Single Amino Acid Change in the Fifth Transmembrane Segment of the TRP Ca2+ Channel Causes Massive Degeneration of Photoreceptors

Abstract

The trp gene encodes subunits of a highly Ca2+-permeable class of light-activated channels ofDrosophila photoreceptors. The recently characterized mutation in this gene, TrpP365, is semidominant and causes massive degeneration of photoreceptors by making the TRP channel constitutively active. We show that a single amino acid change, Phe-550 to Ile, near the beginning of the fifth transmembrane domain of TRP channel subunits is necessary to induce, and sufficient to closely mimic, the original mutant phenotypes ofTrpP365. Hypotheses are presented as to why the amino acid residues at position 550 and its immediate vicinity might be important in influencing the regulation of the TRP channel and why the substitution of Phe for Ile at this position, in particular, could result in constitutive activity of the channel. The trp gene encodes subunits of a highly Ca2+-permeable class of light-activated channels ofDrosophila photoreceptors. The recently characterized mutation in this gene, TrpP365, is semidominant and causes massive degeneration of photoreceptors by making the TRP channel constitutively active. We show that a single amino acid change, Phe-550 to Ile, near the beginning of the fifth transmembrane domain of TRP channel subunits is necessary to induce, and sufficient to closely mimic, the original mutant phenotypes ofTrpP365. Hypotheses are presented as to why the amino acid residues at position 550 and its immediate vicinity might be important in influencing the regulation of the TRP channel and why the substitution of Phe for Ile at this position, in particular, could result in constitutive activity of the channel. electroretinogram Drosophila photoreceptors have two classes of light-activated channels, the highly calcium-permeable TRP channels and the non-specifically cation-selective TRPL channels (1Hardie R.C. Minke B. Neuron. 1992; 8: 613-651Abstract Full Text PDF PubMed Scopus (585) Google Scholar). The TRP channels, the subunits of which are encoded by the trp gene (1Hardie R.C. Minke B. Neuron. 1992; 8: 613-651Abstract Full Text PDF PubMed Scopus (585) Google Scholar, 2Peretz A. Suss-Toby E. Rom-Glas A. Arnon A. Payne R. Minke B. Neuron. 1994; 12: 1257-1267Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 3Peretz A. Sandler C. Kirschfeld K. Hardie R. Minke B. J. Gen. Physiol. 1994; 104: 1057-1077Crossref PubMed Scopus (64) Google Scholar, 4Hardie R.C. Curr. Biol. 1996; 6: 1371-1373Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), are thought to carry most of the phototransduction current (5Reuss H. Mojet M.H. Chyb S. Hardie R.C. Neuron. 1997; 19: 1249-1259Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The TRP protein is widely conserved throughout animal phylogeny (6Birnbaumer L. Zhu X. Jiang M. Boulay G. Peyton M. Vannier B. Brown D. Platano D. Sadeghi H. Stefani E. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202Crossref PubMed Scopus (358) Google Scholar, 7Montell C. Mol. Pharmacol. 1997; 52: 755-763Crossref PubMed Scopus (120) Google Scholar, 8Montell, C. (2001) Science’s STKE, 1–17Google Scholar). However, the mechanisms of activation or regulation of TRP or TRP-related channels are still obscure. Yoon et al. (9Yoon J. Ben-Ami H.C. Hong Y.S. Park S. Strong L.L.R. Bowman J. Geng C. Baek K. Minke B. Pak W.L. J. Neurosci. 2000; 20: 649-659Crossref PubMed Google Scholar) recently described a new mutation in the trp gene,TrpP365, which confers on the mutant a set of phenotypes unlike any seen in previously isolated trpmutants. The TrpP365 mutants do not display photoreceptor responses that terminate prematurely during light stimulus, a phenotype that had been considered to be the defining hallmark of mutants in this gene (10Cosens D.J. Manning A. Nature. 1969; 224: 285-287Crossref PubMed Scopus (465) Google Scholar, 11Minke B., Wu, C.-F. Pak W.L. Nature. 1975; 258: 84-87Crossref PubMed Scopus (188) Google Scholar).TrpP365 responses are small but are maintained throughout the duration of light stimulus. The most striking feature ofTrpP365 mutants, however, is that their photoreceptors undergo rapid degeneration. Moreover, unlike any other known trp mutants, these TrpP365phenotypes are semidominant, i.e. TrpP365 heterozygotes also display mutant phenotypes though they are not as severe as those of homozygotes. From whole cell patch-clamp recordings, Yoon et al. (9Yoon J. Ben-Ami H.C. Hong Y.S. Park S. Strong L.L.R. Bowman J. Geng C. Baek K. Minke B. Pak W.L. J. Neurosci. 