TMEM16A and TMEM16B channel proteins generate Ca2+-activated Cl− current and regulate melatonin secretion in rat pineal glands

Pinealocytes regulate circadian rhythm by synthesizing and secreting melatonin. These cells generate action potentials; however, the contribution of specific ion channels to melatonin secretion from pinealocytes remains unclear. In this study, the involvement and molecular identity of Ca2+-activated Cl− (ClCa) channels in the regulation of melatonin secretion were examined in rat pineal glands. Treatment with the ClCa channel blockers, niflumic acid or T16Ainh-A01, significantly reduced melatonin secretion in pineal glands. After pineal K+ currents were totally blocked under whole-cell patch clamp conditions, depolarization and subsequent repolarization induced a slowly activating outward current and a substantial inward tail current, respectively. Both of these current changes were dependent on intracellular Ca2+ concentration and inhibited by niflumic acid and T16Ainh-A01. Quantitative real-time PCR, Western blotting, and immunocytochemical analyses revealed that TMEM16A and TMEM16B were highly expressed in pineal glands. siRNA knockdown of TMEM16A and/or TMEM16B showed that both channels contribute to ClCa currents in pinealocytes. Conversely, co-expression of TMEM16A and TMEM16B channels or the expression of this tandem channel in HEK293 cells mimicked the electrophysiological characteristics of ClCa currents in pinealocytes. Moreover, bimolecular fluorescence complementation, FRET, and co-immunoprecipitation experiments suggested that TMEM16A and TMEM16B can form heteromeric channels, as well as homomeric channels. In conclusion, pineal ClCa channels are composed of TMEM16A and TMEM16B subunits, and these fluxes regulate melatonin secretion in pineal glands.

Pineal glands regulate the circadian rhythm through the synthesis and secretion of melatonin. This melatonin production can be either positively or negatively regulated by sympathetic and parasympathetic systems, respectively. Norepinephrine (NE) 2 stimulates adrenergic ␤ 1 receptor and promotes cAMP production. The cAMP activates a melatonin-synthesizing enzyme, arylalkylamine-N-acetyltransferase, thus promoting melatonin biosynthesis from tryptophan in pinealocytes. NE also stimulates adrenergic ␣ 1 receptor leading to inositol 1,4,5trisphosphate-induced Ca 2ϩ release, which is thought to enhance the adrenergic ␤ 1 signal pathway (1). In addition to this adrenergic regulation, there is parasympathetic innervation in pineal glands (2). Acetylcholine (ACh) activates nicotinic ACh receptors. This elicits membrane depolarization, and then induces Ca 2ϩ influx through voltage-dependent Ca 2ϩ channels (VDCCs). The resulting increase in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) causes an exocytosis of glutamate (3). This glutamate stimulates metabotropic glutamate receptor type 3 and thus decreases cAMP production, resulting in the reduction of arylalkylamine-N-acetyltransferase activity and melatonin synthesis (4).
Ca 2ϩ -activated Cl Ϫ (Cl Ca ) channels play important roles in many physiological processes, such as epithelial secretion, sensory transduction, neuronal signaling, cardiac excitability, and smooth muscle contraction. Two TMEM16 family proteins, TMEM16A and TMEM16B, have been identified as functional Cl Ca channels (15)(16)(17). TMEM16A is widely expressed in a large variety of tissues, including secretory epithelial cells, smooth muscle cells, interstitial cells of Cajal, and nociceptive neurons. On the other hand, the findings of TMEM16B expression have been limited in sensory nervous systems, such as olfactory neurons and retinal photoreceptors (18 -20).
The present study was undertaken to study the expression of the Cl Ca channel, elucidate their molecular entity, and demonstrate their involvement in melatonin secretion in rat pineal The authors declare that they have no conflicts of interest with the contents of this article. 1 To whom correspondence should be addressed: 3-1 Tanabedori Mizuhoku, Nagoya 467-8603, Japan. glands. To our knowledge, our results are the first to report that TMEM16A and TMEM16B proteins are functionally expressed as Cl Ca channels, and that this Cl Ϫ current contributes to the regulation of melatonin secretion in mammalian pineal glands.
In the presence of ACh, application of 30 M niflumic acid (11.0 Ϯ 2.2 ng/ml, n ϭ 5, p ϭ 0.008 (F ϭ 8.56) versus NE ϩ ACh by Tukey's test) or T16A inh -A01 (5.2 Ϯ 1.2 ng/ml, n ϭ 5, p ϭ 0.00004) further reduced the NE-induced melatonin secretion. These results indicate that the activity of Cl Ca channels is involved in the regulation of melatonin secretion via sympathetic and parasympathetic pathways in rat pineal glands.

