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. Author manuscript; available in PMC: 2015 Mar 17.
Published in final edited form as: Curr Biol. 2014 Mar 6;24(6):621–629. doi: 10.1016/j.cub.2014.02.026

SLEEPLESS is a bi-functional regulator of excitability and cholinergic synaptic transmission

Meilin Wu 1, James E Robinson 1,2, William J Joiner 1,2,3,4
PMCID: PMC4059605  NIHMSID: NIHMS569440  PMID: 24613312

Summary

Background

Although sleep is conserved throughout evolution, the molecular basis of its control is still largely a mystery. We previously showed that the quiver/sleepless (qvr/sss) gene encodes a membrane-tethered protein that is required for normal sleep in Drosophila. SLEEPLESS (SSS) protein functions, at least in part, by upregulating the levels and open probability of Shaker (Sh) potassium channels to suppress neuronal excitability and enable sleep. Consistent with this proposed mechanism, loss-of-function mutations in Sh phenocopy qvr/sss null mutants. However, sleep is more genetically modifiable in Sh than in qvr/sss mutants, suggesting that sss may regulate additional molecules to influence sleep.

Results

Here we show that SSS also antagonizes nicotinic acetylcholine receptors (nAChRs) to reduce synaptic transmission and promote sleep. Mimicking this antagonism with the nAChR inhibitor mecamylamine or by RNAi knockdown of specific nAChR subunits is sufficient to restore sleep to qvr/sss mutants. Regulation of nAChR activity by SSS occurs post-transcriptionally since the levels of nAChR mRNAs are unchanged in qvr/sss mutants. Regulation of nAChR activity by SSS may in fact be direct, since SSS forms a stable complex with and antagonizes fly nAChR function in transfected cells. Intriguingly, lynx1, a mammalian homolog of SSS, can partially restore normal sleep to qvr/sss mutants, and lynx1 can form stable complexes with Shaker-type channels and nAChRs.

Conclusions

Together, our data point to an evolutionarily conserved, bi-functional role for SSS and its homologs in controlling excitability and synaptic transmission in fundamental processes of the nervous system such as sleep.

Introduction

Sleep is an essential process that is highly conserved across evolution, yet its functions and underlying mechanisms of control are poorly understood. One of the most conserved features of sleep is its association with large-scale changes in brain activity relative to the waking state, suggesting that modulation of neuronal activity may be central to the regulation of sleep. In support of this hypothesis, homologous genes in mammals and flies encoding ion channels and ionotropic receptors have been shown to be necessary for normal sleep [1–4]. The requirement for potassium (K) channels in the Shaker (Sh) family is particularly notable. In both mammals and flies, loss-of-function mutations in Sh or its orthologs cause reduced sleep [2, 4]. Although little is known about whether modulators of Sh-type channels might control sleep in mammals, loss-of-function mutations in either of two genes that upregulate Sh, hyperkinetic (Hk) and quiver/sleepless (qvr/sss), result in reduced sleep in flies [5–7]. Unlike Hk, which is a cytosolic protein, SSS is anchored by glycosylphosphatidylinositol (GPI) to the outer leaflet of the plasma membrane, where it may associate with the extracellular surfaces of other membrane proteins. Evidence suggests that one such protein is Sh itself. For example, SSS and Sh are expressed in many of the same regions of the fly brain; SSS can form a stable complex with and upregulate levels of Sh protein; and SSS can increase the activation kinetics and decrease C-type inactivation kinetics of Sh channels in excised patches of transfected cells [6–8].

However, differences between the sleep phenotypes of Sh and qvr/sss mutants suggest that the two genes may not act in exactly the same signaling pathways. For example, over several generations selection pressure appears to favor the accumulation of genetic modifiers in populations of Sh mutants to compensate for loss of sleep [4]. In contrast, loss of sleep in qvr/sss null mutants is not lost over the same time span, if at all (unpublished data), signifying that the qvr/sss phenotype cannot be easily overcome by other naturally occurring alleles in the genome. Furthermore, although Sh mutants exhibit homeostatic recovery sleep following periods of sleep deprivation [4], this process is impaired in qvr/sss mutants [6]. Collectively, these differences suggest that SSS may have other downstream effectors which, in combination with Sh, control sleep.

Clues about the identities of such effectors may be gleaned from the predicted structure of SSS. Modeling of the tertiary structure reveals that SSS is a member of a large family of proteins, including snake α-neurotoxins, which possess a “three-finger” fold that has also been referred to as the ly6 domain [7]. Intriguingly, many of the targets of α-neurotoxins have been identified as ion channels or acetylcholine signaling pathways. For example, FS-2 blocks voltage-gated Ca channels [9]; fasciculin-I blocks acetylcholinesterase [10]; MT2 acts on muscarinic acetylcholine receptors [11]; and α-bungarotoxin inhibits nicotinic acetylcholine receptors (nAChRs) [12]. Although ly6 domain-encoding homologs of qvr /sss have also been identified in the genomes of non-venomous animals [13, 14], in most cases the endogenous binding partners of the corresponding proteins have not been identified.

In mammals, however, evidence strongly suggests that two of these proteins, lynx1 and lynx2, are endogenous antagonists of nAChRs. For example, lynx1 and lynx2 can complex with and accelerate the desensitization rates of α4β2 nAChRs [15, 16]. Consistent with these effects, lynx1 knockout mice have extended critical periods for ocular dominance plasticity that requires nAChR activity [17], and lynx2 knockout mice have increased nicotine-evoked EPSPs in prefrontal cortical pyramidal neurons [18].

