav1.4 channels lacking the ICDI (Cav1.4ΔICDI, previously termed C1884Stop (9Wahl-Schott C. Baumann L. Cuny H. Eckert C. Griessmeier K. Biel M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 15657-15662Crossref PubMed Scopus (73) Google Scholar)), a fragment of the wild-type Cav1.4 expression plasmid was replaced by DNA fragments that are carrying the required stop codon and a restriction site introduced by 3′ primers. In Cav1.4/5A channels, CaM preassociation is disrupted by mutating Ile-1592–Phe-1596 of the IQ motif to alanines, respectively. The mutated sequence (IQDYF) contains isoleucine 1592, glutamine 1593, and two highly conserved aromatic anchors (Tyr-1595 and Phe-1596) that in closely related Cav1.2 channels have been shown to form extensive contacts with CaM (19Van Petegem F. Chatelain F.C. Minor Jr., D.L. Nat. Struct. Mol. Biol. 2005; 12: 1108-1115Crossref PubMed Scopus (201) Google Scholar, 20Fallon J.L. Halling D.B. Hamilton S.L. Quiocho F.A. Structure. 2005; 13: 1881-1886Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). HEK293 cells were transiently transfected with expression vectors encoding calcium channel α subunits together with equimolar amounts of vectors encoding β2a and α2δ1 as described in Ref. 17Baumann L. Gerstner A. Zong X. Biel M. Wahl-Schott C. Invest. Ophthalmol. Vis. Sci. 2004; 45: 708-713Crossref PubMed Scopus (100) Google Scholar. ICa and IBa were measured by using the following solutions. For the pipette solution, we used 112 mm CsCl, 3 mm MgCl2, 3 mm MgATP, 10 mm EGTA, 5 mm HEPES, adjusted to pH 7.4 with CsOH; for experiments with low Ca2+ buffering, EGTA was reduced to 0.5 mm. For the bath solution, we used 82 mm NaCl, 30 mm BaCl2, 5.4 mm CsCl2, 1 mm MgCl2, 20 mm tetraethylammonium, 5 mm Hepes, 10 mm glucose, adjusted to pH 7.4 with NaOH. For experiments with 10 mm Ba2+ or 10 mm Ca2+ in the bath solution, the NaCl concentration was increased to 102 mm. ICa and IBa were measured from the same cell. Bath solution was changed by a local solution exchanger. Currents were recorded at room temperature 2–4 days after transfection by using the whole-cell patch clamp technique. Data were analyzed by using the Origin 6.1 software (OriginLab, Northampton, MA). The peak IV relationship was measured by applying 350-ms or 5-s voltage pulses to potentials between −80 and +70 mV in 10-mV increments from a holding potential of −80 mV at 0.2 Hz. To obtain current densities, the current amplitude at Vmax was normalized to cell membrane capacitance (Cm). For determination of half-maximum activation voltage (V0.5), the chord conductance (G) was calculated from the current voltage curves by dividing the peak current amplitude by its driving force at that respective potential G = I/(Vm − Vrev), where Vrev is the extrapolated reversal potential, Vm is the membrane potential, and I is the peak current. The chord conductance was then fitted with a Boltzmann equation G = Gmax/(1 + e(V0.5−Vm)/kact), where Gmax is the maximum conductance, V0.5 is the half-maximum activation voltage, Vm is the membrane potential, and kact is the slope factor of the activation curve. Cav1.4 channel inactivation was quantified by calculating the fraction of peak Ba2+ and Ca2+ currents remaining after 300 or 5000 ms of depolarization (R300 or R5000) as described (9Wahl-Schott C. Baumann L. Cuny H. Eckert C. Griessmeier K. Biel M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 15657-15662Crossref PubMed Scopus (73) Google Scholar). R300 is used to quantify CDI (fast process). R5000 is used to quantify VDI, a process that is intrinsically very slow in Cav1.4. For all FRET constructs, we used monomeric EYFP(A206K) or ECFP(A206K) (21Zacharias D.A. Violin J.D. Newton A.C. Tsien R.Y. Science. 