Cl ؊ currents and their sensitivity to Ca 2؉ in pinealocytes
Cl Ϫ currents were measured in pinealocytes isolated from rat pineal glands, by use of K ϩ -deficient and Cl Ϫ -rich solutions under whole-cell voltage-clamp conditions (see "Experimental procedures"). Single pinealocytes were depolarized from the holding potential of Ϫ40 mV to selected test potentials (Ϫ80 ϳ ϩ100 mV) by ϩ20 mV increment for 500 ms and, then repolarized to Ϫ80 mV for 250 ms every 15 s. The cell capacitance was 20.6 Ϯ 0.8 pF (n ϭ 65). When Ca 2ϩ concentration in the pipette solution ([Ca 2ϩ ] pip ) was fixed to pCa 6.0, time-dependent outward currents over ϳ400 pA in peak amplitude were detected at membrane potentials positive to ϩ40 mV (I peak ϭ 59.3 Ϯ 9.3 pA/pF at ϩ100 mV, n ϭ 8) (Fig. 2, A and B). Upon repolarization, characteristic inward tail currents were recorded (I tail ϭ 53.1 Ϯ 7.4 pA/pF, n ϭ 8). The current-voltage relationship shows that the reversal potential was ϳ0 mV (Fig.  2B). The amplitude of outward and tail currents were substantially reduced by the decrease in [Ca 2ϩ ] pip to pCa 6.5 or 7.0, in a [Ca 2ϩ ] pip -dependent manner (n ϭ 5ϳ10) (Fig. 2, A-C). The time constant for current activation ( act ) at ϩ100 mV and that for tail current deactivation ( tail ) at Ϫ80 mV after ϩ100 mV stimulation were 80.5 Ϯ 8.4 and 81.0 Ϯ 8.9 ms, respectively (n ϭ 8) (Fig. 2, D and E). The act and tail were also affected by the [Ca 2ϩ ] pip change (n ϭ 5ϳ10) (Fig. 2, D and E). These data indicate that Cl Ϫ channel activity in pinealocytes strongly depends upon [Ca 2ϩ ] i .

Sensitivity to Cl Ca channel blockers on pineal Cl ؊ currents
Effects of Cl Ca channel blockers, niflumic acid, and T16A inh -A01, on both outward and tail currents were examined in rat pinealocytes. When [Ca 2ϩ ] pip was pCa 6.0, 6.5, and 7.0, the application of 100 M niflumic acid significantly reduced the outward peak currents (10.5 Ϯ 3.2 pA/pF at ϩ100 mV and pCa 6.0, n ϭ 10, p ϭ 0.00003 (F ϭ 7.73) versus control of 53.4 Ϯ 6.0 pA/pF by Student's t test, paired) (Fig. 3, A and B). The tail currents were also significantly reduced by 100 M niflumic acid (15.7 Ϯ 3.5 pA/pF at Ϫ80 mV after ϩ100 mV stimulation and pCa 6.0, n ϭ 10, p ϭ 0.00003 (F ϭ 7.71) versus control of 50.2 Ϯ 6.8 pA/pF), except when [Ca 2ϩ ] pip was pCa 7.0. The inhibitory effect of niflumic acid on outward currents at [Ca 2ϩ ] pip of pCa 6.0, was dose-dependent with an IC 50 of 2.6 M and the Hill coefficient of 0.81 (n ϭ 7) (Fig. 3, C, D, and G). In addition, the outward and tail currents were also significantly inhibited by 10 M T16A inh -A01 (n ϭ 3, p ϭ 0.024 (F ϭ 6.38) and p ϭ 0.012 (F ϭ 8.95), respectively, by Student's t test, paired) and the inhibition was removed by washout (Fig. 3, E-G). These data indicate that Cl Ca currents sensitive to niflumic acid and T16A inh -A01 are functionally expressed in rat pinealocytes.