As a result of such studies, we hypothesized that the ly6 domain-containing protein SSS may also antagonize nAChRs to reduce synaptic transmission. Here we present evidence to support this hypothesis and demonstrate that the resulting receptor inhibition is required for normal sleep in Drosophila. Specifically, we show that pharmacological or genetic reduction of nAChR signaling restores sleep to qvr/sss mutants. We also show that SSS regulates nAChRs post-transcriptionally, most likely by direct inhibition since SSS co-immunoprecipitates with the fly nAChR Dα3 and inhibits nAChR activity in transfected cells. Finally, we show that the mammalian SSS homolog lynx1 restores sleep to qvr/sss mutants and co-immunoprecipitates with a murine homolog of Sh, Kv1.2. Collectively our data suggest the existence of an evolutionarily conserved, dual role for SSS-like molecules in reducing excitability and synaptic transmission to control essential nervous system functions like sleep.

Results

SSS-expressing neurons are required for waking

We previously demonstrated that loss-of-function mutations in qvr/sss or Sh lead to increased mEPSP frequency and reduced Sh current in individual muscle fibers at the fly neuromuscular junction (NMJ). We thus concluded that loss of SSS or Sh leads to increased excitability. Since this effect is also accompanied by increased waking, we hypothesized that loss of sss or Sh leads to increased activity of wake-promoting neurons in the central brain. Loss of sleep in qvr/sss and Sh mutants cannot be attributed to seizure activity associated with hyperexcitability since such an effect would reduce locomotor activity, leading to an apparent increase in sleep, rather than the decrease actually observed for these animals. Loss of sleep in qvr/sss and Sh mutants also does not appear to be caused by hyperlocomotion associated with hyperexcitability since waking activity is unchanged in qvr/sss [6]. To determine the role of qvr/sss-expressing neurons in control of the sleep/wake cycle more directly, we used the Gal4/UAS system [19] to express temperature-sensitive TRPA1 channels [20] in neurons that normally express qvr/sss [7]. By activating these neurons with a temperature pulse of 29° C for 6 hours from zeitgeber time (ZT) 18–24, sss-Gal4/UAS-TRPA1 animals were deprived of sleep relative to pulsed controls (Figures 1a, S1a). Thus, activation of qvr/sss-expressing neurons appears to be sufficient for waking.

Figure 1.

Figure 1

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sss neurons promote waking and are sensitive to pharmacological inhibition of nAChRs and K channels. (a) sss neurons are sufficient for waking. sss-Gal4>UAS-TRPA1, UAS-TRPA1>+ and sss-Gal4>+ animals were heat pulsed to 27°C from ZT18-24 from a baseline temperature of 22°C. Change in sleep was calculated as sleep during the heat pulse minus sleep over the equivalent time period 24 hrs before. N > 13 animals for each group. (b) sss neurons are necessary for waking. sss-Gal4>UAS-shits, UAS-shits>+ and sss-Gal4>+ animals were heat pulsed to 28°C from ZT0-6 from a baseline temperature of 22°C. Change in sleep was calculated as in a but over 12 hrs due to a heat-dependent shift in timing of daytime sleep. N > 15 animals for each group. (c) The nAChR antagonist mecamylamine (MCA) dose-dependently restores sleep to qvr/sss and Sh mutants but has little effect on wild-type (WT) control animals (w1118, the genetic background of sssP1 and Shmns mutants). N = 19 - 40 animals for each group. (d) 24 hr sleep profiles of wild-type control (top), sssP1 (middle), and Shmns (bottom) animals used to collect the data in c. N = 19 – 40 animals for each plot. (e) The potassium channel antagonist 4-aminopyridine (4-AP) reduces sleep in wild-type controls but has no effect on sssP1/sssP2 and Shmns mutants. N = 10 – 69 animals for each group. (f) 24 hr sleep profiles of wild-type control (top), sssP1/sssP2 (middle), and Shmns (bottom) animals used to collect the data in e. N = 10 – 69 animals for each plot. * p< .01, *** p < 0.001 by unpaired t-test (a,b) or one-way ANOVA (c,e) with Bonferroni post-test. ns, not significant. Error bars indicate s.e.m. See also Figure S1.

In addition, we expressed temperature-sensitive shibire (shits) in qvr/sss neurons under Gal4/UAS control. shits encodes a dominant mutant version of dynamin ATPase that blocks endocytosis and thus exocytosis at restrictive temperatures [21]. By raising the temperature of sss-Gal4/UAS-shits animals to 28°C for 6 hours from ZT0-6, we found that sleep significantly increased in experimental animals compared to controls that lacked sss-Gal4 or shits expression (Figure 1b, S1b). This change is indeed sleep and not sickness or paralysis since experimental animals were able to right themselves within 10 seconds of being inverted during the temperature pulse (28/31, sss-Gal4>UAS-shits; 32/32, sss-Gal4/+, 32/32, UAS-shits/+). Thus, activation of qvr/sss-expressing neurons also appears to be required for waking.