2002; 296: 913-916Crossref PubMed Scopus (1791) Google Scholar, 22Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4946) Google Scholar) to avoid homo- and heterodimerization. For simplicity, we refer to the different fluorescent proteins as “YFP” and “CFP” throughout the text. All FRET constructs were cloned into the pcDNA3 expression vector (Invitrogen). For CaM interaction assays, the following YFP-tagged C-terminal fragments of Cav1.4 and Cav1.2 (rabbit Cav1.2b subunit (23Biel M. Ruth P. Bosse E. Hullin R. Stühmer W. Flockerzi V. Hofmann F. FEBS Lett. 1990; 269: 409-412Crossref PubMed Scopus (195) Google Scholar); accession number X55763) were used: YFP-CT1.4 (Asp-1445–Leu-1984), YFP-CT1.4ΔICDI (Asp-1445–Thr-1883), YFP-CT1.4R1610Stop (Asp-1445–Gly-1609), and YFP-CT1.2 (Asp-1500–Leu-2166). In all CaM fusion constructs, CFP is fused N-terminally to CaM. In CFP-CaM, CFP is linked to CaM by an AAA linker (24Erickson M.G. Liang H. Mori M.X. Yue D.T. Neuron. 2003; 39: 97-107Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 25Liang H. DeMaria C.D. Erickson M.G. Mori M.X. Alseikhan B.A. Yue D.T. Neuron. 2003; 39: 951-960Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). In CFP227-CaM, CFP has been truncated after residue Ala-227 and linked to CaM by a CGC linker (26Miyawaki A. Llopis J. Heim R. McCaffery J.M. Adams J.A. Ikura M. Tsien R.Y. Nature. 1997; 388: 882-887Crossref PubMed Scopus (2620) Google Scholar). We intentionally limited expression of CFP-CaM and CFP227-CaM by mutating the Kozac sequence from GCC GCC ACC ATG to GCC TCC TTT ATG in a subset of control experiments to avoid spurious FRET (27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 28Erickson M.G. Moon D.L. Yue D.T. Biophys. J. 2003; 85: 599-611Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Cloning of CFPcp174 and CFPcp158 is described in Ref. 29Mank M. Reiff D.F. Heim N. Friedrich M.W. Borst A. Griesbeck O. Biophys. J. 2006; 90: 1790-1796Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar. Briefly, the N and the C terminus of CFP are fused by a GGTGGS linker, and the new N and C termini were created as indicated in Fig. 1A. These permutations show similar spectral properties as the non-permutated fluorescent proteins (29Mank M. Reiff D.F. Heim N. Friedrich M.W. Borst A. Griesbeck O. Biophys. J. 2006; 90: 1790-1796Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). A Ca2+-insensitive CaM mutant harboring aspartate-to-alanine mutations in all four EF-hands (CaM1234) (30Xia X.M. Fakler B. Rivard A. Wayman G. Johnson-Pais T. Keen J.E. Ishii T. Hirschberg B. Bond C.T. Lutsenko S. Maylie J. Adelman J.P. Nature. 1998; 395: 503-507Crossref PubMed Scopus (734) Google Scholar) was fused by an AAA linker to CFP or without a linker to CFPCp174. As negative control, CFP(A206K) and YFP(A206K) were used. For intramolecular FRET experiments, C-terminal fragments of Cav1.4 were N-terminally fused to YFP and C-terminally fused to CFP separated by a triple alanine linker. The fragments used for the respective fusion construct are as follows: YFP-CT1.4-CFP, complete C terminus of Cav1.4 (Asp-1445–Leu-1984); YFP-CT1.4Δ1493–1609-CFP, Asp-1445–Leu-1984 with a deletion of Gln-1493–Gly-1609; YFP-CT1.4Δ1445–1609-CFP, Asp-1445–Leu-1984 with a deletion of Asp-1445–Gly-1609. For the ICDI interaction assays, YFP is fused to C-terminal fragments of Cav1.4. The C-terminal fragments for the respective constructs are as follows: YFP-CT1.4, complete C terminus (Asp-1445–Leu-1984); YFP-CT1.4ΔICDI, Asp-1445–Thr-1883; YFP-CT1.4R1610Stop, Asp-1445–Gly-1609; YFP-EF, Asp-1445–Ile-1492. Fragments of the ICDI domain C-terminally fused to CFP are as follows: ICDI-CFP, Leu-1885–Leu-1984; the proximal part of the ICDI proxICDI-CFP, Leu-1885–Lys-1929; the middle part of the ICDI midICDI-CFP, Gln-1930–Ala-1952; the distal part of the ICDI distICDI-CFP, Gln-1953–Leu-1984; XL-ICDI-CFP, Arg-1610–Leu-1984; L-ICDI-CFP, Ile-1742–Leu-1984. HEK293 cells were grown on coverslips (ibiTreat, ibidi, Martinsried, Germany) and transiently transfected using FuGENE 6 (Roche Diagnostics, Mannheim, Germany). 