2000; 20: 649-659Crossref PubMed Google Scholar) concluded that these phenotypes arise as a result of excessive Ca2+entry caused by constitutive activity of the TRP channel. We now show that the above phenotypes are because of a single amino acid change, Phe-550 to Ile, in the fifth transmembrane segment of the TRP channel. We present, in addition, a possible mechanism by which the phenotypes might arise. Atrp minigene was constructed and subcloned into the pCaSpeR-3 vector for germ line transformation. The minigene consisted of three DNA fragments, the trp promoter and 5′-untranslated regions (−681 to −1 of genomic DNA, where +1 is the trptranslation start site), the coding region (ATG to the stop codon), and the 390-bp region immediately downstream of the stop codon. The minigene was subcloned into the XbaI and StuI sites of the CaSpeR-3 transformation vector. The vector containing the minigene was injected into early embryos in the presence of helper plasmid (12Rubin G.M. Spradling A.C. Science. 1982; 218: 348-353Crossref PubMed Scopus (2340) Google Scholar). All four mutations detected in the TrpP365 mutant are located within a 2444-bpSmaI fragment (from +647 to +3090) in the trpcoding region. The 2444-bp SmaI fragment was isolated from wild-type trp cDNA, subcloned into the M13mp18 vector, and mutagenized as described in Kunkel (13Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4903) Google Scholar). The following primers were used in site-directed mutagenesis: P500T (mutation 1), 5′-GAACACTGCATACGTTTGATCCA;H532N (mutation 2), 5′-CCATTAATCCCAACCTGGGACCCT; F550I (mutation 3), 5′-CATCAAGTTCATCTTCATCTACA; and S867F (mutation 4), 5′-TACTTTCGCCTTCGATCCCATTGG, where the target bases are underlined. Following mutagenesis, the SmaI fragments were sequenced to ascertain only the intended mutations had been induced. Each mutagenized SmaI fragment was then swapped with the corresponding SmaI fragment in the trp minigene, which had previously been cloned into pCaSpeR-3. The following nine mutant trp minigene constructs were generated, and transgenic flies carrying each mutant minigene were generated by P-element-mediated germ line transformation in a wild-type background: P[trp (1Hardie R.C. Minke B. Neuron. 1992; 8: 613-651Abstract Full Text PDF PubMed Scopus (585) Google Scholar)], with mutation 1; P[trp (2Peretz A. Suss-Toby E. Rom-Glas A. Arnon A. Payne R. Minke B. Neuron. 1994; 12: 1257-1267Abstract Full Text PDF PubMed Scopus (125) Google Scholar)], with mutation 2; P[trp (3Peretz A. Sandler C. Kirschfeld K. Hardie R. Minke B. J. Gen. Physiol. 1994; 104: 1057-1077Crossref PubMed Scopus (64) Google Scholar)], with mutation 3; P[trp (4Hardie R.C. Curr. Biol. 1996; 6: 1371-1373Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar)], with mutation 4; P[trp (123)], with mutations 1, 2, and 3; P[trp (124)], with mutations 1, 2,and 4; P[trp (134)], with mutations 1, 3, and 4; P[trp (234)], with mutations 2, 3, and 4; and P[trp(1234)], with all four mutations. In addition to these transgenic lines in a wild-type background, three of the lines, that carrying mutation 3, that carrying mutations 1, 2, and 4, and that carrying all four mutations, were also placed in atrp-null (trpP343) background by appropriate genetic crosses. The trpP343 mutant was generated in this laboratory and was shown by others (14Scott K. Sun Y. Beckingham K. Zuker C.S. Cell. 1997; 91: 375-383Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) to be functionally null. Fly eyes were dissected in a fixative (4% formaldehyde in phosphate-buffered saline with 0.3% Triton X-100), and the dissected retinas (photoreceptor layers) were allowed to remain in the same fixative for 1 h. After incubation in phosphate-buffered saline that contained 4% normal goat serum, the fixed retinas were stained with phalloidin-tetramethylrhodamine B isothiocyanate (Sigma) to label filamentous actin in the rhabdomeres. Transverse optical sections of ∼1 μm thickness were taken ∼6 μm from the distal tips of the rhabdomeres. Yoon et al. (9Yoon J. Ben-Ami H.C. Hong Y.S. Park S. Strong L.L.R. Bowman J. Geng C. Baek K. Minke B. Pak W.L. J. Neurosci. 