Possible contribution of Cl Ca channel activity to the resting membrane potential
Effect of Cl Ca channel blocker on the resting membrane potential was examined in rat pinealocytes under whole-cell current-clamp mode. The mean resting membrane potential was Ϫ34.5 Ϯ 3.1 mV (n ϭ 8) (Fig. 4). Application of 100 M niflumic acid caused a significant hyperpolarization to Ϫ40.0 Ϯ 2.8 mV (p ϭ 0.007 (F ϭ 3.77), n ϭ 8, by Student's t test, paired) and it was recovered by removal of niflumic acid (to Ϫ30.5 Ϯ 4.6 mV, n ϭ 8, p ϭ 0.043 (F ϭ 2.47) versus niflumic acid, p ϭ 0.163 (F ϭ 1.56) versus control). The experimental conditions of the pipette solution used here provide Cl Ϫ reversal potential of 0 mV and also the [Ca 2ϩ ] i lower than 100 nM. These may result in somewhat over and under estimation of the contribution, respectively. Thus, niflumic acid-sensitive Cl Ca channel activity may be involved in the regulation of resting membrane potential in rat pinealocytes, particularly when [Ca 2ϩ ] i is elevated.

Expression of TMEM16A and TMEM16B in pineal glands
To identify the molecular components of Cl Ca channels in rat pinealocytes, expression analyses of the TMEM16 family were performed by quantitative real-time PCR, Western blotting, and immunocytochemical methods. Among the TMEM16 family, Tmem16B mRNA was highly expressed (0.074 Ϯ 0.009 of ␤-actin, n ϭ 10), and Tmem16A and Tmem16K mRNAs were also identified in pineal glands (0.043 Ϯ 0.007 and 0.061 Ϯ 0.008, respectively, n ϭ 10) (Fig. 5A). Western blot analysis showed the expression of TMEM16A and TMEM16B in the plasma membrane fraction from pineal glands (n ϭ 6ϳ8; Fig.  5B). In addition, immunocytochemical staining showed that TMEM16A and TMEM16B proteins were abundantly ex-pressed at the plasma membrane of pinealocytes (n ϭ 10) (Fig.  5C). Taken together, TMEM16A and TMEM16B are both expressed at the plasma membrane of rat pinealocytes.

Figure 2. Macroscopic Cl Ca currents in rat pinealocytes.
A, in whole-cell voltage-clamp experiments, single pinealocytes were depolarized from the holding potential of Ϫ40 mV to test potentials (Ϫ80 ϳ ϩ100 mV) by ϩ20 mV increment for 500 ms and subsequently repolarized to Ϫ80 mV for 250 ms every 15 s. Representative current traces of pCa 7.0, 6.5, and 6.0 in the pipette solution. Note that time-dependent outward currents and tail currents, which are characteristic of Cl Ca currents, were observed at pCa 6.5 and 6.0 in pinealocytes. B, current-voltage relationships of outward currents at pCa 7.0, 6.5, and 6.0. Note that these currents reversed around 0 mV, which is a theoretical equilibrium potential of Cl Ϫ under the experimental conditions. C, Ca 2ϩ dependence of outward currents. D, act of outward currents. E, tail of tail currents. Experimental data were obtained from 5 to 10 pinealocytes.