1–2 days later, the cells were washed and maintained in buffer solution composed of 140 mm NaCl, 5 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 10 mm glucose, 10 mm Na-HEPES, pH 7,4 at room temperature. Cells were placed on an inverted epifluorescence microscope equipped with an oil immersion 60× objective (UPlanSApo 60× OL/1.35, Olympus, Tokyo, Japan) and a built-in dual-emission system (iMIC 2010 FRET module; TILL Photonics). For simultaneous recording of CFP and YFP emission, a multiband FRET filter set (CFP/YFP-A-000, Semrock, NY) was used consisting of a dual-band excitation filter (excitation bands: 416 ± 12.5 and 501 ± 9 nm), emission filter (emission bands: 464 ± 11.5 and 547 ± 15.5 nm), and dichroic beam splitter (reflection bands: 405 ± 22 and 502 ± 9 nm; transmission bands: 466 ± 17 and 549.5 ± 19.5 nm). Samples were excited with light from a Polychrome 5000 (TILL Photonics; center wave length ± 7.5 nm). The illumination time was set to 10 ms. For the single channels (CFP, YFP), mean intensity values derived from a selective background region near the investigated cell were used for background correction. After this correction, mean values for each cell were calculated from a region of interest drawn around a cell of interest. Images were recorded by a CCD camera (IMAGO-QE). The setup was controlled with the software TILLvisION (version 4.0) and a stand-alone DSP controller. TILLvisION was also used for the image analysis. All imaging equipment was supplied by TILL Photonics (part of Agilent Technologies) unless noted otherwise. Measurements of single-cell FRET based on aggregate (nonspatial) fluorescence recordings were performed using three-cube FRET as described previously (24Erickson M.G. Liang H. Mori M.X. Yue D.T. Neuron. 2003; 39: 97-107Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). The notation and abbreviations used follow the definition of Refs. 24Erickson M.G. Liang H. Mori M.X. Yue D.T. Neuron. 2003; 39: 97-107Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar and 27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar. The degree of FRET in an individual cell was quantified using the FRET ratio (FR), which is defined as the fractional increase in YFP emission caused by FRET. The FR was calculated using FR = [SFRET − (RD1)(SCFP)]/[(RA1)(SYFP − (RD2)(SCFP))]. Fluorescence measurements for the determination of SFRET, SCFP, and SYFP were performed in cells coexpressing CFP-tagged and YFP-tagged peptides or intramolecular FRET constructs dually labeled by CFP and YFP using the following parameters: SCFP, excitation at 416 ± 7.5 nm and emission at 464 ± 11.5 nm (donor excitation; donor emission); SFRET, excitation at 416 ± 7.5 nm and emission at 547 ± 15.5 nm (donor excitation; acceptor emission); and SYFP, excitation at 501 ± 7.5 nm and emission at 547 ± 15.5 nm (acceptor excitation; acceptor emission). RD1, RA1, and RD2 are experimentally predetermined constants from measurements applied to single cells expressing only CFP- or YFP-tagged molecules. These constants are used to correct for bleed-through of CFP into the YFP channel (RD1), direct excitation of YFP by CFP excitation (RA1), and the small amount of CFP excitation at the YFP excitation wavelength (RD2) as described by Ref. 24Erickson M.G. Liang H. Mori M.X. Yue D.T. Neuron. 2003; 39: 97-107Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar. Kinetic measurements were performed using the setup described above and a 20× objective (UPlanSApo, N.A. 0.75, Olympus, Tokyo, Japan). Cells were maintained in buffer solution composed of 140 mm NaCl, 5 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 10 mm glucose, 10 mm Na-HEPES, pH 7,4 at room temperature. 