Pharmacological antagonism of nAChR and Sh activities influences sleep

We previously demonstrated that a reduction in either SSS or Sh causes a reciprocal reduction in the other protein [6, 7]. This observation is consistent with the idea that the two proteins can form a complex and mutually stabilize each other. These results led us to hypothesize that restoring the expression of Sh in qvr/sss mutants or vice-versa using the Gal4/UAS system might rescue the severe sleep defects observed in both groups of animals. However, expressing UAS-Sh in sssP1 mutants or UAS-sss in Shmns mutants under the control of sss-Gal4 was not sufficient to restore sleep to normal levels (Figure S1c), suggesting that additional factors might be required.

Interestingly, SSS is homologous to the endogenous nAChR antagonists lynx1 and lynx2 as well as to snake α-neurotoxins, some of which also affect acetylcholine signaling. Thus, we hypothesized that SSS might normally reduce nAChR activity in wake-promoting neurons to enable animals to sleep. According to this hypothesis, nAChR activity should be elevated in qvr/sss loss-of-function mutants and perhaps in Sh mutants as well, since SSS upregulates Sh. To test this hypothesis, we fed wild type, sssP1 and Shmns flies increasing concentrations of the non-competitive nAChR antagonist mecamylamine (MCA) and measured sleep 24–48 hours later. We found that treatment of wild-type animals with drug caused only a small increase in sleep. In contrast, treatment of both sssP1 and Shmns mutants with MCA caused a dose-dependent restoration of sleep (Figures 1c and 1d). We interpret these changes as true sleep rather than sickness or paralysis because the behavioral state was acutely reversible with brief mechanical agitation, and waking activity with drug treatment was as high as controls (Figure S2a,b). These findings strongly suggest that the ability of SSS and Sh to promote sleep under normal conditions is mediated in part by a suppression of nAChR activity, and that loss of this suppression leads to reduced sleep in qvr/sss and Sh mutants. This mechanism seems to be at least somewhat selective for qvr/sss and Sh mutants since mecamylamine was not able to increase sleep in Clkjrk mutants (Fig S3), which have been reported to have low sleep [22–24].

Although we previously reported that SSS enhances both Sh protein levels as well as gating kinetics, at that time we did not demonstrate a direct role for Sh in mediating the low sleep phenotype of qvr/sss mutants. To examine this relationship, we tested whether acute pharmacological inhibition of Sh activity in wild-type animals could reduce sleep, as observed in sssP1 and Shmns mutants. To accomplish this task, we fed animals the voltage-gated potassium channel blocker 4-aminopyridine (4-AP). 4-AP treatment was able to dose-dependently reduce sleep in a wild-type background to levels similar to those observed in Shmns mutants but did not affect waking activity (Figures 1e, 1f and S2c). 4-AP appeared to act selectively on Sh rather than on other ion channels to reduce sleep since there was no effect of the drug on Shmns mutants (Figures 1e and 1f). Tellingly, in trans-heterozygous sssP1/sssP2 mutants in which sleep was reduced to levels equivalent to those observed in Shmns mutants, 4-AP also had no acute effect. These results suggest that the reduced sleep in qvr/sss mutants is at least partially mediated by a reduction in Sh activity, which in turn leads to increased neuronal excitability in arousal-promoting neurons, as previously hypothesized [6, 7].

qvr/sss genetically interacts with nAChR subunits to control sleep

After determining that acute antagonism of nAChR activity could restore sleep to sssP1 mutants, we asked which nAChR subunits might be aberrantly upregulated in the absence of SSS. To address this question we first determined which nAChR subunits are expressed in the adult fly brain. In mammals, nAChRs are homopentamers of alpha subunits or heteropentamers of alpha and beta subunits. In the fly genome there are 7 genes encoding alpha subunits (Dα1–7) and 3 genes encoding beta subunits (Dβ1–3). The combinations of receptor subunits that can form functional channels in flies is unknown, largely because it has been difficult to measure activity of cloned fly nAChRs in heterologous expression systems [25]. Using quantitative PCR (qPCR) to measure the levels of nAChR transcripts, we found that all but one subunit is enriched in the adult fly brain relative to the body (Figure S4a).

To determine which of these subunits might be responsible for the increased waking time of qvr/sss mutants, we reduced expression of each alpha or beta subunit in sss-expressing neurons by RNAi knockdown. Consistent with our hypothesis that nAChR activity is upregulated in qvr/sss mutants, knockdown of Dα3 in particular and to a lesser extent, Dβ3, partly restored sleep to sssP1 mutants (Figure 2a and 2b) without affecting waking activity (Figure S2d) but had no effect on sleep in control animals with normal levels of qvr/sss expression (Figure S4b). qPCR analysis of Dα3 transcripts from heads of pan-neuronal RNAi knockdown flies confirms a ~65% reduction in Dα3 expression levels (Figure S4c). These results suggest that excessive nAChR activity may be at least partly responsible for the increased waking time of sssP1 mutants. These results also indicate that nAChR activity does not normally need to be reduced by withdrawal of endogenous cholinergic signaling to allow wild-type animals to sleep.

Figure 2.

Figure 2

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RNAi knockdown of nAChR subunits restores sleep to qvr/sss mutants. (a) Sleep in sss-Gal4/+;UAS-RNAi/UAS-dicer animals compared to UAS-RNAi/+ controls (all in a sssP1 background). The low sleeping phenotype of sssP1 mutants is rescued by knockdown of Dα3 and to a lesser extent Dβ3. N > 26 for each group. (b) 24 hr sleep profiles of sssP1 alone or sssP1 animals in which Dα3 has been knocked down in sss neurons. (c) Overexpression of Dα3 in sss neurons reduces total daily sleep. N > 15 for each group. *** p<0.001 by one-way ANOVA with Bonferroni post-test. Error bars indicate s.e.m. See also Figure S2.

