Abstract
Mammalian T-type Ca2+ channels are encoded by three separate genes (Cav3.1, 3.2, 3.3). These channels are reported to be sleep stabilizers important in the generation of the delta rhythms of deep sleep, but controversy remains. The identification of precise physiological functions for the T-type channels has been hindered, at least in part, by the potential for compensation between the products of these three genes and a lack of specific pharmacological inhibitors. Invertebrates have only one T-type channel gene, but its functions are even less well-studied. We cloned Ca-α1T, the only Cav3 channel gene in Drosophila melanogaster, expressed it in Xenopus oocytes and HEK-293 cells, and confirmed it passes typical T-type currents. Voltage-clamp analysis revealed the biophysical properties of Ca-α1T show mixed similarity, sometimes falling closer to Cav3.1, sometimes to Cav3.2, and sometimes to Cav3.3. We found Ca-α1T is broadly expressed across the adult fly brain in a pattern vaguely reminiscent of mammalian T-type channels. In addition, flies lacking Ca-α1T show an abnormal increase in sleep duration 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.
Highlights
Unlike mammals, Drosophila melanogaster has only one T-type Ca2+ channel, Ca-α1T, which is known as DmαG
Electrophysiological characterization of Ca-α1T in Xenopus oocytes showed that Ca-α1T has the hallmark properties of a T-type channel: low-threshold activation at around − 60 mV, a maximal current output at − 20 mV, transient current kinetics elicited by a step-pulse protocol producing a “criss-crossing” pattern, and slow deactivation of tail currents (Fig. 1)
In terms of current kinetics, Ca-α1T is more similar to mammalian Cav3.1 and Cav3.2 than Cav3.3, which exhibits considerably slower kinetics
Summary
Drosophila melanogaster has only one T-type Ca2+ channel, Ca-α1T, which is known as DmαG. A recent study found that motor neurons in flies lacking Ca-α1T show reduced LVA and reduced high-voltage-activated (HVA) Ca2+ currents, suggesting that Ca-α1T seems to be a genuine T-type channel, it may have interesting biophysical properties[5]. We cloned a single isoform of Ca-α1T, 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 generated several Ca-α1T 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, their role in sleep physiology
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