TMEM16A and TMEM16B currents in HEK293 cells
To obtain new information concerning the comparative contributions of TMEM16A and TMEM16B to Cl Ca currents in pinealocytes, the electrophysiological parameters of cloned rat TMEM16A and TMEM16B channels were measured in HEK293 cells, in which heterologous expression was performed. In TMEM16A-transfected HEK293 cells, outward and tail currents were not detected, when [Ca 2ϩ ] pip was pCa 7.0 (n ϭ 3), but were consistently observed in a concentration-dependent manner at pCa 6.5 (I peak ϭ 28.3 Ϯ 7.2 pA/pF and I tail ϭ 16.6 Ϯ 5.3 pA/pF, n ϭ 5) and 6.0 (81.5 Ϯ 28.6 and 63.6 Ϯ 18.9 pA/pF, respectively, n ϭ 5) (Fig. 7, A, B, and G). In contrast, in TMEM16B-transfected cells, these currents were not detected at pCa 7.0 and 6.5 (n ϭ 4), and observed at pCa 6.0 (I peak ϭ 65.9 Ϯ 4.6 pA/pF and I tail ϭ 24.5 Ϯ 3.8 pA/pF, n ϭ 4) (Fig. 7, C,  D, and G). When TMEM16A and TMEM16B cDNAs were co- A, under whole-cell voltage-clamp configuration, single pinealocytes were depolarized from the holding potential of Ϫ40 mV to test potentials (Ϫ80 ϳ ϩ100 mV) by ϩ20 mV increment for 500 ms and subsequently repolarized to Ϫ80 mV for 250 ms every 15 s. Representative current traces of pCa 7.0, 6.5, and 6.0 in the pipette solution in the absence of drug (Control). Application of 100 M niflumic acid (NFA) was inhibited outward and tail currents. The inhibitory effects of niflumic acid were removed by washout. B, effect of 100 M niflumic acid on outward currents at ϩ100 mV and tail currents at Ϫ80 mV following ϩ100 mV depolarization. C, time course showing a dose-dependent inhibition of niflumic acid (0.01ϳ1000 M) on outward (at ϩ100 mV; peak) and tail (at Ϫ80 mV following ϩ100 mV depolarization) currents at pCa 6.0 in the pipette solution. D, representative current traces in the absence and presence of 1, 10, and 100 M niflumic acid. E, time course showing an inhibitory effect of 10 M T16A inh -A01 (T16A) on outward and tail currents. F, representative current traces in the absence and presence of 10 M T16A inh -A01. The inhibitory effect of T16A inh -A01 was removed by washout. G, dose-response curves for niflumic acid and T16A inh -A01 on outward currents. The IC 50 value of niflumic acid was 2.6 M and the Hill coefficient of 0.81. T16A inh -A01 also blocked outward currents in a concentration-dependent manner. Experimental data were obtained from 3 to 10 pinealocytes. *, p Ͻ 0.05; **, p Ͻ 0.01 by Student's t test (paired).

Molecular interaction between TMEM16A and TMEM16B in living HEK293 cells
Although it has been reported that the TMEM16 family can form heterodimers mainly based on co-immunoprecipitation assays using a heterologous expression system (21), little is known about this interaction in living cells. Bimolecular fluorescent complementation (BiFC) analysis can detect a direct interaction between two molecules fused, respectively, with the N or C terminus of Venus (VN173 or VC155) by the generation of reconstructed Venus fluorescence (22)(23)(24). As shown in Fig. 8, the strong fluorescent signals of Venus were observed at the plasma membrane in homomeric TMEM16A-VN/ TMEM16A-VC and TMEM16B-VN/TMEM16B-VC co-expressing HEK293 cells (n ϭ 10) (Fig. 8, A and B). Similarly, the consistent Venus signals in heteromeric TMEM16A-VN/ TMEM16B-VC and TMEM16A-VC/TMEM16B-VN HEK293 cells were detected at the plasma membrane (n ϭ 10) (Fig. 8C). In contrast, there were no fluorescent signals in HEK293 cells expressing TMEM16A-VN, TMEM16B-VN, TMEM16A-VC, or TMEM16B-VC alone (n ϭ 10). In addition, fluorescent signals were not detected in HEK293 cells co-expressing TMEM16A-VC/TASK1-VN or TMEM16B-VC/TASK1-VN (n ϭ 10) (Fig. 8D). These data support that TMEM16A directly interacts with TMEM16B to form a heteromeric in addition to homomeric channels in living cells.

TMEM16A and TMEM16B Cl Ca channels in pineal glands
can form heteromeric channels with efficiency comparative to that of homomeric channels.

Evidence for heteromeric TMEM16A/B channels in pineal glands
To our knowledge, there is no report showing the heteromeric channel formation of TMEM16A and TMEM16B in native tissues. As shown in Fig. 10, our co-immunopre-cipitation results demonstrate heteromeric interaction of TMEM16A and TMEM16B in rat pineal glands (n ϭ 4).