To determine whether upregulation of nAChR activity in qvr/sss-expressing neurons could reduce sleep, we coupled sss-Gal4 to a P-element (d08339) inserted ~40 bp upstream of the predicted transcriptional start site of the Dα3 gene. Since this P-element carries UAS sequences pointing in both directions along the X chromosome, animals bearing it in combination with sss-Gal4 should express elevated levels of Dα3 transcript. When we tested these animals we found that they expressed very high levels of Dα3 mRNA and slept less than controls (Figures S4d, 2c). Thus, upregulation of Dα3 nAChRs in qvr/sss-expressing neurons is sufficient to account for at least part of the low-sleeping phenotype of sssP1 mutants.

nAChRs are post-transcriptionally regulated by SSS and co-localize with SSS and Sh in the mushroom bodies

To determine if SSS can indirectly regulate nAChRs by transcriptional feedback, we performed qPCR on Dα3 and Dβ3, two of the nAChR subunits that genetically interacted with qvr/sss (Figure 2a and 2b). Just as we previously showed for regulation of Sh transcript [7], levels of fly brain nAChR transcripts are unchanged in sssP1 mutants. We also found that fly brain nAChR transcripts are similarly unchanged in Shmns mutants (Figure 3a). Collectively, these data support a post-transcriptional role for regulation of nAChR activity by SSS, although we cannot exclude the possibility that upregulation of nAChR transcript in qvr/sss mutants occurs in a small subset of the neurons in our whole brain preparations and is therefore undetectable in our assays.

Figure 3.

Figure 3

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Dα3 and Dβ3 are not transcriptionally regulated by SSS or Sh but co-express with them in the mushroom bodies. (a) Quantitative PCR analyses of Dα3 and Dβ3 transcripts show no dependence on qvr/sss or Sh for normal expression. N = 3 for each group. ns, not significant by one-way ANOVA with Bonferroni post-test. Error bars indicate s.e.m. (b) Coexpression of sss, Sh and Dα3 in the mushroom bodies. Representative immunostaining (N = 8) of tdTomato expressed under the control of sss-Gal4 (upper panel), native Sh protein (center panel), and native Dα3 protein (lower panel). Scale bar = 25μm. (c) MB-Gal80 suppresses rescue of sleep by sss-Gal4>UAS-sss in sssP1 mutants. N > 30 for all groups. *** p<0.001 by one-way ANOVA with Bonferroni post-test. Error bars indicate s.e.m.

If, however, SSS is acting directly on both K channels and nAChRs to suppress neuronal activity and promote sleep, then SSS and both effector proteins should be expressed in the same set of neurons. To determine if this is indeed the case, we labeled qvr/sss-expressing neurons with UAS-tdTomato expressed under the control of sss-Gal4. In the same brains we also stained for the presence of Sh and Dα3 using established antibodies for each molecule [7, 26]. We found overlapping expression of all 3 molecules in the mushroom bodies (MBs) (Figure 3b), which we and others previously showed to be important sleep-regulatory loci [27, 28]. Our cell labeling was likely to be specific for the intended antigens since Sh labeling disappeared in Sh mutants [7]; Dα3 labeling was significantly reduced when antibody was pre-incubated with lysate from COS cells transfected with Dα3 cDNA (Figure S5a–c); and Dα3 labeling increased in brains of animals in which sss-Gal4 was combined with UAS-Dα3 (i.e. P-element d08339) (Figure S5d). Thus, the sleep-regulating proteins SSS, Sh and Dα3 are expressed together in a known sleep-regulating locus in the fly brain.

To determine whether the MBs might contribute to sleep regulation by qvr/sss, we first coupled the sss-Gal4 driver to UAS-sss in a sssP1 mutant background. As we previously demonstrated [7], we found that the qvr/sss transgene was capable of fully rescuing the loss of sleep in sssP1 mutants. However, when we blocked expression of the qvr/sss transgene with MB-Gal80, a repressor of Gal4 activity that expresses in the MBs, restoration of sleep to otherwise genetically identical animals was reduced (Figure 3c). These data suggest that qvr/sss utilizes the MBs to regulate sleep, though they do not exclude the possibility that other brain regions may also be involved.

SSS forms stable complexes with both Sh and Dα3

Our findings that SSS upregulates Sh and antagonizes nAChR activity in vivo suggest that SSS might mediate these effects directly. We previously demonstrated that SSS can form a complex with Sh channels in Xenopus oocytes, which express K currents very robustly following injection of Sh cRNA. To determine if SSS can form complexes with Sh channels as well as nAChRs in transfected cells, we first added a MYC epitope near the C-terminus of SSS, just before the attachment site of glycosylphosphatidylinositol. We also fused green fluorescent protein (GFP) and hemagglutinin (HA) epitopes to the N- and C-termini of Sh and Dα3, respectively. These constructs were expressed in HEK-tsa cells, and complexes were immunoprecipitated with antibodies against either GFP (i.e. Sh) or HA (i.e. Dα3). Subsequent western blot analyses revealed that SSS can be co-immunoprecipitated with Sh (Figure 4a) or Dα3 (Figure 4b), but not in the absence of the channel or receptor (Figure 4a and 4b). These results demonstrate that SSS can form stable complexes with both Sh and Dα3.