2000; 20: 649-659Crossref PubMed Google Scholar) showed that theTrpP365 mutant carries four protein sequence-altering mutations within its trp gene, P500T, H531N, F550I, and S867F. To simplify notations, these mutations will be referred to as mutations 1, 2, 3, and 4, respectively. To determine which of these four mutations is responsible for theTrpP365 phenotype, site-directed mutagenesis was carried out on a trp minigene to generate mutants carrying each of the four mutations singly, three of the four in all possible triplet combinations, or all four mutations together. The resulting mutant minigene constructs were introduced into a wild-type background by P-element-mediated germ line transformation (see “Experimental Procedures”). A single copy of the trp minigene, which has no introns, was found to fully complement the trp mutant phenotype when introduced into the null trpP343background (not shown). Shown in Fig. 1 are electroretinograms (ERGs),1 extracellularly recorded, light-evoked mass responses of the eye, obtained from transgenic flies each carrying a single copy of the recombinanttrp minigene harboring mutation 1, 2, 3, or 4,i.e. P[trp (1Hardie R.C. Minke B. Neuron. 1992; 8: 613-651Abstract Full Text PDF PubMed Scopus (585) Google Scholar)]/+, P[trp(2)]/+, P[trp(3)]/+, or P[trp(4)]/+, all in a wild-type background. Thus, these transgenic flies had two copies of the endogenous wild-type trp gene and one copy of the mutagenized recombinant trp minigene. ERGs of P[trp(1)]/+, P[trp(2)]/+, or P[trp(4)]/+ were indistinguishable from those of wild-type (Fig. 1, a, b, and d). Only those obtained from P[trp(3)]/+ were distinctly different from wild-type ERGs in that they showed markedly slower kinetics of decay at stimulus off-set (Fig. 1c). There was little variation in the ERG decay times among individuals of P[trp(3)]/+ so that the P[trp(3)]/+ ERG could always be distinguished from that of wild-type by inspection. Yoonet al. (9Yoon J. Ben-Ami H.C. Hong Y.S. Park S. Strong L.L.R. Bowman J. Geng C. Baek K. Minke B. Pak W.L. J. Neurosci. 2000; 20: 649-659Crossref PubMed Google Scholar) have shown previously that one copy ofTrpP365 introduced into a wild-type background causes the ERGs of the transgenic host to become somewhat smaller in amplitude and slower in the kinetics of decay than those of wild-type. Thus, the slow time courses of decay observed in this study were consistent with the previous findings, although little or no change in amplitudes was detected in the present study. We next made the P[trp (3Peretz A. Sandler C. Kirschfeld K. Hardie R. Minke B. J. Gen. Physiol. 1994; 104: 1057-1077Crossref PubMed Scopus (64) Google Scholar)] transgene homozygous so that the transgenic flies now carried two copies of P[trp(3)] and two copies of the endogenous wild-type trp gene. The main effect of making P[trp(3)] homozygous on the ERG was to slow down the kinetics of response decay even further (Fig.2a). Thus, mutation 3 was the only one of the four that had any effect at all on the ERG, suggesting that mutation 3, i.e. F550I, might be the primary contributor to the TrpP365 mutant ERG phenotype. To examine to what extent the other three mutations might play a role in producing the TrpP365phenotype, we generated transgenic flies carrying various triplet combinations of the four mutations (123, 124, 134, and 234) or all four mutations together (1234). These transgenic lines, too, were made homozygous for the transgene so that each carried two copies of the transgene and two copies of the endogenous trp+gene. ERGs obtained from three of the triplet combination lines, P[trp(123)], P[trp(134)], and P[trp(234)], were very similar to each other, with slow decay time courses much like that seen in P[trp(3)] flies (compare Fig. 2, c and a). For this reason, an ERG obtained from only one of these three lines, P[trp(123)], is shown in Fig. 2. On the other hand, ERGs of transgenic flies of the P[trp(124)] line, the only line that did not carry mutation 3, were completely wild-type (Fig.2d). Thus, whenever the transgenic flies carried mutation 3, whether by itself or in combination with the other mutations, their ERGs were mutant, and whenever they did not carry mutation 3 their ERGs were wild-type. The results were clear-cut in suggesting that 1) mutation 3 alone was sufficient to generate the mutant phenotype, and 2) the other mutations contributed very little to the mutant phenotype. All transgenic lines tested to this point were generated in a wild-type background. Therefore, they were not genotypically comparable withTrpP365/+ because of the presence of endogenoustrp+, even though the ratio of mutant to wild-type trp gene copy number was one, as inTrpP365/+ (2:2 in the transgenic flies and 1:1 in TrpP365/+). In fact, ERGs of all transgenic flies carrying two copies of mutation 3 tested were not nearly as severely mutant as that of TrpP365/+ (compare Fig. 2, a–c and Fig.3b), suggesting that the presence of the extra wild-type copies of the trp gene might be influencing the ERG phenotype. To assess properly whether or not mutation 3 corresponds toTrpP365/+, we needed to carry out a complementation test in the absence