Discussion
The pineal gland is a melatonin-secreting organ in the brain. It regulates circadian rhythm through the synthesis and secretion of melatonin. This regulation of melatonin production depends upon a balance of activity, modulated by sympathetic  experiments). B, single pinealocytes were depolarized from the holding potential of Ϫ40 mV to test potentials (Ϫ80 ϳ ϩ100 mV) by ϩ20 mV increment for 500 ms and subsequently repolarized to Ϫ80 mV for 250 ms every 15 s. Representative current traces of Cl Ca currents in rat pinealocytes transfected with control, Tmem16A, Tmem16B, or Tmem16A ϩ Tmem16B siRNA. C, current-voltage relationships of outward currents in pinealocytes transfected with control, Tmem16A, Tmem16B, or Tmem16A ϩ Tmem16B siRNA. D, outward (at ϩ100 mV: peak) and tail (at Ϫ80 mV following ϩ100 mV depolarization) current densities. E, act of outward currents. F, tail of tail currents. Experimental data were obtained from 5 to 10 pinealocytes. *, p Ͻ 0.05; **, p Ͻ 0.01 by Tukey's test.

TMEM16A and TMEM16B Cl Ca channels in pineal glands
and parasympathetic innervation (1,2). Although meaningful electrophysiological studies have been done, no comprehensive analyses of the functional expressions of ion channels has been reported in mammalian pineal glands. Accordingly, the physiological roles of specific ion channels in the regulation of melatonin secretion are not known. Our new results show that TMEM16A and TMEM16B proteins are the predominant Cl Ca channel subtype in pinealocytes, and demonstrate that this Cl Ϫ conductance is involved in melatonin secretion in pineal glands.
In rat pinealocytes, substantial voltage-dependent Cl Ϫ currents have been recorded by a whole-cell patch clamp technique, after K ϩ currents were totally blocked by Cs ϩ and tetraethylammonium. Depolarizing voltage steps to positive membrane potentials elicited a slow time-dependent outward current; and subsequent repolarization produced a characteristic inward tail current. The amplitude of these currents was highly dependent on [Ca 2ϩ ] i in the range of pCa 7.0ϳ6.0 in the pipette solution. This current reversed near 0 mV, which is a theoretical equilibrium potential of Cl Ϫ (E Cl ϭ Ϫ0.9 mV) under our experimental conditions. Furthermore, these currents were strongly inhibited by a specific blocker of TMEM16A and TMEM16B channels, T16A inh -A01, as well as a classical Cl Ca channel blocker, niflumic acid. To our knowledge, this is first demonstration of Cl Ca current in pineal glands.
Cl Ca channels are ubiquitously expressed in epithelia, smooth muscles, interstitial cells of Cajal, and some neurons (18 -20). In neurons and smooth muscles, an increase in Cl Ca conductance significantly shifts the resting membrane potential in the depolarizing direction. This facilitates Ca 2ϩ influx Figure 7. Current properties of TMEM16A and TMEM16B cloned from rat pinealocytes. A-F, rat TMEM16A (3 g), TMEM16B (3 g), or TMEM16A ϩ TMEM16B (1 ϩ 2 g) cDNA was transfected into HEK293 cells. HEK293 cells were depolarized from the holding potential of Ϫ40 mV to test potentials (Ϫ80 ϳ ϩ100 mV) by ϩ20 mV increment for 500 ms and subsequently repolarized to Ϫ80 mV for 250 ms every 15 s. Representative traces of Cl Ca currents and their current-voltage relationships at pCa 7.0, 6.5, and 6.0 in the pipette solution from HEK293 cells expressing TMEM16A (A and B), TMEM16B (C and D), or TMEM16A ϩ TMEM16B (E and F). G, outward current density at ϩ100 mV at pCa 6.5. H, act of outward currents in pinealocytes and HEK293 cells transfected with TMEM16A, TMEM16B, TMEM16A ϩ TMEM16B (1:1), TMEM16A ϩ TMEM16B (1:2), or TMEM16B-TMEM16A tandem form. I, tail of tail currents. Note that the predominant electrophysiological properties of pinealocytes are very similar to these of TMEM16A/TMEM16B (1:2) co-expressing HEK293 cells. Data from pinealocytes have been replotted from Fig. 2. Experimental data were obtained from 4 to 8 cells.