Figure 4.

Figure 4

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SSS forms complexes with nAChRs and inhibits their activity. Representative immunoprecipitations of (a) GFP-tagged Sh or (b) HA-tagged Dα3 from transiently transfected Cos-7 cells. Co-transfected Myc-tagged SSS co-immunoprecipitates with Sh and Dα3 (lane 3, both blots) but not in the absence of Sh (lane 1, panel a) and Dα3 (b, lane 3). IP, immunoprecipitations. WB, western blots. (c) Concentration-response curves depicting α4β2 nAChR activity by the agonist epibatidine in the absence (solid line) and presence of SSS (dashed line). nAChR activity was determined by baseline-subtracted (i.e. agonist minus no agonist) FRET between the CFP and YFP domains of the genetically encoded Ca2+ sensor TN-XXL in HEK-tsa cells transiently transfected with mouse α4 and β2 nAChR subunits, TN-XXL, and either sss cDNA or empty vector (N = 4 for each point). (d) SSS suppresses maximal α4β2 nAChR activity. Quantitation of maximal values measured in c. All numbers were normalized to values obtained for activated α4β2 nAChRs in the absence of SSS (N=4). **p < 0.01 by paired t-test. Error bars indicate s.e.m in c and d.

SSS reduces nAChR activity

Regulation of Sh kinetics by SSS has previously been demonstrated in multiple expression systems [7, 8]. To determine if complexes formed by SSS and nAChRs also result in altered receptor activity, we examined the contribution of SSS to nAChR function in transfected HEK-tsa cells. For these experiments we used nAChRs from mice because fly nAChRs have been recalcitrant to functional expression on their own. In the absence of clear murine orthologs of fly nAChRs we chose α4/β2 for our assay because these receptors express well in heterologous expression systems. In our assay we measured agonist-dependent influx of Ca2+ through receptors using the ratiometric FRETable cytosolic Ca2+ reporter TN-XXL [29]. In the presence of α4/β2 and TN-XXL, the nicotinic agonist epibatidine elicited an increase in FRET ratio that was well-fit by a sigmoidal concentration-response curve with a half-maximal effective concentration (EC50) similar to previous reports [29]. In cells in which the qvr/sss cDNA was included in the transfection mixture, however, the maximal nAChR response was reduced by 75% (Figures 4c and 4d). Thus, in transfected cells, SSS suppresses nAChR function, just as in vivo. Taken together with our data showing that SSS and fly nAChRs can form a stable complex, it is likely that the functional suppression of nAChR activity by SSS is direct.

The molecular functions of SSS homologs are conserved across evolution

SSS structurally resembles both snake α-neurotoxins such as α-bungarotoxin, which blocks nAChRs, and other ly6-domain containing proteins such as the endogenous mammalian nAChR antagonists lynx1 and lynx2. While SSS and lynx1 share only 18% amino acid identity, they both contain a series of conserved cysteines that are thought to form disulfide bonds that maintain the characteristic “three-finger” tertiary structure of the ly6 domain [7]. Thus, it is possible that the tertiary structures of SSS and lynx1 are similar enough that the two proteins could function interchangeably.

To test this hypothesis, we first performed co-immunoprecipitation experiments with MYC-tagged lynx1 and HA-tagged Dα3 in transiently transfected HEK-tsa cells. Lynx1 is known to associate with mammalian nAChRs, and we found that like SSS, it could also form stable complexes with Dα3 (Figure 5a). We further tested to see if, like SSS, lynx1 was also able to associate with voltage-gated K channels. We fused GFP to the N-terminus of Sh as well as its mammalian homolog, Kv1.2, and co-transfected cells with each construct along with the MYC-tagged lynx1 cDNA. In both cases we found that lynx1 could be co-immunoprecipitated with the K channel (Figure 5b). The formation of these complexes cannot be attributed to a tendency for lynx1 to associate with other proteins non-specifically since lynx1could not be co-immunoprecipitated with the membrane protein GRID2 (Figure 5b). These results suggest that the targeting of voltage-gated K channels and nAChRs by ly6 proteins is conserved across evolution.

Figure 5.

Figure 5

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The molecular function of mammalian qvr/sss homologs is conserved. (a) Myc-tagged lynx1 co-immunoprecipitates with HA-tagged Dα3 in lysates from transiently transfected Cos-7 cells (lane 2) but not in the absence of Dα3 (lane 1). (b) Lynx1 also co-immunoprecipitates with GFP-tagged Kv1.2 (lane 3) and Sh (lane 7). Lynx1 does not co-immunoprecipitate with the GFP-tagged transmembrane protein Grid2 (lane 5). (c) Expression of UAS-lynx1 under the control of sss-Gal4 partially restores sleep to sssP1 mutants (N > 18 for each group). *** p < 0.001 by one-way ANOVA with Bonferroni post-test. Error bars indicate s.e.m.

Based on this conservation of molecular function, we tested whether lynx1 could replace SSS and allow flies to sleep normally. We generated UAS-lynx1 transgenic flies and expressed lynx1 under the control of sss-Gal4. Remarkably, we found that lynx1 can restore sleep to sssP1 mutants without altering waking activity (Figures 5c, S2e), demonstrating that lynx1 can function sufficiently like SSS to substitute for the latter in vivo.

