In Vivo Light-induced and Basal Phospholipase C Activity in Drosophila Photoreceptors Measured with Genetically Targeted Phosphatidylinositol 4,5-Bisphosphate-sensitive Ion Channels (Kir2.1)
2004; Elsevier BV; Volume: 279; Issue: 46 Linguagem: Inglês
10.1074/jbc.m407525200
ISSN1083-351X
AutoresRoger Hardie, Yuchun Gu, Fernando Martín, Sean T. Sweeney, Padinjat Raghu,
Tópico(s)Photoreceptor and optogenetics research
ResumoThe phosphatidylinositol 4,5-bisphosphate (PIP2)-sensitive inward rectifier channel Kir2.1 was expressed in Drosophila photoreceptors and used to monitor in vivo PIP2 levels. Since the wild-type (WT) Kir2.1 channel appeared to be saturated by the prevailing PIP2 concentration, we made a single amino acid substitution (R228Q), which reduced the effective affinity for PIP2 and yielded channels generating currents proportional to the PIP2 levels relevant for phototransduction. To isolate Kir2.1 currents, recordings were made from mutants lacking both classes of light-sensitive transient receptor potential channels (TRP and TRPL). Light resulted in the effective depletion of PIP2 by phospholipase C (PLC) in approximately three or four microvilli per absorbed photon at rates exceeding ∼150% of total microvillar phosphoinositides per second. PIP2 was resynthesized with a half-time of ∼50 s. When PIP2 resynthesis was prevented by depriving the cell of ATP, the Kir current spontaneously decayed at maximal rates representing a loss of ∼40% loss of total PIP2 per minute. This loss was attributed primarily to basal PLC activity, because it was greatly decreased in norpA mutants lacking PLC. We tried to confirm this by using the PLC inhibitor U73122; however, this was found to act as a novel inhibitor of the Kir2.1 channel. PIP2 levels were reduced ∼5-fold in the diacylglycerol kinase mutant (rdgA), but basal PLC activity was still pronounced, consistent with the suggestion that raised diacylglycerol levels are responsible for the constitutive TRP channel activity characteristic of this mutant. The phosphatidylinositol 4,5-bisphosphate (PIP2)-sensitive inward rectifier channel Kir2.1 was expressed in Drosophila photoreceptors and used to monitor in vivo PIP2 levels. Since the wild-type (WT) Kir2.1 channel appeared to be saturated by the prevailing PIP2 concentration, we made a single amino acid substitution (R228Q), which reduced the effective affinity for PIP2 and yielded channels generating currents proportional to the PIP2 levels relevant for phototransduction. To isolate Kir2.1 currents, recordings were made from mutants lacking both classes of light-sensitive transient receptor potential channels (TRP and TRPL). Light resulted in the effective depletion of PIP2 by phospholipase C (PLC) in approximately three or four microvilli per absorbed photon at rates exceeding ∼150% of total microvillar phosphoinositides per second. PIP2 was resynthesized with a half-time of ∼50 s. When PIP2 resynthesis was prevented by depriving the cell of ATP, the Kir current spontaneously decayed at maximal rates representing a loss of ∼40% loss of total PIP2 per minute. This loss was attributed primarily to basal PLC activity, because it was greatly decreased in norpA mutants lacking PLC. We tried to confirm this by using the PLC inhibitor U73122; however, this was found to act as a novel inhibitor of the Kir2.1 channel. PIP2 levels were reduced ∼5-fold in the diacylglycerol kinase mutant (rdgA), but basal PLC activity was still pronounced, consistent with the suggestion that raised diacylglycerol levels are responsible for the constitutive TRP channel activity characteristic of this mutant. Phototransduction in Drosophila is mediated by a G-protein-coupled phospholipase C (PLC) 1The abbreviations used are: PLC, phospholipase C; TRP, transient receptor potential; TRPL, TRP-like; DAG, diacylglycerol; InsP3, inositol 1,4,5-trisphophate; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; GFP, green fluorescent protein; eGFP, enhanced GFP; WT, wild-type; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; LIC, light-induced