of the endogenous wild-typetrp. Accordingly, we placed the P[trp(3)] and P[trp(1234)] transgenes in a trp-null (trpP343) background. As a positive control, the P[trp(124)] transgene was also placed in atrp-null background. If mutation 3, indeed, is the same asTrpP365, one would expect that transgenic flies homozygous for a transgene harboring mutation 3 would display phenotypes closely resembling those of TrpP365. If mutation 3 is absent from the transgene, on the other hand, one would expect the transgene to fully complement the trp-null background. Fig. 3 compares the ERGs obtained from TrpP365heterozygotes and homozygotes (Fig. 3, b and e, respectively) with those obtained from transgenic flies homozygous for the transgenes harboring mutation 3 alone (P[trp(3)]) (Fig. 3c), all four mutations together (P[trp(1234)]) (Fig. 3d), or the only triplet combination that does not include mutation 3 (P[trp(124)]) (Fig. 3a), all placed in a trpP343background. ERGs were tested at three different ages, 0, 3, and 7 days post-eclosion. Because ERGs showed very little age-dependent variations, particularly between 3 and 7 days post-eclosion, results from 7 days were not included in Fig. 3. They were very similar to those at 3 days post-eclosion. At all three ages tested, the ERGs obtained from transgenic flies carrying (P[trp(124)]) were completely wild-type (Fig.3a). That is, the transgene completely complemented the nulltrp background when it harbored all the other three mutations but not mutation 3. The ERGs of transgenic flies carrying transgenes P[trp(3)] and P[trp(1234)], on the other hand, were both much more severely mutant than those ofTrpP365/+ heterozygotes and approached those ofTrpP365 homozygotes in severity. There was a tendency for ERGs of flies carrying mutation 3 alone to be somewhat less severe than those of flies carrying all four mutations (compare Fig. 3, c and d), but even the latter did not quite match ERGs of TrpP365 in severity (compare Fig. 3, d and e). At least four different transgenic lines were tested for each genotype, and the above generalizations held true across different transgenic lines, making it unlikely that the minor differences in phenotype between the transgenic flies and the original mutant are due to differences in the sites of insertion of transgenes. Thus, the results of the complementation test were consistent with the idea that mutation 3, and it alone, corresponds to the originalTrpP365 mutation, although mutation 3 alone never quite matched the severity of TrpP365 in ERG phenotypes. The slight disparity between behaviors of the transgenes and TrpP365 will be discussed To determine whether the ERG phenotypes of these mutants are by photoreceptor degeneration and whether mutation 3 alone is sufficient to we carried out was on the retinas of the same classes of flies that were tested for their ERGs (Fig. 3), as as wild-type and null trp mutant (trpP343) at 0, 3, and 7 days post-eclosion. The retinas were stained with to label filamentous actin in the rhabdomeres. As be seen in the normal in both wild-type flies (Fig. null mutants (Fig. as as transgenic flies carrying mutations 1, 2, and 4, at all three however, the of by 3. in and to be in two (Fig. most were that they were no and most no showed a complement of only of a isolated were even at (Fig. The degeneration phenotypes of the two classes of transgenic P[trp(3)] and P[trp(1234)] and were between those of TrpP365 homozygotes and heterozygotes but more closely that of homozygotes than that of The to was much than was from ERG Thus, the of degeneration in transgenic flies carrying all four mutations was indistinguishable from that of particularly at days 3 and 7 (compare Fig. and in the transgenic flies carrying only mutation 3 was somewhat At post-eclosion, most nearly However, the label was very and to be (Fig. 3, most were no and only a was in toTrpP365/+, in which most distinctly even at flies carrying mutations 1, 2, and had completely normal at all three ages tested These results were completely consistent with ERG results (Fig. that transgenic flies carrying mutation 3 alone more closely the original than in ERG whole cell from photoreceptor Yoon et al. (9Yoon J. Ben-Ami H.C. Hong Y.S. Park S. Strong L.L.R. Bowman J. Geng C. Baek K. Minke B. Pak W.L. J. Neurosci. 