Cellular Processing of Cone Photoreceptor Cyclic GMP-gated Ion Channels
2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês
10.1074/jbc.m400035200
ISSN1083-351X
AutoresMaría Paula Faillace, Ramón Bernabeu, Juan I. Korenbrot,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoWe examined cellular protein processing and functional expression of photoreceptor cyclic nucleotidegated (CNG) ion channels. In a mammalian cell line, wild type bovine cone photoreceptor channel α subunits (bCNGA3) convert from an unglycosylated state, at 90 kDa, to two glycosylated states at 93 and 102 kDa as they transit within the cell to their final location at the plasma membrane. Glycosylation per se is not required to yield functional channels, yet it is a milestone that distinguishes sequential steps in channel protein maturation. CNG ion channels are not gated by membrane voltage although their structure includes the transmembrane S4 motif known to function as the membrane voltage sensor in all voltage-gated ion channels. S4 must be functionally important because its natural mutation in cone photoreceptor CNG channels is associated with achromatopsia, a human autosomal inherited loss of cone function. Point mutation of specific, not all, charged and neutral residues within S4 cause failure of functional channel expression. Cellular channel protein processing fails in every one of the non-functional S4 mutations we studied. Mutant proteins do not reach the 102-kDa glycosylated state and do not arrive at the plasma membrane. They remain trapped within the endoplasmic reticulum and fail to transit out to the Golgi apparatus. Coexpression of cone CNG β subunit (CNGB3) does not rescue the consequence of S4 mutations in CNGA3. It is likely that an intact S4 is required for proper protein folding and/or assembly in the endoplasmic reticulum membrane. We examined cellular protein processing and functional expression of photoreceptor cyclic nucleotidegated (CNG) ion channels. In a mammalian cell line, wild type bovine cone photoreceptor channel α subunits (bCNGA3) convert from an unglycosylated state, at 90 kDa, to two glycosylated states at 93 and 102 kDa as they transit within the cell to their final location at the plasma membrane. Glycosylation per se is not required to yield functional channels, yet it is a milestone that distinguishes sequential steps in channel protein maturation. CNG ion channels are not gated by membrane voltage although their structure includes the transmembrane S4 motif known to function as the membrane voltage sensor in all voltage-gated ion channels. S4 must be functionally important because its natural mutation in cone photoreceptor CNG channels is associated with achromatopsia, a human autosomal inherited loss of cone function. Point mutation of specific, not all, charged and neutral residues within S4 cause failure of functional channel expression. Cellular channel protein processing fails in every one of the non-functional S4 mutations we studied. Mutant proteins do not reach the 102-kDa glycosylated state and do not arrive at the plasma membrane. They remain trapped within the endoplasmic reticulum and fail to transit out to the Golgi apparatus. Coexpression of cone CNG β subunit (CNGB3) does not rescue the consequence of S4 mutations in CNGA3. It is likely that an intact S4 is required for proper protein folding and/or assembly in the endoplasmic reticulum membrane. Cyclic nucleotide-gated ion channels (CNG) 1The abbreviations used are: CNG, cyclic nucleotide-gated ion channels; ER, endoplasmic reticulum; CNGA3, cone photoreceptor channel α subunit; bCNGA3, bovine cone photoreceptor channel α subunit; HA, hemagglutinin; EGFP, epidermal growth factor protein; wt, wild type. 1The abbreviations used are: CNG, cyclic nucleotide-gated ion channels; ER, endoplasmic reticulum; CNGA3, cone photoreceptor channel α subunit; bCNGA3, bovine cone photoreceptor channel α subunit; HA, hemagglutinin; EGFP, epidermal growth factor protein; wt, wild type. are localized in the plasma membrane