10 s after the start of the recording, 5 μm ionomycin was added. After 30 s, the cells were perfused with buffer solution containing 0 Ca2+, 5 μm ionomycin, and 5 mm EGTA. The illumination time was set to 100 ms. For ratiometric analysis, the ratio (R) of YFP and CFP fluorescence intensities was calculated as the following, R = SFRET/SCFP. The baseline ratio (R0) was calculated as an average of the first 10 s before stimulation. The ratio change (ΔR/R) is ΔR/R = (R − R0)/R0 (26Miyawaki A. Llopis J. Heim R. McCaffery J.M. Adams J.A. Ikura M. Tsien R.Y. Nature. 1997; 388: 882-887Crossref PubMed Scopus (2620) Google Scholar, 31Mank M. Santos A.F. Direnberger S. Mrsic-Flogel T.D. Hofer S.B. Stein V. Hendel T. Reiff D.F. Levelt C. Borst A. Bonhoeffer T. Hübener M. Griesbeck O. Nat. Methods. 2008; 5: 805-811Crossref PubMed Scopus (380) Google Scholar). For the single channels (CFP, YFP), mean intensity values derived from a selective background region near the investigated cell was used for background correction. After this correction, mean values for each cell were calculated from a region of interest drawn around a cell of interest. The ratio R was formed from the ratio of the background-corrected single channels. The complete C terminus of Cav1.4 containing the 5A mutation within the IQ motif (CT1.4/5A see above) or C-terminal fragments of Cav1.4 (CT1.4R1610Stop (Asp-1445–Gly-1609); CT1.4R1610Stop/5A) were amplified by PCR and cloned into the pcDNA3 expression vector. All sequences were fused with a Myc tag at the N terminus. CaM1234 was constructed in the same manner by using a 5′ primer containing the triple FLAG sequence. For expression of recombinant proteins, HEK293 cells were transfected by using the calcium phosphate method. Immunoprecipitation was performed 3 days after transfection. A detailed protocol has been published previously (9Wahl-Schott C. Baumann L. Cuny H. Eckert C. Griessmeier K. Biel M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 15657-15662Crossref PubMed Scopus (73) Google Scholar). Each coimmunoprecipitation was repeated at least three times. All values are given as mean ± S.E.; n is the number of experiments. An unpaired t test was performed for the comparison between two groups. Significance was tested by analysis of variance followed by Dunnett's test whether multiple comparisons were made. Values of p < 0.05 were considered significant. To test whether CaM is bound to Cav1.4 channels, we carried out FRET experiments in living cells using the full-length C terminus of Cav1.4 and CaM (Fig. 1, A and B). This approach has been successfully applied to other HVA Ca2+ channels (24Erickson M.G. Liang H. Mori M.X. Yue D.T. Neuron. 2003; 39: 97-107Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 25Liang H. DeMaria C.D. Erickson M.G. Mori M.X. Alseikhan B.A. Yue D.T. Neuron. 2003; 39: 951-960Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). We first fused wild type CFP and CaM by a flexible AAA linker (CFP-CaM). This construct yielded significant FRET with YFP-CT1.2, whereas there was no FRET for YFP-CT1.4 (Fig. 1B). A possible explanation for the lack of FRET could be that CaM does not bind to Cav1.4 as suggested previously (10Singh A. Hamedinger D. Hoda J.C. Gebhart M. Koschak A. Romanin C. Striessnig J. Nat. Neurosci. 2006; 9: 1108-1116Crossref PubMed Scopus (109) Google Scholar). Alternatively, CaM may bind, but FRET could be prevented by structural constraints in the steric environment of the CaM binding region in Cav1.4. We addressed this problem using fusion proteins where CaM is attached to structurally modified CFPs (Fig. 1, A and B). A fusion protein where the last 11 residues of CFP have been truncated (CFP227-CaM) showed strong FRET in CT1.2 and in CT1.4ΔICDI, a CT1.4 variant lacking the ICDI domain. Importantly, in the wild type Cav1.4 C terminus, a very small FRET signal just below the threshold of significance was obtained. To further optimize the geometric orientation of the fluorophores, we fused CFP to CaM at different angles using circularly permutated CFPs (29Mank M. Reiff D.F. Heim N. Friedrich M.W. Borst A. Griesbeck O. Biophys. J. 2006; 90: 1790-1796Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 32Baird G.S. Zacharias D.A. Tsien R.Y. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 11241-11246Crossref PubMed Scopus (740) Google Scholar, 33Topell S. Hennecke J. Glockshuber R. FEBS Lett. 1999; 457: 283-289Crossref PubMed Scopus (110) Google Scholar, 34Nagai T. Yamada S. Tominaga T. Ichikawa M. Miyawaki A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10554-10559Crossref PubMed Scopus (861) Google Scholar) (CFPcp158-CaM and CFPcp174-CaM; Fig. 1, A and B). To this end, the C and the N termini of wild type CFP were fused by a linker, and a new C and N termini were introduced at different positions. Using circularly permutated CFPs, CaM binding to CT1.2 and CT1.4ΔICDI could be detected (Fig. 1B). Notably, coexpression of CFPcp174-CaM and YFP-CT1.4 resulted in a significant FRET. The specificity of this interaction was demonstrated by the lack of FRET in the negative control (Fig. 1B). The FRET response for CFPcp174-CaM indicated that CaM binds to a target sequence in the C terminus and that binding of CaM is not prevented in the presence of the ICDI. To test whether the interaction observed is independent of resting Ca2+ levels and Ca2+ activation of CaM, we next used CaM mutant (CaM1234) that is deficient for Ca2+ binding (30Xia X.M. Fakler B. Rivard A. Wayman G. Johnson-Pais T. Keen J.E. Ishii T. Hirschberg B. Bond C.T. Lutsenko S. Maylie J. Adelman J.P. Nature. 1998; 395: 503-507Crossref PubMed Scopus (734) Google Scholar). CaM1234 serves as a surrogate for apocalmodulin (apoCaM) and is known to preassociate with the C termini of other HVA Ca2+ channels (24Erickson M.G. Liang H. Mori M.X. Yue D.T. Neuron. 2003; 39: 97-107Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 25Liang H. DeMaria C.D. Erickson M.G. Mori M.X. Alseikhan B.A. Yue D.T. Neuron. 2003; 39: 951-960Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Clear FRET could be observed between CFPcp174-CaM1234 and YFP-CT1.4 similar to that observed for CFPcp174-CaM and YFP-CT1.4 (supplemental Fig. S1). FRET between CaM and CT1.2 or CT1.4 could be significantly increased by raising the intracellular Ca2+ concentration above resting limits (Fig. 2, A and B). This increase was reversible because clamping intracellular Ca2+ to nominal zero Ca2+ decreased binding of CaM to resting levels. For CaM1234, no Ca2+-dependent changes in FRET could be observed. To study the functional effects that CaM exerts on Cav1.4, we generated a Cav1.4 mutant that is deficient for CaM binding (Cav1.4/5A; Fig. 3). In this mutant, five amino acid residues within the IQ motif are replaced by alanines (CT1.4/5A and CT1.4R1610Stop/5A). Indeed, coimmunoprecipitation experiments confirmed that CaM1234 binding to the mutated full-length C terminus (CT1.4/5A) or to CT1.4 that is truncated after the IQ (CT1.4R1610Stop/5A) was totally abolished. As a positive control, we used CT1.4R1610Stop that binds CaM (9Wahl-Schott C. Baumann L. Cuny H. Eckert C. Griessmeier K. Biel M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 15657-15662Crossref PubMed Scopus (73) Google Scholar). We next compared the electrophysiological properties of wild type Cav1.4 channels with the properties of Cav1.4 channels deficient for CaM (Cav1.4/5A) in HEK293 cells (Fig. 3, B–F). Currents induced for wild type Cav1.4 channels consistently gave higher peak Ba2+ current densities (−4.91 ± 0.79 pA/pF; n = 12) than Cav1.4/5A channels (−2.37 ± 0.34 pA/pF; n = 17; Fig. 3D)," @default.
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• W2054539671 date "2009-10-01" @default.
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• W2054539671 title "Calmodulin Is a Functional Regulator of Cav1.4 L-type Ca2+ Channels" @default.
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