Ca-\(\alpha\)1T, a fly T-type Ca2+ channel, negatively modulates sleep
Kyunghwa Jeong1, Soyoung Lee2, Haengsoo Seo2, Yangkyun Oh1, Donghoon Jang1, Joonho Choe1, Daesoo Kim1, Jung-Ha Lee2,\(+\), Walton D. Jones1,\(\ast\)
Mammalian T-type Ca2+ channels are encoded by three separate genes (Cav3.1, 3.2, 3.3). In mammals, T-type channels are reported to be sleep stabilizers that are important in the generation of the delta rhythms of deep sleep, but controversy remains. Progress in identifying the precise physiological functions of the T-type channels has been hindered by many factors, including possible compensation between the products of these three genes and a lack of specific pharmacological inhibitors. Invertebrates have only one T-type channel gene and its physiological functions are less well-studied. We cloned Ca-\(\alpha\)1T, the only Cav3 channel gene in the Drosophila melanogaster genome, expressed it in Xenopus oocytes or HEK-293 cells, and verified that it is capable of passing typical T-type currents. Voltage-clamp analysis revealed that the biophysical properties of Ca-\(\alpha\)1T show mixed similarity, sometimes falling closer to Cav3.1, sometimes to Cav3.2, and sometimes to Cav3.3. We found that Ca-\(\alpha\)1T is broadly expressed across the adult fly brain in a pattern vaguely reminiscent of mammalian T-type channels. In addition, flies lacking Ca-\(\alpha\)1T show an abnormal increase in sleep duration that is most pronounced during subjective day under continuous dark conditions despite normal oscillations of the circadian clock. Thus, our study suggests invertebrate T-type Ca2+ channels promote wakefulness rather than stabilizing sleep.
T-type Ca2+ channels are a subfamily of voltage-dependent Ca2+ channels (VDCCs) that produce low-voltage-activated (LVA) Ca2+ currents implicated in NREM sleep in mammals(Lee 2004). Three different genes encode the pore-forming alpha subunits of mammalian T-type channels, Cav3.1, 3.2, and 3.3. Of these, Cav3.1 and 3.3 are highly expressed in the thalamus, where the oscillations required for NREM sleep are generated(Steriade 1991, Dossi 1992, Talley 1999). Mice lacking Cav3.1 show reduced delta-wave activity and reduced sleep stability, suggesting that mammalian T-type currents have a sleep-promoting or stabilizing function(Lee 2004).
Unlike mammals, Drosophila melanogaster has only one T-type Ca2+ channel, Ca-\(\alpha\)1T, which is also known as Dm\(\alpha\)G and Ca-\(\alpha\)1T. A recent study found that motor neurons in flies lacking Ca-\(\alpha\)1T show reduced LVA but also reduced high-voltage-activated (HVA) Ca2+ currents, suggesting that although Ca-\(\alpha\)1T seems to be a genuine T-type channel, it may have interesting biophysical properties(Ryglewski 2012). We therefore cloned a single isoform of Ca-\(\alpha\)1T, expressed it in Xenopus oocytes or HEK-293 cells, and compared its biophysical properties with those of the rat T-type channel Cav3.1. We also generated several Ca-\(\alpha\)1T mutant alleles and identified a defect in their sleep/wake cycles. Contrary to results in mammals, the fly T-type Ca2+ channel destabilizes sleep. We anticipate that our findings will help clarify species-dependent differences in the in vivo functions of T-type Ca2+ channels, particularly their role in sleep physiology.