Discussion

We previously demonstrated that SSS couples Sh levels and gating kinetics to reduced membrane excitability to allow sleep in Drosophila. Here we show that SSS interacts with and antagonizes nAChRs to promote sleep as well, and that the activity of sss-expressing neurons is both necessary and sufficient for this process in Drosophila. The molecular bi-functionality of SSS is unexpected since K channels and nAChRs are functionally and structurally unrelated. For example, Sh-type channels are gated by voltage, have 6 transmembrane domains and multimerize to form functional tetramers. In contrast, nAChRs are gated by synaptic release of acetylcholine, have 4 transmembrane domains and multimerize to form functional pentamers.

It is unclear which structural features SSS recognizes on each class of membrane protein, especially in the case of Sh, which is thought to expose little surface area outside the plasma membrane. X-ray structures of α-cobtratoxin (α-Cbtx) bound to Lymnaea stagnalis acetylcholine binding protein (LS-AChBP) show interactions between two loops of α-Cbtx and both the agonist binding pocket and the cis-loop of AChBP [30]. Using this information and an NMR structure of water-soluble lynx1, Lyukmanova et. al, modeled a possible interaction of lynx1 with the same site, although the loops of lynx1 were shorter than those of α-Cbtx, and did not form as many contacts with the AChBP [31].

It is also unclear whether such interactions, even if translated to SSS, would result in acute and direct antagonism of nAChR activity or, alternatively, reduction in targeting of receptors at the cell surface. Both potential mechanisms of action could account for the reduced α4/β2 activity we observed in cells transfected with qvr/sss. In addition, it is unknown whether a single SSS molecule can interact with both Sh and nAChRs simultaneously. However, our data suggest that all three proteins are co-expressed in some regions of the brain, particularly the mushroom bodies, which we and others have shown to play an important role in controlling sleep [27, 28, 32]. We previously showed that SSS is enriched in these structures as well as the antennal nerves, superior protocerebrum and the lobula plate of the optic lobes [7], and here we show that MB-Gal80 reduces the ability of sss-Gal4>UAS-sss to restore sleep to sssP1 mutants. Nonetheless, the contribution of the MBs relative to other brain loci in regulating sleep via SSS, Sh and Dα3 still needs to be determined.

A particularly intriguing feature of SSS is its ability to reduce both membrane excitability and synaptic transmission, which endows the protein with unusual gain control over neuronal activity. In the present context we propose that SSS reduces the activity of wake-promoting neurons through two pathways to permit sleep (Figure 6). In one, it enhances Sh K channel protein levels and open channel probability to reduce neuronal excitability. In another pathway, it inhibits nAChR signaling to reduce synaptic transmission. In qvr/sss mutants the processes are reversed in wake-promoting neurons: K channel activity is reduced, leading to increased excitability, and nAChR activity is increased, leading to increased synaptic transmission. In these as well as Sh mutants, reduction of nAChR expression by RNAi knockdown or of nAChR activity by pharmacological antagonism reduces the activity of wake-promoting neurons within a range in which sleep can once again occur (Figure 6).

Figure 6.

Figure 6

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Model for control of sleep by SSS in wake-promoting neurons. SSS reduces neuronal activity in arousal-promoting neurons through two pathways to permit sleep. SSS enhances Sh K channel activity to reduce intrinsic excitability (left side). SSS also suppresses nAChR activity to reduce synaptic transmission (right side). Pharmacological or genetic antagonism of nAChR activity functionally substitutes for SSS to restore sleep to sssP1 mutants (right side). Bi-directional arrow (left side) reflects mutual dependence of Sh and SSS for elevated expression levels of both proteins.

Our finding that the molecular properties of SSS are shared by the mammalian homolog lynx1 suggests that ly6 proteins in general may have evolved a multitude of roles in regulating basic properties of the nervous system. It is notable that fundamental plastic processes are perturbed in loss-of-function mutants of qvr/sss and lynx1. For example, in sssP2 hypomorphs sleep homeostasis is impaired [6], and in lynx1 knockouts the critical period for ocular dominance plasticity is extended to adulthood [17]. Although phenotypes have been described for additional ly6 mutants in both flies and mice, only in the case of SSS, lynx1 and lynx2 have molecular effectors been identified in vivo, and these are either K channels or nAChRs. Since over 20 ly6 genes have been described in Drosophila [14], and there appear to be many homologous genes in mammals, additional roles for these molecules may remain to be discovered.

In summary, we have identified a role for SSS in regulating synaptic transmission, in addition to its established role in regulating membrane excitability. Both functions appear to be important for the ability of SSS to regulate sleep in Drosophila. The ability of lynx1 to substitute for SSS as well as to form complexes with effectors of SSS, namely Sh-type K channels and nAChRs, suggests that lynx1 and perhaps other mammalian ly6 proteins possess similar, multifunctional roles in controlling neuronal activity. Potassium channels, nAChRs and their modulators are key regulators or targets for molecular intervention in various human disorders, including ataxias, congenital deafness, epilepsy, cardiac arrhythmias, type II diabetes, autoimmune diseases such as multiple sclerosis and rheumatoid arthritis, cognitive decline in Alzheimer's Disease, loss of motor coordination in Parkinson's Disease, nicotine addiction and its associated risk of developing cancer and cardiovascular disease from smoking [33–39]. Understanding the functions of ly6 proteins may provide insights into these disorders as well as new screening strategies for more selective and efficacious pharmacotherapeutic regulators of neuronal function.