TMEM16A and TMEM16B Cl Ca channels in pineal glands
through VDCCs, resulting in the enhancement of cell excitability in the form of Ca 2ϩ -dependent action potentials. TMEM16A and TMEM16B are currently the preferred candidates for Cl Ca channel conductances in native tissues. Specifically, among neurons, TMEM16A is expressed in small dorsal root ganglion neurons associated with nociception (29) and thermoreceptors (30). On the other hand, TMEM16B is expressed in presynaptic terminals of retinal photoreceptors (31) and the cilia of olfactory sensory neurons (32,33), where it appears to play a special role in sensory transduction. In addition, TMEM16B can regulate action potential waveform, and synaptic responses in hippocampus neurons (34). Our results demonstrate that both TMEM16A and TMEM16B proteins are abundantly expressed at the plasma membrane of rat pinealocytes. Although the Tmem16K transcript was also expressed in pinealocytes, it is rather unlikely that TMEM16K per se forms a functional Cl Ca channel (20). Therefore, the present study focused on physiological functions of TMEM16A and TMEM16B in pinealocytes.
The biophysical characteristics of TMEM16B channels show significant differences from those of TMEM16A channels. First, the single-channel conductance of the TMEM16B channel (0.8ϳ1.2 pS) has been reported to be smaller than that of the TMEM16A channel (8.3 pS) (17,32,35). However, a recent report suggests that there are no significant differences between both channels (TMEM16A ϭ 3.5 pS versus TMEM16B ϭ 3.9 pS) (36). Second, the [Ca 2ϩ ] i level needed for activation of the TMEM16B channel (Ͼ1 M) is higher than that of the TMEM16A channel (ϳ0.3 M) (17, 32, 36 -38). Third, the kinetics of activation and deactivation ( deact ) of the TMEM16B current are much faster than those of the TMEM16A current. The act and deact of TMEM16B are 4 -24 at ϩ100 mV and 3-7 ms at Ϫ60 ϳ Ϫ100 mV, respectively (35,36,38). In contrast, those of TMEM16A are ranged between 120ϳ400 and 55ϳ150 ms, respectively (36,38,39). In the pres-   . Co-immunoprecipitation assay for TMEM16A and TMEM16B in rat pineal glands. In co-immunoprecipitation assays, lysates from rat pineal glands were precipitated with TMEM16A antibody and blotted using TMEM16B antibody. N indicates a negative control lane loaded with protein treated with the Control Agarose Resin (see "Experimental procedures"). Similar results were obtained from four independent experiments.