current; PI, phosphatidylinositol.1The abbreviations used are: PLC, phospholipase C; TRP, transient receptor potential; TRPL, TRP-like; DAG, diacylglycerol; InsP3, inositol 1,4,5-trisphophate; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; GFP, green fluorescent protein; eGFP, enhanced GFP; WT, wild-type; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; LIC, light-induced current; PI, phosphatidylinositol. cascade, resulting in activation of two classes of light-sensitive channels TRP and TRPL. These are the prototypical members of the large and diverse family of "transient receptor potential" (TRP) channels many of which, including all of the most closely related "canonical" TRPC subfamily, are also regulated by PLC (reviewed in Refs. 1Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (947) Google Scholar, 2Minke B. Cook B. Physiol. Rev. 2002; 82: 429-472Crossref PubMed Scopus (526) Google Scholar, 3Hardie R.C. Annu. Rev. Physiol. 2003; 65: 735-759Crossref PubMed Scopus (199) Google Scholar). Although in most cases the exact mechanism of TRP channel gating remains unresolved, mounting evidence, both in Drosophila and in at least a subset of mammalian TRP homologues, now suggests that diacylglycerol (DAG) rather than inositol 1,4,5-trisphophate (InsP3) is the critical product of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis (4Chyb S. Raghu P. Hardie R.C. Nature. 1999; 397: 255-259Crossref PubMed Scopus (358) Google Scholar, 5Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Crossref PubMed Scopus (1237) Google Scholar), whereas some evidence suggests that the reduction in PIP2 itself may also be important (3Hardie R.C. Annu. Rev. Physiol. 2003; 65: 735-759Crossref PubMed Scopus (199) Google Scholar, 6Estacion M. Sinkins W.G. Schilling W.P. J. Physiol. 2001; 530: 1-19Crossref PubMed Scopus (126) Google Scholar, 7Hardie R.C. Raghu P. Moore S. Juusola M. Baines R.A. Sweeney S.T. Neuron. 2001; 30: 149-159Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Because of its central role in phototransduction and PLC signaling generally, it is important to understand the dynamics of PIP2 turnover. In recent years a number of attempts have been made to monitor PIP2 levels in vivo, many making use of the GFP-tagged PIP2 binding PH domain from PLCδ (8Stauffer T.P. Ahn S. Meyer T. Curr. Biol. 1998; 8: 343-346Abstract Full Text Full Text PDF PubMed Google Scholar). While this has provided valuable insight into dynamic and spatial aspects of PIP2 mobilization, the PLCδ PH domain also binds InsP3 with high affinity, and it is not always clear whether PIP2 or InsP3 levels are being monitored (Ref. 9Hirose K. Kadowaki S. Tanabe M. Takeshima H. Iino M. Science. 1999; 284: 1527-1530Crossref PubMed Scopus (452) Google Scholar, but see also Ref. 10van der Wal J. Habets R. Varnai P. Balla T. Jalink K. J. Biol. Chem. 2001; 276: 15337-15344Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Fluorescence-based technologies are also of restricted value in photoreceptors, because the excitation light represents a saturating and usually damaging stimulus for the cell. Therefore we adopted an alternative approach by using an electrophysiological biosensor in the guise of the well characterized PIP2-sensitive ion channel, Kir2.1 (7Hardie R.C. Raghu P. Moore S. Juusola M. Baines R.A. Sweeney S.T. Neuron. 2001; 30: 149-159Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Like all members of the inwardly rectifying Kir family, these channels require phosphoinositide binding for their activity, but Kir2.1 has the highest specificity for PIP2 showing, for example, no detectable activation by PI or phosphatidylinositol 3,4-bisphosphate and <5% activation by phosphatidylinositol 4-phosphate (PIP) or phosphatidylinositol 3,4,5-trisphosphate (11Rohacs T. Chen J. Prestwich G.D. Logothetis D.E. J. Biol. Chem. 1999; 274: 36065-36072Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 12Rohacs T. Lopes C.M. Jin T. Ramdya P.P. Molnar Z. Logothetis D.