of sensory transduction neurons, such as retinal photoreceptor and olfactory sensory cells where they play a central role in signal transduction (for review see Refs. 1Trudeau M.C. Zagotta W.N. J. Biol. Chem. 2003; 278: 18705-18708Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 2Kaupp U.B. Seifert R. Physiol. Rev. 2002; 82: 769-824Crossref PubMed Scopus (917) Google Scholar, 3Kramer R.H. Molokanova E. J. Exp. Biol. 2001; 204: 2921-2931PubMed Google Scholar). Photoreceptor CNG channels are heteromers constituted by α and β subunits in 3:1 stoichiometry (4Zheng J. Trudeau M.C. Zagotta W.N. Neuron. 2002; 36: 891-896Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 5Zhong H. Molday L.L. Molday R.S. Yau K.W. Nature. 2002; 420: 193-198Crossref PubMed Scopus (189) Google Scholar, 6Weitz D. Ficek N. Kremmer E. Bauer P.J. Kaupp U.B. Neuron. 2002; 36: 881-889Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Whereas specific details of CNG protein cellular processing have previously not been reported, it is likely these channels follow the cellular processing pattern typical of other channel proteins (reviewed in Ref. 7Deutsch C. Annu. Rev. Physiol. 2002; 64: 19-46Crossref PubMed Scopus (96) Google Scholar). Nascent channel proteins first attach to endoplasmic reticulum (ER) membranes, targeted to these membranes by a signal sequence. In the ER, proteins fold and translocate across the lipid bilayer and are core glycosylated by linking of N-acetylglucosamine (GlcNAc) to Asn residues in the consensus sequence NX-S or-T. Frequently, core-glycosylated channel proteins are processed by additional glycosylation in the ER and/or Golgi apparatus.Various single point mutations in the α subunit of CNG channels of cone photoreceptors have been identified in humans afflicted by achromatopsia, also known as total color blindness or rod monochromic, an autosomal, inherited disease characterized by complete loss of cone photoreceptor function (8Wissinger B. Gamer D. Jagle H. Giorda R. Marx T. Mayer S. Tippmann S. Broghammer M. Jurklies B. Rosenberg T. Jacobson S.G. Sener E.C. Tatlipinar S. Hoyng C.B. Castellan C. Bitoun P. Andreasson S. Rudolph G. Kellner U. Lorenz B. Wolff G. Verellen-Dumoulin C. 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Its three-dimensional structure has been recently proposed (9Jiang Y. Ruta V. Chen J. Lee A. MacKinnon R. Nature. 2003; 423: 42-48Crossref PubMed Scopus (710) Google Scholar). The motif is present in every known voltage-gated ion channel (K+,Na+, and Ca2+), and its existence in CNG ion channels is one of the arguments first used to identify CNG channels as members of the same gene superfamily as voltage-gated K+ channels (10Jan L.Y. Jan Y.N. Nature. 1990; 345: 672Crossref PubMed Scopus (199) Google Scholar, 11Heginbotham L. Abramson T. MacKinnon R. Science. 1992; 258: 1152-1155Crossref PubMed Scopus (357) Google Scholar).In voltage-gated channels S4 functions as a voltage sensor (12Papazian D.M. Timpe L.C. Jan Y.N. Jan L.Y. Nature. 1991; 349: 305-310Crossref PubMed Scopus (429) Google Scholar, 13Perozo E. Santacruz-Toloza L. Stefani E. Bezanilla F. Papazian D.M. Biophys. J. 1994; 66: 345-354Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 14Seoh S.A. Sigg D. Papazian D.M. 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In addition to its biophysical function, however, the S4 structure also plays a role in the cellular biogenesis of voltage-gated channels (for review see Refs. 7Deutsch C. Annu. Rev. Physiol. 2002; 64: 19-46Crossref PubMed Scopus (96) Google Scholar, 21Papazian D.M. Neuron. 1999; 23: 7-10Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, and 22Deutsch C. Neuron. 2003; 40: 265-276Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In voltage-gated K+ channels, neutralization of some charged amino acid residues in S4 change the voltage dependence of gating, whereas mutation of others cause channel functional failure (23Papazian D.M. Shao X.M. Seoh S.A. Mock A.F. Huang Y. Wainstock D.H. Neuron. 