2000; 20: 649-659Crossref PubMed Google Scholar) showed that the TRP channels are constitutively suggesting that the excessive of the TRP channels causes the massive photoreceptor degeneration observed in the These that the in the trp gene ofTrpP365 the regulation of the TRP channel to an that the channels and remain in an age-dependent results that the amino acid residues that are in the TRP channel of TrpP365 might be important for the regulation of the TRP channel We have shown in the present of the four amino acid detected in the TRP channel of TrpP365 (9Yoon J. Ben-Ami H.C. Hong Y.S. Park S. Strong L.L.R. Bowman J. Geng C. Baek K. Minke B. Pak W.L. J. Neurosci. 2000; 20: 649-659Crossref PubMed Google Scholar), the F550I change in the fifth transmembrane segment is the mutation for the TrpP365 phenotype. The ERG and degeneration phenotypes of transgenic flies homozygous for mutation 3 alone or all four mutations together nearly the phenotypes of TrpP365 homozygotes when the transgenes are placed in a trp-null background (3Peretz A. Sandler C. Kirschfeld K. Hardie R. Minke B. J. Gen. Physiol. 1994; 104: 1057-1077Crossref PubMed Scopus (64) Google and 3 However, these phenotypes do not quite the severity ofTrpP365 homozygotes 3 and Moreover, the phenotypes of transgenic flies carrying only mutation 3 to be than those of transgenic flies carrying all four mutations (compare Fig. 3, c and Fig. and Because these behaviors of transgenes are observed in different lines of transgenic flies for each transgene, they are not to be due to differences in from different sites of transgene On the other hand, the transgenes had a trp promoter of only and were of (see “Experimental Procedures”). of gene constructs in the transgenes could for the minor differences in between the mutation transgenes and the original TrpP365 The gene for the of protein or the of gene The to be because the observed differences are only On the other hand, the slight in ERG and degeneration phenotypes between transgenic flies carrying mutation 3 alone and those carrying all four mutations be by differences in sites of transgene or the gene constructs used in transgenes. be that mutations 1, 2, and have minor on mutation 3, even though alone they do not any phenotype. The however, that mutation 3 be present for the mutant phenotypes to at all 1, 2, Moreover, mutation 3 alone is sufficient to phenotypes closely those of 1, 2, 3, That is, mutation 3 alone is necessary for the ERG and degeneration phenotypes of TrpP365 and sufficient for phenotypes closely those ofTrpP365. We in all mutation 3 corresponds to the original The amino acid caused by mutation 3 is the effect it has on the mutant phenotype. the substitution of one for of the TRP with those of other TRP-related channel subunits that Phe-550 of TRP is conserved in of all but not all other TRP-related tested (Fig. In the TRP-related this position is by Phe in one, as in and in the On the other hand, in all TRP-related tested of TRP a conserved this position (Fig. In no however, is this position by The following are from these and the previous 1) position is important for the regulation of TRP channel 2) The amino acid for this position to be different for different Phe for Drosophila and for because of different channel Ile to be particularly at this In the we why this position might be important for the regulation of TRP channel and why Ile, in particular, be a to this In a et al. J. A. R. Science. PubMed Scopus Google Scholar) the of the channel of by The channel is by four each of which two transmembrane one the and the other the these the of the channel. The four are like the of an and in a to a small near the of the channel. of Y. M. G. Neuron. 1997; 19: Full Text Full Text PDF PubMed Scopus Google Scholar) on channels that the by the as the for these of the channel to other channels as various channels and TRP-related of these channels, however, have transmembrane than in each or For these channels, the transmembrane corresponds to the and the fifth transmembrane corresponds to the of the channel. Thus, the do not the channel they are immediately to and are in a position to the of Phe-550 near the of corresponding to the of the is also the region of the channel at which are thought to to the Thus, the residues at 550 or in its immediate vicinity be in a position to channel would Ile at this position constitutive activity of the channel but not the residues found at this out that Ile has a that it does not with or Ile is at the position, of the other three has at the at is to the of the main by the of Scholar). The presence of a to the the of the the and could the in its et al. K. M. S. Ben-Ami H.C. B. Kirschfeld K. Minke B. J. Neurosci. 2000; 20: PubMed Google Scholar) showed that the of the TRP and TRPL channels is the and that a from an is to the channels in the absence of light stimulus. Thus, constitutive of the TRP channel the channel in the i.e. its We of the of and of for