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 745-750Crossref PubMed Scopus (173) Google Scholar). In an initial study we expressed eGFP-tagged Kir2.1 channels in Drosophila photoreceptors under the control of the rhodopsin promoter and found them to be specifically targeted to the light-transducing microvillar membrane. In whole cell recordings from dissociated cells, the channels generated large constitutive currents, which could be rapidly and reversibly suppressed by light, representing PIP2 hydrolysis by PLC and its subsequent resynthesis (7Hardie R.C. Raghu P. Moore S. Juusola M. Baines R.A. Sweeney S.T. Neuron. 2001; 30: 149-159Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar).In the present study we have developed this technology further by expressing a mutant version of the Kir2.1 channel with reduced affinity for PIP2 with a dynamic range that more effectively covers the range of physiologically relevant PIP2 levels. We expressed the channels in several mutant backgrounds, including flies lacking the light-sensitive TRP and TRPL channels so that Kir2.1 currents could be recorded in isolation. Our results indicate that activated PLC can deplete PIP2 at rates well in excess of 100% s–1. In addition our data provide in vivo measurements of basal rates of PLC activity that, although they are orders of magnitude less than that of activated PLC, can still deplete all detectable PIP2 within minutes if PIP2 resynthesis is blocked. As well as providing unique quantitative information on the in vivo activity of PLC, the results also shed light on some recent studies concerning the mechanism of phototransduction.EXPERIMENTAL PROCEDURESFlies—Drosophila melanogaster were raised in the dark at 25 °C. The eGFP-KiR2.1R228Q construct was generated from the wild-type (WT) eGFP-Kir2.1 construct previously described (7Hardie R.C. Raghu P. Moore S. Juusola M. Baines R.A. Sweeney S.T. Neuron. 2001; 30: 149-159Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 13Baines R.A. Uhler J.P. Thompson A. Sweeney S.T. Bate M. J. Neurosci. 2001; 21: 1523-1531Crossref PubMed Google Scholar) using the QuikChange mutagenesis system (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Following mutagenesis, the presence of the R228Q mutation and no other was confirmed by sequencing of the full gene. The eGFP-KiR2.1R228Q gene was then subcloned into pUAST and injected into yw embryos to obtain transformants as previously described (7Hardie R.C. Raghu P. Moore S. Juusola M. Baines R.A. Sweeney S.T. Neuron. 2001; 30: 149-159Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 13Baines R.A. Uhler J.P. Thompson A. Sweeney S.T. Bate M. J. Neurosci. 2001; 21: 1523-1531Crossref PubMed Google Scholar). Expression was driven by the Gal4-UAS system (14Brand A.H. Perrimon N. Development. 1993; 118: 401-415Crossref PubMed Google Scholar) under control of the rhodopsin promoter (p[Rh1-Gal4] flies provided by J. Treismann and F. Pichaud). p[UAS Kir2.1], p[Rh1-Gal4] recombinants for both WT and R228Q Kir2.1 channels were generated on both second and third chromosomes to facilitate crossing into a variety of mutant backgrounds, including trp343, a null mutant of the dominant light-sensitive and Ca2+ permeable channel (15Niemeyer B.A. Suzuki E. Scott K. Jalink K. Zuker C.S. Cell. 1996; 85: 651-659Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar); trpl302, null mutant of the second class of light-sensitive channel (15Niemeyer B.A. Suzuki E. Scott K. Jalink K. Zuker C.S. Cell. 1996; 85: 651-659Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar); rdgA1, the most severe allele of the photoreceptor DAG kinase (16Masai I. Okazaki A. Hosoya T. Hotta Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11157-11161Crossref PubMed Scopus (136) Google Scholar); norpAP24, a null or near-null allele of the photoreceptor phospholipase C (17Pearn M.T. Randall L.L. Shortridge R.D. Burg M.G. Pak W.L. J. Biol. Chem. 1996; 271: 4937-4945Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar); and Gαq1, a severe allele of the photoreceptor Gq protein with 80%. Data were collected and analyzed using Axopatch 1-D or 200 amplifiers and pCLAMP 8 or 9 software (Axon Instruments, Foster City, CA). Cells were stimulated via one of two green light-emitting diodes, with maximum effective intensity of ∼2 × 105 and 8 × 107 photons s–1 per photoreceptor, respectively. Relative intensities were calibrated using a linear photodiode and converted to absolute intensities in terms of effectively absorbed photons by counting quantum bumps at low intensities in WT flies (e.g. Ref. 20Henderson S.R. Reuss H. Hardie R.C. J. Physiol.