1995; 14: 1293-1301Abstract Full Text PDF PubMed Scopus (321) Google Scholar, 24Tiwari-Woodruff S.K. Schulteis C.T. Mock A.F. Papazian D.M. Biophys. J. 1997; 72: 1489-1500Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 25Tu L. Wang J. Helm A. Skach W.R. Deutsch C. Biochemistry (Mosc.). 2000; 39: 824-836Crossref Scopus (56) Google Scholar). In HCN channels, which are gated by voltage and modulated by cyclic nucleotides, S4 plays a similar dual role: some residues affect the voltage dependence in gating, whereas others interfere with their arrival at the plasma membrane (19Mannikko R. Elinder F. Larsson H.P. Nature. 2002; 419: 837-841Crossref PubMed Scopus (165) Google Scholar, 26Chen J. Mitcheson J.S. Lin M. Sanguinetti M.C. J. Biol. Chem. 2000; 275: 36465-36471Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar).Although CNG channels contain an S4 domain they are not voltage-gated. That is, their probability of opening is independent of membrane voltage and depends only on cyclic nucleotide concentration. This is despite the fact that the CNG channels' S4 domain can perform as a voltage sensor when placed in a permissive structural environment. Chimera experiments demonstrate that replacing the authentic S4 motif in ether-ago-go K+ channel with the olfactory S4 motif (CNGA2) maintains the voltage-dependent behavior of the K+ channel (27Tang C.Y. Papazian D.M. J. Gen. Physiol. 1997; 109: 301-311Crossref PubMed Scopus (49) Google Scholar). On the other hand, CNG channels do exhibit several voltage-dependent functions. 1) The ligand sensitivity is voltage dependent. The ligand concentration necessary to half-maximally activate the channels is about 10 μm higher when measured at -50 mV than at +50 mV (in cones 28Picones A. Korenbrot J.I. J. Gen. Physiol. 1992; 100: 647-673Crossref PubMed Scopus (54) Google Scholar, in rods 29Zimmerman A.L. Baylor D.A. J. Physiol. (Lond.). 1992; 449: 759-783Crossref Scopus (72) Google Scholar). 2) The lifetime of the channel open state in rod CNG is slightly longer at depolarized than at hyperpolarized voltages, causing slight outward rectification in the open channel I-V curve (30Benndorf K. Koopmann R. Eismann E. Kaupp U.B. J. Gen. Physiol. 1999; 114: 477-490Crossref PubMed Scopus (29) Google Scholar). 3) Open channels are blocked by divalent cations, but the extent of block is voltage-dependent: block is relieved as the voltage moves away from the reversal voltage (in cones 31Picones A. Korenbrot J.I. Biophys. J. 1995; 69: 120-127Abstract Full Text PDF PubMed Scopus (77) Google Scholar and 32Haynes L.W. J. Gen. Physiol. 1995; 106: 507-523Crossref PubMed Scopus (28) Google Scholar, in rods 29Zimmerman A.L. Baylor D.A. J. Physiol. (Lond.). 1992; 449: 759-783Crossref Scopus (72) Google Scholar and 33Colamartino G. Menini A. Torre V. J. Physiol. (Lond.). 1991; 440: 189-206Crossref Scopus (71) Google Scholar). Ion permeation is well described by an Eyring rate theory model that assumes ions bind to sites in the pore of the channel and binding is dependent on membrane voltage (in rods 29Zimmerman A.L. Baylor D.A. J. Physiol. (Lond.). 1992; 449: 759-783Crossref Scopus (72) Google Scholar, 30Benndorf K. Koopmann R. Eismann E. Kaupp U.B. J. Gen. Physiol. 1999; 114: 477-490Crossref PubMed Scopus (29) Google Scholar, 31Picones A. Korenbrot J.I. Biophys. J. 1995; 69: 120-127Abstract Full Text PDF PubMed Scopus (77) Google Scholar, 32Haynes L.W. J. Gen. Physiol. 1995; 106: 507-523Crossref PubMed Scopus (28) Google Scholar, 33Colamartino G. Menini A. Torre V. J. Physiol. (Lond.). 1991; 440: 189-206Crossref Scopus (71) Google Scholar, 34Wells G.B. Tanaka J.C. Biophys. J. 1997; 72: 127-140Abstract Full Text PDF PubMed Scopus (20) Google Scholar, in cones 35Ohyama T. Picones A. Korenbrot J.I. J. Gen. Physiol. 