Experimental Procedures

Fly stocks and transgenic fly lines

sssP1, sss-Gal4 and UAS-sss flies were described previously [6, 7]. Other lines were provided as follows: Shmns (Chiara Cirelli), UAS-shits (Toshi Kitamoto), UAS-TRPA1 (Paul Garrity). UAS-Dα3 (stock d03389) was from the Exelixis collection at Harvard. Other fly lines were obtained from the Bloomington Stock Center (UAS-dicer [24650]; UAS-tdTomato [32221]; UAS-nAChR RNAi's [28688, 27493, 27671, 31985, 25943, 25835, 27251, 31883, 28038 and 25927]). UAS-lynx1 flies were generated by targeting UAS-lynx1 in pUAST-attB to the attP site of y1,w67c23;;attP2 flies (Rainbow Transgenics, Camarillo, CA) and outcrossing transgenic animals into a w1118 iso31 background for 2 generations.

Behavior assays

1–5 day old flies were loaded into glass tubes containing 5% sucrose and 2% agarose and entrained to a 12hr:12hr light:dark (LD) cycle for 2 days before measuring sleep/wake patterns using the Drosophila Activity Monitoring System (Trikinetics). Sleep was defined as 5 minutes of inactivity and measured as previously described [22]. Experiments were carried out at 25°C except those involving 6 hour heat pulses to 27°C to activate TRPA1 or to 28°C to activate shits in which case baseline temperature was maintained at 22°C.

Prior to drug treatment, sucrose/agarose mixtures were equilibrated to 50°C and the appropriate amounts of concentrated 4-aminopyridine (pH adjusted to 7.0, Acros Organics) or mecamylamine (R&D, Tocris) were added. Animals that had been entrained for 2 days on sucrose/agarose alone were switched to activity tubes containing the drug mixture at ZT0, and sleep was measured beginning one day later for a period of 24 hours. Animals that died within 4 days of drug intake were excluded from analysis.

Quantitative PCR

For each sample, 30–50 brains or 7–12 heads from 5–9 day old flies were lysed in Trizol (Life Technologies), and first strand cDNA was synthesized from extracted RNA using High Capacity cDNA Reverse Transcription (ABI). Quantitative PCR was performed on each cDNA sample using the primers listed in Table 1 (Supplemental Experimental Procedures), and results were normalized to levels measured for RP49. Relative expression was further normalized to brain Dα1 levels (Figure S4a) or levels of Dα3 or Dβ3 transcript measured in w1118 controls (Figure 3). All primer pairs were validated for amplification efficiencies (R2) greater than 0.98.

Molecular Biology

To generate GFP-tagged Shaker, eGFP was amplified from eGFP-C3 (Clontech) by PCR using Hind-eGFP-F and eGFP-Xho-R primers (Table 2, Supplemental Experimental Procedures) and subcloned in frame at the 5' end of ShD in pcDNA3 [7] using HindIII and XhoI. GFP-tagged Kv1.2 was generated by replacing ShD with Kv1.2 that had been amplified by PCR with Xho-Kcna2-F and Kcna2-Not-R2 primers (Table 2, Supplemental Experimental Procedures) and cut with XhoI-NotI. GFP-GRID2 was similarly generated by amplifying eGFP with EcoR1-eGFP-F and eGFP-Kpn1-R primers (Table 2, Supplemental Experimental Procedures) and subcloning the fragment into the EcoRI and KpnI sites of Flag-GRID2 (kind gift from Z.Yue, Mt. Sinai).

To generate Myc-tagged SSS, EcoRV and Xho1 sites were first inserted into sss in pcDNA3 [7] by PCR using SSS-Cmyc-S and SSS-Cmyc-AS primers (Table 2, Supplemental Experimental Procedures). Cmyc-S and Cmyc-AS oligos encoding the Myc epitope (Table 2, Supplemental Experimental Procedures) were then annealed and subcloned into the newly engineered EcoRV and Xho1 sites. To generate Myc-tagged lynx1, an EST (I.M.A.G.E. ID 5325123, Open Biosystems) encoding full-length mouse lynx1 was first amplified by PCR with primers EcoR1-Lynx1-F and Lynx1-Not1-R (Table 2, Supplemental Experimental Procedures) and subcloned between the EcoRI and NotI sites of pcDNA3. A fragment of Myc-tagged SSS was then amplified by PCR with the primers Lynx1-Myc-F and Lynx1-Myc-R (Table 2, Supplemental Experimental Procedures), and the resulting product was used for primer extension using lynx1 in pcDNA3 as a template. The product of this reaction was then amplified with EcoR1-Lynx1-F and Lynx1-Not1-R primers (Table 2, Supplemental Experimental Procedures) and subcloned between the EcoR1 and Not1 sites of pcDNA3.

To generate hemagglutanin (HA)-tagged Dα3, we first subcloned oligos Not1-HA-Xba1-S and Not1-HA-Xba1-AS (Table 2, Supplemental Experimental Procedures) between the NotI and XbaI sites of pcDNA3 to make pcDNA3-HA. To fuse Dα3 to the HA epitope, Dα3 was amplified by PCR from an EST (DGRC clone number 12624) using primers Dα3-Kpn1-F and Dα3-Not-R (Table 2, Supplemental Experimental Procedures) and subcloned into the KpnI and NotI sites of pcDNA3-HA.