-Lond. 2000; 524: 179-194Crossref PubMed Scopus (118) Google Scholar).With normal bath solutions, the constitutive currents generated by Kir2.1 channels were often very large (>5 nA), potentially leading to unacceptable series resistance errors. We therefore reduced the current amplitudes by recording in the presence of 4 mm Cs+, which partially blocks Kir2.1 channels. Furthermore, the block is voltage-dependent such that the current voltage (I-V) relationship reaches a maximum inward current at voltage of ∼–84 mV (Fig. 1). The current at this unique voltage can always be found by applying voltage ramps (typically from –100 to –40 mV) and determining the maximum current level, which will always occur at the same absolute voltage irrespective of any series resistance error. More usually, cells were unambiguously clamped at –84 mV using a manual offset voltage adjustment to find the maximum current, which was regularly checked throughout the recording.Estimating Resting PIP2 Levels—To compare PIP2 levels in different genetic backgrounds, dark-adapted Kir current levels were normalized to the background-subtracted GFP fluorescence measured from at least 10 randomly selected ommatidia from the same preparations using a microfluorimetry system (Photon Technology International) incorporating a photomultiplier, which measured the fluorescence above 510 nm induced by 485-nm excitation from a 75-watt xenon arc lamp.RESULTSThe Dynamic Range of Kir2.1R228Q Matches Endogenous PIP2 Levels—To track PIP2 levels in vivo we previously expressed the wild-type eGFP-tagged Kir2.1 channel (Kir2.1WT) in Drosophila photoreceptors under control of the rhodopsin (Rh1) promoter. The channels localize almost exclusively to the light-transducing microvilli, where they generate large constitutive inwardly rectifying currents, which can be suppressed by light as PIP2 is hydrolyzed by PLC, and recover in the dark as PIP2 is resynthesized. Most notably, when Ca2+ influx via TRP channels was prevented by blocking them with La3+, modest stimulation by light resulted in near complete suppression of Kir currents, indicating essentially total depletion of PIP2 as well as PI and PIP. This suggested that Ca2+ influx via TRP channels is normally required to inhibit PLC and/or facilitate PIP2 resynthesis (7Hardie R.C. Raghu P. Moore S. Juusola M. Baines R.A. Sweeney S.T. Neuron. 2001; 30: 149-159Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). We concluded that the resulting light-induced depletion of PIP2 in the trp mutant was the underlying cause of the long-debated trp ("transient receptor potential") phenotype (21Cosens D.J. Manning A. Nature. 1969; 224: 285-287Crossref PubMed Scopus (445) Google Scholar), whereby the response to maintained light in trp mutants decays to baseline, and thereafter the photoreceptors remain insensitive to further stimulation, recovering their sensitivity slowly in the dark as the PIP2 is resynthesized.The loss of PIP2 inferred from the suppression of the Kir current correlated in general with the loss of sensitivity of the light-induced current (LIC); however, there were significant quantitative discrepancies. In particular, suppression of the Kir current (representing PIP2 loss) required higher light intensities than the suppression of the LIC, whereas recovery of the Kir current was faster than the recovery of the light response, although both presumably reflect resynthesis of PIP2 as recovery of both was blocked in mutants of the PIP2 recycling pathway (7Hardie R.C. Raghu P. Moore S. Juusola M. Baines R.A. Sweeney S.T. Neuron. 