2002; 119: 341-354Crossref PubMed Scopus (11) Google Scholar).S4 mutations in CNGA3, hence, can cause channel malfunction because of either defective protein biogenesis or a defect in a voltage-dependent function. To explore the role of S4 in CNG channel function, we investigated the consequences of point mutations in the S4 domain of the CNG channel α subunit cloned from bovine cone photoreceptors (bCNGA3). We measured the electrical properties of transfected mammalian cells, as well as the cell-processing and expression patterns of the channel proteins. We have found that normal channels exist in various states of glycosylation as they transit within cells to the plasma membrane. S4 plays a crucial role in channel biogenesis. Specific, not all, point mutations in S4 cause failure of cellular channel protein processing. Mutant channel proteins are not glycosylated and do not reach the surface plasma membrane; they remain trapped within the endoplasmic reticulum, likely misfolded.EXPERIMENTAL PROCEDURESSite-directed Mutagenesis and Hemagglutinin (HA) Tag Insertion— The cDNA of bovine CNGA3 (accession NM_174279, Refs. 36Weyand I. Godde M. Frings S. Weiner J. Muller F. Altenhofen W. Hatt H. Kaupp U.B. Nature. 1994; 368: 859-863Crossref PubMed Scopus (231) Google Scholar and 37Biel M. Zong X. Distler M. Bosse E. Klugbauer N. Murakami M. Flockerzi V. Hofmann F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3505-3509Crossref PubMed Scopus (124) Google Scholar) and mouse CNGB3 were the kind gift of Dr. M. Biel (University of Munich). The cDNA was inserted in-frame with the cytomegalovirus promoter in a bacterial plasmid (pcDNA 3, Invitrogen, Carlsbad, CA). Site-directed mutagenesis of the cDNA was executed using the QuikChange® kit (Stratagene, San Diego, CA).To track cellular processing of bCNGA3 we tagged the protein with HA peptide (YPYDVPDYA) by inserting the coding sequence of the peptide in-frame with the channel sequence at three specific locations using the QuikChange kit. 1) bCNGA3-N-HA in the amino terminus, between Leu32 and Ser33. 2) bCNGA3-C-HA in the carboxyl terminus, between Pro699 and Gln700 (seven residues from the end of the protein). 3) bCNGA3-S1S2-HA in the extracellular loop that connects transmembrane helices S1 and S2, between residues Leu214 and Gln215. All mutants and chimeras were sequenced in full to ensure that unintended, random mutations did not occur because of polymerase errors.Cell Transfection—tsA-201 cells (transformed from HEK 293 cells to constitutively expressing T-antigen) were grown on plastic wells in Dulbecco's modified Eagle's H-21 media (with 10% fetal bovine serum and penicillin/streptomycin) at 37 °C under 5% CO2. When they reached 70% confluence they were transfected with plasmid cDNA introduced as a calcium phosphate precipitate using a commercial kit (Specialty Media, Phillipsburg, NJ). 9.6-cm2 wells were simultaneously transfected with 12.5 μg of channel cDNA plasmid and 3 μg of pEGFP, a pcDNA 3 plasmid that promotes cytoplasmic expression of EGFP. 20 h later the DNA-calcium phosphate precipitate was washed away. Cells were cultured for another 48 h before subjecting them to electrophysiological, biochemical, or imaging studies.Electrophysiological Measurements—Cells were suspended in a simple Ringer's solution consisting of (in mm): NaCl (140), KCl (2.5), NaHCO3 (3.5), Na2HPO4 (1), CaCl2 (1), MgCl2 (1), glucose (10), minimal essential medium amino acids and vitamins (1×), and HEPES (10), pH 7.4, osmotic pressure: 310 mosmol. To produce these suspensions, cell layers growing on plastic were gently trypsinized (0.05% trypsin, 0.02% Versene), washed, and maintained in Ringer's solution at 4 °C for at least 30 min and for as long as 6 h.About 60,000 cells in 200 μl were plated on a poly-l-lysine-coated (0.1 mg/ml) glass coverslip that formed the bottom of an electrophysiological recording chamber. The chamber was held on the stage of a modified upright microscope that permitted observation of the cells with DIC contrast enhancement under deep red light illumination, as described in detail elsewhere (38Ohyama T. Hackos D.H. Frings S. Hagen V. Kaupp U.B. Korenbrot J.I. J. Gen. Physiol. 