UAS-lynx1 was generated by subcloning the previously described full-length mouse EST into pUAST-attB between the EcoRI and NotI sites.

Immunohistochemistry

3–7 day old female brains were dissected in ice cold PBS before fixation in 4% paraformaldehyde. Brains were blocked in PBST (PBS, 0.3% Triton X-100) containing 5% normal donkey serum (Jackson Laboratory) and 5% normal goat serum (Life Technologies). Brains were incubated with 1:200 rat anti-Sh [40] pre-cleared with ShDf lysate followed sequentially by incubation with 1:250 rabbit anti-Dα3 bleed 88 [26]. Brains were washed with PBST and stained with 1:1000 Alexa 633 anti-rabbit (Life Technologies) and 1:1000 anti-rat Alexa 488 (Jackson ImmunoResearch). After additional washes, brains were equilibrated and mounted in Vectashield (Vector Labs). Images were taken at 40× magnification on a Leica SP5 confocal microscope using 0.5μm stack intervals. 5μm Z-projection images were generated, rotated and brightness/contrast adjustments were made across the entirety of the images using Fiji [41].

Cell Culture and Co-immunoprecipitations

Cos-7 and HEK-tsa cells were maintained at 25°C and 7.5% CO2 in culture medium consisting of 10% fetal bovine serum (Omega), 1% penicillin/streptomycin (Mediatech) and 1% L-glutamine (Sigma) in low glucose DMEM with 2 mM L-Glutamine (Mediatech). Cells were grown to 50–80% confluence for transfection with X-tremegene HP reagent (Roche) at a 2:1 ratio of transfection reagent to DNA in Optimem (Life Technologies). Transfection mixture was removed 24 hours after transfection and replaced with normal growth medium. Cells were lysed 48 hours after transfection in SDS lysis/IP buffer (10 mM Tris [pH 7.5], 100 mM NaCl, 5mM EDTA, 1% Triton X-100, and 0.05% SDS) with Complete protease inhibitors (Roche). For co-immunoprecipitation assays, 500 μg of total cell lysate was incubated in a total volume of 1 ml SDS lysis/IP buffer with primary antibodies overnight at 4°C on a rotating platform. The following day, protein-G conjugated magnetic beads (NEB) were added and incubated at 4°C for 2 – 5 hours. Beads were washed 3 times in a 1 ml volume of IP wash buffer (10 mM Tris [pH 7.5], 100 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, and 0.05% SDS) and resuspended in 2X LDS sample buffer (Life Technologies). Antibodies used were rabbit anti-GFP (Life Technologies) or rabbit anti-HA (Rockland).

Western Blot Analyses

Western blot analyses of Cos-7 cell lysates and co-immunoprecipitation complexes were performed as previously described [40]. For 20% input conditions, 100 μg of protein was loaded per lane. Proteins were detected using the following antibodies: rabbit anti-GFP (Life Technologies), mouse anti-HA (Covance), mouse anti-Myc (Santa Cruz Biotechnologies), and mouse anti-actin (Millipore EMD).

FRET-based measurements of nAChR activity

Measurements of α4β2 nAChR activity were performed as previously described [29] with the following alterations: HEK-tsa cells were transiently transfected with α4-pciNeo and β2-dm-pciNeo (gifts from H. Lester) plus TN-XXL (gift from P. Taylor) supplemented with either sss-pcDNA3 or pcDNA3 alone at a ratio of 1:1:2:10, respectively. Cells were pre-incubated in growth media containing 1 μM nicotine (Tocris, R&D) for 20 hours prior to assay in artificial cerebral spinal fluid (ACSF: 121 mM NaCl, 5 mM KCl, 26 mM NaHCO3, 1.2 mM NaH2PO4H2O, 10 mM Glucose, 5 mM HEPES, 2.4 mM Ca2+, 1.3 mM Mg2+, pH 7.4). Maximum responses were calculated from concentration-response curves averaged over 4 experiments, each performed in triplicate, with results normalized to the maximum response of α4β2 nAChR without sss. One-way ANOVA repeated measures analysis with Dunnett's multiple comparison post-test was used for statistical analysis.

Supplementary Material

01

Highlights

  • Reduction of nAChR activity restores normal sleep to qvr/sss mutants.

  • SSS complexes with certain nAChRs and K channels, and all three are expressed in mushroom bodies.

  • SSS inhibits nAChR activity in vitro.

  • The mammalian SSS homolog lynx1 can substitute for SSS in regulating sleep.

Acknowledgments

We thank H. Lester for the mouse α4 and β2 nAChR cDNAs, P. Taylor for TN-XXL, Z. Yue for the Flag-GRID2 expression construct, U. Thomas and E. Gundelfinger for the Dα3 antibody, and A. Sehgal for the Sh antibody. We also thank A. Sehgal and M. Shi for sharing data prior to publication. We are also grateful for technical assistance provided by J. Yamauchi and K. Gomez for FRET assays and by B. Yip for qPCR. We also thank members of the Joiner lab for helpful discussions. This work was supported by grants from the NIH (NS072431), the Whitehall Foundation (WF20110560), and a Pharma Foundation Research Starter Grant to WJJ. This work was also supported by the UCSD Neuroscience Microscopy Shared Facility Grant P30 NS047101. JER was supported by funding from the National Science Foundation Graduate Research Fellowship Program (DGE-1144086) and the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752).

Footnotes

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