2001; 30: 149-159Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). We attributed these differences to the known very high affinity of the Kir2.1 channel for PIP2 (22Zhang H.L. He C. Yan X.X. Mirshahi T. Logothetis D.E. Nature Cell Biol. 1999; 1: 183-188Crossref PubMed Scopus (293) Google Scholar). To confirm this and in an attempt to generate a more accurate probe for PIP2, we generated a point mutation (R228Q) in Kir2.1, which had previously been reported to substantially reduce the effective affinity of the channel for PIP2 (22Zhang H.L. He C. Yan X.X. Mirshahi T. Logothetis D.E. Nature Cell Biol. 1999; 1: 183-188Crossref PubMed Scopus (293) Google Scholar). We first confirmed a reduced effective affinity of ∼4-fold by expressing the channels in Drosophila S2 cells and measuring the dose response function to exogenously applied PIP2 in inside-out patches (Supplemental Fig. S1). We then expressed the channels in the photoreceptors and found that, like Kir2.1WT, the channels were also almost exclusively targeted to the microvillar membrane and mediated large inwardly rectifying K+ currents. After normalization for expression levels, the Kir currents in cells expressing Kir2.1R228Q channels were only 36% (±4% S.E., n = 27) of those expressing Kir2.1WT, suggesting that the endogenous PIP2 levels in the dark were only sufficient to partially activate Kir2.1R228Q. As will be described in more detail elsewhere, 2Y. Gu, M. Postma, G. Thomas, P. Raghu, and R. C. Hardie, manuscript in preparation. this was confirmed by showing that Kir2.1R228Q channel activity in the photoreceptors could be further increased at least 2-fold by application of exogenous PIP2 while Kir2.1WT channels seemed to be saturated (i.e. additional PIP2 did not significantly increase currents). Furthermore, like the WT channel (23Lopes C.M. Zhang H. Rohacs T. Jin T. Yang J. Logothetis D.E. Neuron. 2002; 34: 933-944Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar), Kir2.1R228Q channels have a PIP2 dose response function with a Hill coefficient close to 1.0 (Supplemental Fig. S1); together with the low (<50%) level of activation, this means that the current should be approximately directly proportional to physiological PIP2 levels.We quantitatively compared the ability of calibrated light stimuli to suppress Kir currents on the one hand and to inactivate the LIC on the other. With Kir2.1WT channels, increasing intensities of light, delivered to cells in the presence of La3+ to block TRP channels, progressively suppressed both Kir current and the LIC; however, ∼9× higher intensities were required to suppress the Kir current (Fig. 2). By contrast, in flies expressing Kir2.1R228Q, suppression of sensitivity and Kir current were much more closely matched with only a 2.5-fold shift. A very similar behavior (2-fold shift) was also seen in trp mutants expressing Kir2.1R228Q and recorded in the absence of La3+.Fig. 2Comparison of Kir2.1WT and Kir 2.1R228Q channels. In the presence of La3+, a 5-s light stimulus (bar, ∼60,000 photons) resulted in a partial "trp decay" and an associated ∼70% suppression of the responses to dim test flashes (arrows). In a cell expressing Kir2.1WT (A) the Kir current was only suppressed by ∼10%; however, in a cell expressing Kir2.1R228Q (B), the Kir current was reduced by ∼50%. The upper dotted lines (zero current) represent the resting current after total suppression. The graphs (C and D) plot the intensity of the "depleting" stimulus versus normalized suppression of both Kir current (open symbols) and the response to test flashes (LIC: closed symbols) (mean ± S.E. n = 6–7 cells). For the Kir 2.1WT channel the fitted curves (Equation 1) indicate an 8.6-fold mismatch between sensitivity of the LIC and the Kir current to suppression, whereas with Kir2.1R228Q there is only a relative shift of ∼2.5-fold. Similar experiments using the trp mutant expressing Kir2.1R288Q in the absence of La3+ revealed a similar behavior (shift of 1.98; n = 5; data not shown).View Large Image Figure ViewerDownload (PPT)We