TMEM16A and TMEM16B Cl Ca channels in pineal glands
ent study, pineal Cl Ca currents were significantly activated by 0.3 M [Ca 2ϩ ] i , which suggests TMEM16A involvement. The kinetic parameters of pineal Cl Ca currents were intermediate with respect to those of TMEM16A and TMEM16B.
In addition, siRNA experiments revealed that treatment with either siTMEM16A or siTMEM16B resulted in the remaining much smaller Cl Ca current having characteristics of the remaining TMEM16A/B channels. As expected, therefore, pineal Cl Ca currents were completely abolished by double siRNA knockdown of Tmem16A and Tmem16B. In separate experiments, the most prominent biophysical properties of pineal Cl Ca currents were mimicked by HEK293 cells that co-expressed TMEM16A and TMEM16B, which were cloned from rat pineal glands. A proportion of 1:2 for TMEM16A and TMEM16B co-transfection to HEK293 cells was determined based on the mRNA expression level in rat pineal glands. This 1:2 expression ratio rather than 1:1 resulted in the functional expression of the more closely mimicked Cl Ca current with respect to kinetics parameters to those of native Cl Ca current in rat pinealocytes. In combination, these results strongly suggest that both of TMEM16A and TMEM16B functionally contribute to Cl Ca current in pinealocytes.
It is known that the expression pattern of TMEM16A proteins is distinct from that of TMEM16B proteins; therefore, each protein is presumed to form a homodimer as a functional Cl Ca channel in native cells (27,40,41). In heterologous expression systems, TMEM16A protein can interact with TMEM16B protein, resulting in heterodimeric channels (21). Therefore, TMEM16A and TMEM16B heterodimers may form in native cells co-expressing both proteins. The expression of both proteins has been reported in rodent tissues, but the expression patterns are different; TMEM16A is found mainly in secretory epithelia versus TMEM16B in chemosensory neurons (42). In murine olfactory epithelium, the TMEM16A channel regulates Cl Ϫ homeostasis in supporting cells, whereas the TMEM16B channel contributes to the olfactory signal transduction in sensory neurons (43). Although TMEM16A and TMEM16B proteins are expressed in mammalian uterine smooth muscles (44) and dorsal root ganglion (45), heteromeric channel formation has not been demonstrated. Our results show that, in rat pinealocytes, TMEM16A and TMEM16B were co-expressed and that these proteins can form heteromeric assembly as well as homomeric channels. The heteromeric formation in living cells was clearly detected by BiFC analyses in HEK293 cells. The results of FRET analyses suggest that the efficiency of heterodimerization appears to be comparative to those of homodimerization. In addition, Cl Ca currents were observed in HEK293 cells transfected with the TMEM16B-TMEM16A tandem form. Taken together, it appears that pineal Cl Ca channels are composed of heteromeric TMEM16A and TMEM16B complexes, in addition to homomers.
Melatonin assays revealed that Cl Ca channel blockers can reduce the NE-induced melatonin secretion. The intracellular Cl Ϫ concentration varies widely, 10 -60 mM, depending on tissues (46). Although the intracellular Cl Ϫ concentration in pinealocytes is not known, Cl Ca channel block caused a consistent hyperpolarization under our experimental conditions, where the equilibrium potential of Cl Ϫ was set at 0 mV. Under physi-ological conditions, the equilibrium potential of Cl Ϫ in pinealocytes may be between Ϫ20 and Ϫ70 mV. Taken together, when action potentials occur spontaneously (5)(6)(7)(8)(9) or in response to endogenous stimulation, Cl Ca channel activation by Ca 2ϩ influx may increase the repolarization current and reduce the action potential duration. Further experiments are necessary for elucidating the molecular mechanism underlying the modulation of melatonin secretion by Cl Ca channel activity.
In conclusion, pineal Cl Ca currents flow through homomeric and heteromeric channels based on TMEM16A and TMEM16B subunits. The functional activity of Cl Ca channels significantly contributes to the regulation of melatonin secretion. Thus this study provides novel information concerning the molecular mechanism that regulates circadian rhythm through melatonin secretion in pineal glands.

Ethical approval
All experiments were approved by the Ethics Committee of Nagoya City University and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the Japanese Pharmacological Society.

Melatonin assay
Pineal glands were removed from male Wistar/ST rats (6ϳ9 weeks; Japan SLC, Hamamatsu, Japan). The freshly dissected pineal glands were incubated for 1 h at 37°C in phosphatebuffered saline (PBS) and then exposed to 1 M NE or vehicle (control) for 2 h. Test compounds were added into PBS at the beginning of incubation prior to NE addition. The amount of melatonin secreted from the whole pineal gland was quantitatively determined using a melatonin ELISA kit (IBL International, Hamburg, Germany).

Cell culture
Pineal glands were incubated in PBS containing 0.1% collagenase (Wako Pure Chemical Industries, Osaka, Japan) and 0.02% trypsin (Type I; Sigma) for 25 min at 37°C (9). After incubation, these tissues were dispersed mechanically in PBS. The pinealocytes were cultured on coverslips coated with 5 g/ml of poly-L-lysine (Sigma) in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen/Gibco), 20 units/ml of penicillin, and 20 g/ml of streptomycin (Wako Pure Chemical Industries). Experiments were performed at 24ϳ96 h after cell culture.