2000; 116: 735-754Crossref PubMed Scopus (34) Google Scholar). The microscope was equipped with a high-resolution, cooled CCD camera and follow up hardware and software (MicroMax1300 and WinView32, Roper Scientific). The microscope included two epi-illumination pathways. One permitted us to select EGFP-expressing cells by their fluorescence under appropriate spectral filters, whereas the second allowed the delivery of bright, brief flashes of light to the cell under investigation. These flashes were generated by a Xenon arc flash lamp (200 Joules, 170-μs half-bandwidth) (Chadwick-Helmuth Co., El Monte, CA).All experiments were conducted at room temperature (20-21 °C). After identifying an EGFP-expressing cell, tight-seal electrodes were applied onto the cell under deep red light (to avoid uncaging). Electrodes were produced from alumino silicate glass (1724, 1.5 × 1.0 mm outer diameter × inner diameter, Sutter Instruments, Novato, CA). We measured membrane currents under voltage-clamp with a patch clamp amplifier (Axopatch 1D, Axon Instruments, Foster City, CA). Analog signals were low pass filtered below 100 Hz with an eight pole Bessel filter (Kronh-Hite, Avon, MA) and digitized on line at 200 Hz (pClamp, Axon Instruments, Union City, CA).Electrode filling solution was freshly prepared under dim red light and used within 3 h or discarded. We used a salt solution consisting of (in mm): gluconic acid (130), triethanolamine-Cl (20), HEPES (10), MgCl2 (3.7), GTPNa3 (1), ATPNa2 (2), 1,2-bis(2-aminophenoxyl)ethane-N,N,N′,N′-tetraacetic acid (2) titrated to pH 7.3 with CsOH. Under deep red light we added MCM-caged 8-pCPT-cGMP ((7-methoxycoumarin-methyl-4-yl) methyl-caged 8-(4-chlorophenylthio)guanosine cyclic 3′,5′-monophosphate)) from a 25 mm in Me2SO stock to a final concentration of 15 μm. The compound was the kind gift of Dr. V. Hagen, who has described its synthesis (39Hagen V. Bendig J. Frings S. Wiesner B. Schade B. Helm S. Lorenz D. Kaupp U.B. J. Photochem. Photobiol. B Biol. 1999; 53: 91-102Crossref PubMed Scopus (43) Google Scholar).Immunoblot Analysis of HA-tagged Channel Proteins—Transfected cells were lysed with non-denaturing lysis buffer: 100 mm Tris/HCl, pH 8.0, 100 mm NaCl, 0.5% Triton X-100, and 1 tablet/10 ml of solution of a protease inhibitor mixture (Complete Mini EDTA-free, Roche Applied Science). 25-μg samples of total protein were separated by size using SDS-PAGE on 6% gels, and then transferred to nitrocellulose membranes by semidry electroblotting. Membranes were incubated overnight in blocking buffer: 100 mm phosphate-buffered saline, 0.1% Tween 20, 5% nonfat dried milk, and 1% goat serum at 4 °C with continuous agitation. They were then incubated for 1 h at room temperature in 15 ml of the same buffer, now containing anti-HA rat monoclonal antibody at 100 ng/ml (3F10 clone, Roche Applied Science). Bound primary antibody was detected by reaction with a secondary anti-rat goat antibody conjugated to horseradish peroxidase (1:1000 in blocking buffer) (Jackson ImmunoResearch Laboratories, West Grove, PA). Horseradish activity was exposed either with diaminobenzidine and nickel substrate or with ECL reagents (Amersham Biosciences). Immunoblots were repeated at least three times.Immunocytochemistry—In these experiments tsA-201 cells were grown on poly-d-lysine-coated (1 mg/ml) glass number 1 coverslips placed in plastic wells. Cells were grown on 2-cm2 wells and transformed with plasmid DNA scaled in concentration to maintain the weight ratios described above for cells grown on larger wells. 48 h after washing away the DNA-calcium-phosphate precipitate, cells were fixed with 4% paraformaldehyde for 10 min at room temperature, washed thoroughly with phosphate-buffered saline, and then blocked by incubation in phosphate-buffered saline containing 0.1% Tween 20, 2% bovine serum albumin, and 2% goat serum for at least 1 h at room temperature. Coverslips were then incubated overnight with anti-HA rat monoclonal antibody at 200 ng/ml added to the blocking solution (3F10 clone). The anti-HA antibody was detected with a fluorescent secondary goat antibody conjugated with rhodamine (1:500) (Jackson ImmunoResearch