also compared the time courses of the recovery of Kir current (representing PIP2 resynthesis) and the LIC following stimuli inducing substantial PIP2 depletion (Fig. 3). Currents mediated by Kir2.1WT channels recovered relatively rapidly (t½ ∼ 30 s), but significant sensitivity to light only began to be restored after the Kir current had almost fully returned to pre-stimulation levels. By contrast, using Kir2.1R228Q the LIC and Kir current recovered over a similar time course (t½ ∼ 50 s). We also discovered that PIP2 synthesis could be routinely monitored at the start of any recording made in the presence of La3+ or the absence of Ca2+, because without Ca2+ influx via the TRP channels even the red light used for observation during electrode approach (equivalent to ∼200–400 effectively absorbed photons per second) was sufficient to significantly deplete PIP2. Consequently sensitivity was often very low immediately after establishing the whole cell configuration (break-in) but then increased over a period of 2–3 min in the dark. Similarly, the Kir current was typically relatively small at first but then increased over a similar time course, starting as soon as (but not before) the red light was turned off. In cells expressing Kir2.1WT channels there was only a modest (28 ± 7%, n = 7; mean ± S.E.) increase in Kir current, which was not well correlated with the increase in sensitivity to light. However, over the same experimental time period in cells expressing Kir2.1R228Q the current typically increased over 3-fold (369 ± 38%, n = 34) over the first ∼3 min in the dark and, when measured, closely paralleled a similar increase in the sensitivity to light (Fig. 3C). A similar "run-up" behavior was first described over ten years ago in recordings made in Ca2+ free Ringer (24Hardie R.C. Minke B. Neuron. 1992; 8: 643-651Abstract Full Text PDF PubMed Scopus (574) Google Scholar, 25Hardie R.C. J. Comp. Physiol. A. 1995; 177: 707-721Crossref PubMed Scopus (20) Google Scholar), but at that time it was speculated that it might represent refilling of intracellular Ca2+ stores.Fig. 3PIP2 resynthesis time courses. Following a flash (bar) that induced ∼50–75% depletion (recorded in WT flies in the presence of 20 μm La3+) the Kir2.1WT current (A) recovered with a t½ of ∼30 s, whereas Kir2.1R228Q recovery (B) was slower. The simultaneously recorded responses to brief test flashes (vertical deflections) recovered with similar time course in cells expressing Kir2.1R228Q, but the Kir2.1WT current almost completely recovered before significant recovery of the light response (note the initial phase of resynthesis in B is masked by the slow component of the Kir2.1 response to PIP2 depletion). C, the increase of sensitivity to light flashes and Kir2.1R228Q current recorded during "run up" shortly after establishing the whole cell recording configuration (time after w-c) were closely matched. Dotted lines represent zero current (i.e. after complete suppression of Kir current). Normalized time courses of Kir current recovery (solid symbols) and light response (open symbols) are shown on the accompanying graphs. D, half times (t½) for 50% recovery of the light response (LIC), Kir2.1WT and Kir2.1R228Q currents following 50–75% suppression by PIP2 "depleting" stimuli in the presence of 20–40 μm La3+ and (for Kir2.1R228Q only) also following a maximum intensity stimulus under "physiological" conditions in the absence of La3+ (see also Fig. 4) (mean ± S.E., n = 7–11 cells).View Large Image Figure ViewerDownload (PPT)In summary, the activity of Kir2.1R228Q channels shows a close quantitative correlation with the sensitivity to light over a range of PIP2 levels, and the channels' operating range thus appears to effectively cover the PIP2 levels relevant to phototransduction. Although Kir2.1WT channels are more sensitive to low levels of PIP2, e.g. during the initial period of PIP2 resynthesis following depletion, they seem to be largely saturated by the dark-adapted resting level of PIP2. We also note that the large differences in sensitivity