Electrophysiological recording
Electrophysiological studies were carried out using a wholecell patch clamp technique with a CEZ-2400 (Nihon Kohden, Tokyo, Japan) amplifier, an analog digital converter (Digidata 1440A; Molecular Devices/Axon, Foster City, CA), and pCLAMP software (version 10; Molecular Devices/Axon) in single pinealocytes and HEK293 cells (25,47). The pipette resistance ranged from 3 to 5 M⍀ when filled with the pipette solution. The seal resistance was ϳ30 G⍀. Series resistance was between 5 and 8 M⍀ and was partly compensated. Under whole-cell voltage-clamp mode, cells were step-clamed from TMEM16A and TMEM16B Cl Ca channels in pineal glands the holding potential of Ϫ40 mV to test potentials (Ϫ80 ϳ ϩ100 mV) by ϩ20 mV increment for 500 ms and subsequently returned to Ϫ80 mV for 250 ms every 15 s. Electrophysiological data were acquired at 1 kHz. The HEPES-buffered solution was used as an extracellular solution: 137 mM NaCl, 5.9 mM KCl, 2.2 mM CaCl 2 , 1.2 mM MgCl 2 , 14 mM glucose, and 10 mM HEPES. The pH was adjusted to 7.4 with 10 N NaOH. The pipette solution for Cl Ca current measurement had the following ionic composition: 120 mM CsCl, 20 mM tetraethylammonium-Cl, 2.8 mM MgCl 2 , 2 mM ATPNa 2 , 10 mM HEPES, 5 mM EGTA, and 1.79 (pCa 7.0), 3.19 (pCa 6.5), or 4.25 mM (pCa 6.0) CaCl 2 . The pH was adjusted to 7.2 with 1 N CsOH. For the recording of membrane potential under whole-cell current-clamp mode, the pipette solution had the following ionic composition: 140 mM KCl, 4 mM MgCl 2 , 2 mM ATPNa 2 , 10 mM HEPES, and 0.05 mM EGTA. The pH was adjusted to 7.2 with 1 N KOH. Electrophysiological recordings were performed at room temperature (23-25°C).

FRET analysis
Single-molecule imaging was performed with a TIRF imaging system, which consisted of a fluorescent microscope (ECLIPSE TE2000-U; Nikon), an objective lens (CFI Apo TIRF ϫ60/1.45, oil immersion; Nikon), an EM-CCD camera (C9100 -12; Hamamatsu Photonics, Hamamatsu, Japan), and AQUACOSMOS software (version 2.6; Hamamatsu Photonics), as previously reported (25). In brief, cells were fixed with PBS containing 4% paraformaldehyde for 10 min. E FRET was evaluated based on the acceptor photobleaching method. The fluorescence of YFP was photobleached for 2 min. TIRF images were acquired for 4.65 s. E FRET was calculated using the following equation: E FRET (%) ϭ [(CFP after Ϫ CFP before )/CFP after ] ϫ 100, where CFP after and CFP before are CFP emissions after and before YFP photobleaching, respectively.

Co-immunoprecipitation assay
Co-immunoprecipitation was performed using a co-immunoprecipitation kit (Pierce Biotechnology) as reported previously (23). In brief, seven pineal glands were lysed in immunoprecipitation lysis/wash buffer with a protease inhibitor mixture (Sigma). Homogenates were centrifuged (15,000 ϫ g, 25 min, 4°C), and supernatant was precleared with control resin (1 h, 4°C). Precleared lysates (ϳ100 g of protein) were incubated with AminoLink Plus Coupling Resin, with which TMEM16A antibody was immobilized for 12 h at 4°C. As a negative control, Control Agarose Resin, which was composed of the same support material as the AminoLink Plus Coupling Resin but was not amine-reactive, was used. The incubated lysates were finally subjected to 7.5% SDS-PAGE. The blots were incubated with TMEM16B antibody (1:200 dilution) for 12 h at 4°C, and then treated with anti-rabbit horseradish peroxidase-conjugated IgG (1:2000 dilution) for 1 h at 4°C, and finally exposed to an enhanced chemiluminescence detection system. The luminescence images were analyzed using a LAS-3000 system.

Drugs
Pharmacological reagents were obtained from Sigma, except for EGTA and HEPES (Dojin, Kumamoto, Japan). All hydrophobic compounds were dissolved in dimethyl sulfoxide at a concentration of 10 -1000 mM as a stock solution.

Statistics
Pooled data are shown as the mean Ϯ S.E. Statistical significance between two groups was determined by Student's t test. Statistical significance among groups was determined by Tukey's test after one-way analysis of variance.