Laboratories). Glass coverslips were mounted onto glass slides using Vectashield (Vector Laboratories, Burlingame, CA) and the edges sealed with rubber cement.Specific Membrane Compartment Labeling of Transformed Cells— Cells grown on poly-d-lysine-coated coverslips in 2-cm2 wells were simultaneously transfected with channel cDNA (2.6 μg) and 0.65 μg each of one of several DNA constructs designed to express EGFP fusion protein at specific subcellular compartments.To label the plasma membrane we transformed cells with pEGFP-F plasmid (BD Biosciences, Clontech, Palo Alto, CA). This construct promotes the expression of a farnesylation signal in fusion with EGFP, the protein product is palmitoylated and targeted specifically to the cell plasma membrane. To label the endoplasmic reticulum we transformed cells with pEGFP-ER, a construct consisting of the residues -30 to 8 of bovine preprolactin (40Sasavage N.L. Nilson J.H. Horowitz S. Rottman F.M. J. Biol. Chem. 1982; 257: 678-681Abstract Full Text PDF PubMed Google Scholar) in fusion with EGFP. To label Golgi apparatus we transformed with pEGFP-TG, a construct consisting of residues -77 to -31 of human β-1,4-galactosyltransferase (41Masri K.A. Appert H.E. Fukuda M.N. Biochem. Biophys. Res. Commun. 1988; 157: 657-663Crossref PubMed Scopus (130) Google Scholar) in fusion with EGFP. The later two constructs were the kind gift of Dr. A. Verkman (University of California, San Francisco), who has described their construction and application (42Kneen M. Farinas J. Li Y. Verkman A.S. Biophys. J. 1998; 74: 1591-1599Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar).Microscopy and Image Analysis—Fluorescent cell images were captured with a confocal microscope (Zeiss, model LSM 510) using a ×63/1.3 NA objective. Spatial resolution was further improved by deblurring the confocal images with the use of deconvolution algorithms (C-Imaging Systems, Compix Inc., Cranberry Township, PA). The same software was used for image analysis and overlay in pixel-by-pixel operations at 12 bit resolution.RESULTSWe investigated the electrical properties and cellular protein processing of the bCNGA3. Electrical function was assayed in transformed tsA-201 cells by assessing whether, under voltage clamp, membrane currents were activated upon photo release of cyclic nucleotide from a caged precursor compound. The analog we used, 8-pCPT-cGMP is the most potent CNG channel agonist so far described (about 80-fold higher than cGMP (43Wei J.Y. Cohen E.D. Genieser H.G. Barnstable C.J. J. Mol. Neurosci. 1998; 10: 53-64Crossref PubMed Scopus (52) Google Scholar)), thus yielding a highly sensitive assay of function. Caged nucleotide was loaded into cells by allowing time for its diffusion from the lumen of a tight-seal electrode into the cell cytoplasm. Typically, the effect of photoreleased 8-pCPT-cGMP was assessed 3.5 min after attaining the whole cell mode, a time sufficient for equilibration between the cytoplasm of the cell and electrode filling solution. At this time, cells transformed with wt α subunits of bCNGA3 exhibited a mean holding current at -40 mV of -14.3 ± 17.6 pA (n = 22). Because endogenous K+ channels in tsA-201 were partially blocked by the ionic solutions we used in these experiments, holding current magnitude principally reflects cell integrity and the quality of the seal resistance and offers an excellent tool to discard damaged cells. Indeed, cells were accepted for analysis only if their holding current was within the statistical range reported above.Uncaging pCPT-cGMP by bright flash illumination caused a large inward current at -75 mV in cells transfected with wt bCNGA3 (Fig. 1A). In cells tested at holding voltages between -65 and -25 mV, 27 of 29 exhibited uncaging flash-activated currents. The few failures almost certainly reflect cells in which EGFP was expressed (the criterion to identify transformed cells), but channels were not. The absolute amplitude of the current varied from cell to cell, likely reflecting variance in the level of channel protein expression. At -75 mV mean cyclic nucleotide-dependent current amplitude was -74 ± 50.5 (range -36 to -170, n = 6).Three independent controls make evident that the current generated by uncaging the cyclic nucleotide analog reflects specific activation of bCNGA3 channels. 