to suppression by light and the time course of the subsequent recovery, following a point mutation in Kir2.1 known to reduce its effective affinity for PIP2, represent strong additional confirmation that Kir2.1 channels are directly monitoring PIP2 levels under these conditions.Light-induced PLC Activity—We previously found that, under control "physiological" conditions with both TRP and TRPL function intact, even very bright stimuli only suppressed the Kir2.1WT current by ∼20%. It could also not be excluded that even this modest suppression might have represented modulation by the large light-induced ion fluxes (which include Na+, K+, Ca2+, and Mg2+) rather than PIP2 depletion. We therefore repeated these experiments using flies expressing Kir2.1R228Q, reasoning that if PIP2 levels were in fact reduced under these conditions, there should now be substantially more suppression of the Kir current. Indeed, the Kir2.1R228Q channels were significantly more sensitive to suppression by light; nevertheless, even the brightest stimuli tested (∼106 photons, roughly equivalent to full daylight intensities) failed to suppress more than ∼40% of the current (Fig. 4). The currents then recovered with the typical time course seen in the presence of La3+ (t½ ∼ 50 s: Figs. 3D and 4B). These results indicate that there is indeed a significant, though not debilitating loss of PIP2 during light adaptation under physiological conditions.Fig. 4Depletion of PIP2 under "physiological" conditions.A, the current mediated by Kir2.1WT channels expressed on a WT background and recorded without La3+ was maximally suppressed by only ∼10–20% with stimuli approximating full daylight intensities (∼106 photons delivered with a 5-s stimulus). B, by contrast Kir2.1R228Q channels were suppressed by ∼40% (shown on a longer time scale in the inset below). Vertical deflections (B only) are responses to brief test flashes. C, response intensity function for Kir suppression using either Kir2.1WT or Kir2.1R228Q channels. Also shown are data from Kir2.1R228Q channels expressed in the trp mutant and recorded without La3+ (n = 4–12 cells).View Large Image Figure ViewerDownload (PPT)In experiments described thus far, the Kir current was simultaneously recorded with the LIC. Although this approximates physiological conditions, it compromises accurate measurement of the Kir current, because it cannot be cleanly separated from the LIC and because the Kir current may be indirectly influenced by ion fluxes associated with the LIC. To isolate the Kir current we expressed and recorded from Kir2.1 channels in trp;trpl double mutants or in trpl mutants in the presence of La3+. In either case this results in total elimination of all native light-sensitive currents (15Niemeyer B.A. Suzuki E. Scott K. Jalink K. Zuker C.S. Cell. 1996; 85: 651-659Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 26Reuss H. Mojet M.H. Chyb S. Hardie R.C. Neuron. 1997; 19: 1249-1259Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), and the electrophysiological response to light now consisted solely of a suppression of the constitutive Kir current uncontaminated by any other conductance. Suppression and recovery of the Kir current should thus now provide basic quantitative information on rates of PIP2 hydrolysis and synthesis in the absence of feedback by Ca2+. Both the overall suppression and the rate of suppression increased with brief flashes of increasing intensity (Fig. 5). In terms of the overall suppression reached, the data could be reasonably well fitted by a simple equation that assumes that each absorbed photon effectively depletes a number (n) of microvilli of their PIP2; namely in Equation 1 (see Ref. 7Hardie R.C. Raghu P. Moore S. Juusola M. Baines R.A. Sweeney S.T. Neuron. 2001; 30: 149-159Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar),I/Imax=e(−p/(m/n))(Eq. 1) where p is the number of effectively absorbed photons, m is the total number of microvilli (estimated at 45,000), and I/Imax is the normalized re
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