1) Flash illumination of transformed cells not loaded with caged nucleotides or untransformed cells loaded with the nucleotides did not generate currents. 2) The I-V characteristics of the flash-activated current are the same as those of the native cone CNG channels under similar ionic solutions (Fig. 1D). The composition of intra- and extracellular solutions were designed to minimize the amplitude of small delayed rectifier K+ and Ca2+-dependent Cl- currents endogenous to tsA-201 cells. The 8-pCPT-cGMP-dependent difference I-V curve is typical of CNGA3 channels and demonstrates the well documented voltage-dependent relief of channel blocking by divalent cations. 3) Currents were of identical features whether activated by uncaging caged-8-pCPT-cGMP or caged-cGMP (not shown).Functional Consequence of Various Point Mutations in S4—We transformed cells with bCNGA3 cDNA modified to mutate R296Q, this is the second Arg in the S4 motif of the channel (Table I). In these cells the holding current at -40 mV was -9.6 ± 9.6 pA (n = 10). None of these cells exhibited detectable changes in current upon cytoplasmic flash release of cyclic nucleotides (Fig. 1B). The failure of functional channel expression is specifically because of the point mutation, because cells transformed with the very same plasmid mutated back to the wt sequence resulted in electrical behavior identical to that of wt channels (Fig. 1C). In cells transformed with revertant Q296R cDNA, the holding current at -40 mV was -5.6 ± 3.7 pA and peak uncaging flash-generated current at -75 mV was -59.3 ± 36.8 pA (range -20 to -96 pA, n = 3). That is, our mutagenesis protocol introduced the designed point mutations in S4 without causing unintended changes in the cDNA and neutralizing the charge of one specific amino acid in S4 caused failure of functional channel expression.Table IElectrophysiological characterization of the consequence of point mutations in S4 in cone bCNGA3 ion channels and its tagged derivative bCNGA3-C-HAPositionCOOH-taggedMutationI hold at −40 mVΔI flash at −75 mVRangeResponsive/total cellsaCounting cells transformed with wt bCNGA3 and tested at voltages other than −75 mV, 27/29 EGFP-positive cells exhibited currents upon flash release of pCPT-cGMP.pAwt−6.8 ± 6.4−74.0 ± 50.5−36 to −1706/7wtHA−13.8 ± 13.9−84.6 ± 94−15 to −28011/11Arg293Gln−11.3 ± 10.100/4Arg293HAGln−5.0 ± 3.500/4Phe294Leu, Ala−12.1 ± 11.1−119.3 ± 143.4−28 to −4006/7Asn295Gln−10.7 ± 8.400/7Arg296Gln−9.6 ± 9.600/10Arg296HAGln-6.0 ± 0.800/4Arg296CysbSame mutations as found in congenital achromatopsia.−10.6 ± 4.500/3Arg296HACys−3.0 ± 1.000/5Leu297Ala−7.4 ± 9.400/8Lys299Gln−30 ± 2200/2Lys299HAGln−11.5 ± 16.900/4Leu300Phe−7.8 ± 6.1−54.4 ± 26.9−16 to −905/6Arg302TrpbSame mutations as found in congenital achromatopsia.−600/1Arg302HATrp−9.6 ± 5.500/5Arg302HAGln−7.0 ± 2.600/6Leu303Ala−9.6 ± 7.4−103 ± 61.8−34 to −1775/5a Counting cells transformed with wt bCNGA3 and tested at voltages other than −75 mV, 27/29 EGFP-positive cells exhibited currents upon flash release of pCPT-cGMP.b Same mutations as found in congenital achromatopsia. Open table in a new tab We explored the functional consequence of individually mutating most of the residues in the S4 motif. The wt sequence in bCNGA3 and the mutations we tested are listed in Table I. The data presented are the mean holding current at -40 mV, the mean and range of current amplitude activated by flash uncaging at -75 mV, and the ratio of the number of cells that exhibited flash-activated current over the total number of EGFP-positive cells sampled. The holding current was similar in value in every one of the cells analyzed, independently of CNG channel function. Effects of the mutatio
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