Familial advanced sleep phase syndrome (FASPS) is an autosomal dominant circadian rhythm variant; affected individuals are “morning larks” with a 4-hour advance of the sleep, temperature, and melatonin rhythms. Here we report localization of the FASPS gene near the telomere of chromosome 2q. A strong candidate gene (h Per2 ), a human homolog of the period gene in Drosophila , maps to the same locus. Affected individuals have a serine to glycine mutation within the casein kinase I ɛ (CKI ɛ ) binding region of hPER2, which causes hypophosphorylation by CKI ɛ in vitro. Thus, a variant in human sleep behavior can be attributed to a missense mutation in a clock component, hPER2, which alters the circadian period.

The serine/threonine protein kinase casein kinase I ε (CKIε) is a key regulator of metazoan circadian rhythm. Genetic and biochemical data suggest that CKIε binds to and phosphorylates the PERIOD proteins. However, the PERIOD proteins interact with a variety of circadian regulators, suggesting the possibility that CKIε may interact with and phosphorylate additional clock components as well. We find that CRY1 and BMAL1 are phosphoproteins in cultured cells. Mammalian PERIOD proteins act as a scaffold with distinct domains that simultaneously bind CKIε and mCRY1 and mCRY2 (mCRY). mCRY is phosphorylated by CKIε only when both proteins are bound to mammalian PERIOD proteins. BMAL1 is also a substrate for CKIε in vitro, and CKIε kinase activity positively regulates BMAL1-dependent transcription from circadian promoters in reporter assays. We conclude that CKIε phosphorylates multiple circadian substrates and may exert its effects on circadian rhythm in part by a direct effect on BMAL1-dependent transcription.Circadian rhythms allow organisms to optimize their metabolic and physiologic behavior in response to the 24 h day. The rhythm is generated by cell-autonomous transcription-translation feedback loops highly conserved in outline if not in detail in most eukaryotes studied to date (recently reviewed in Refs. 1-4). Extensive genetic investigation coupled with molecular studies has uncovered many of the essential elements of the metazoan clock. The central feature of the clock is transcription, regulated by a heterodimeric transcription factor that drives expression of genes whose protein products are negative regulators of their own transcription. This negative feedback loop establishes stable molecular oscillations with a period of ~24 h. In mammals, the transcription factors are the PAS domain-containing proteins CLK 1 and BMAL1, whereas the negative factors include the PERIOD proteins PER1 and PER2 and the cryptochromes CRY1 and CRY2. The stability and period of the circadian oscillations are likely to be dependent on multiple factors, including the rate at which these regulators accumulate in the cell (i.e. the sum of their synthesis and degradation rates) and the rate at which they accumulate in the nucleus. Finally, the activities of circadian regulators are modified by post-translational modifications.Analysis of animals and humans with altered circadian rhythms demonstrates the importance of phosphorylation in the regulation of the molecular clock. Mutations in the casein kinase Iε

Slow activation and rapid C-type inactivation produce inward rectification of the current-voltage relationship for human ether-a'-go-go-related gene (hERG) channels. To characterize the voltage sensor movement associated with hERG activation and inactivation, we performed an Ala scan of the 32 amino acids (Gly 514 -Tyr 545 ) that comprise the S4 domain and the flanking S3-S4 and S4 -S5 linkers. Gating and ionic currents of wild-type and mutant channels were measured using cut-open oocyte Vaseline gap and two microelectrode voltage clamp techniques to determine the voltage dependence of charge movement, activation, and inactivation. Mapping the position of the charge-perturbing mutations (defined as ͦ⌬⌬Gͦ > 1.0 kcal/mol) on a threedimensional S4 homology model revealed a spiral pattern. As expected, mutation of these residues also altered activation. However, mutation of residues in the S3-S4 and S4 -S5 linkers and the C-terminal end of S4 perturbed activation (ͦ⌬⌬Gͦ > 1.0 kcal/mol) without altering charge movement, suggesting that the native residues in these regions couple S4 movement to the opening of the activation gate or stabilize the open or closed state of the channel. Finally, mutation of a distinct set of residues impacted inactivation and mapped to a single face of the S4 helix that was devoid of activation-perturbing residues. These results define regions on the S4 voltage sensor that contribute differentially to hERG activation and inactivation gating. The human ether-a'-go-go-related gene (hERG)1 channels are primarily expressed in the brain and the heart (1) but are also up-regulated in tumors from a variety of tissues (2). In the heart, hERG channels conduct the rapidly activating, delayed rectifier potassium current, I Kr (3, 4). Unlike most voltagegated K ϩ (Kv) channels, the fully activated current-voltage relationship of hERG channels exhibits inward rectification, a property that limits efflux of K ϩ during the plateau phase of the cardiac action potential.Inward rectification of hERG channels results from a rapid and voltage-dependent C-type inactivation that proceeds at a rate much faster than activation. At membrane potentials between Ϫ20 and ϩ20 mV, channels activate over hundreds of milliseconds but inactivate in milliseconds (5-7). The voltage dependence of hERG inactivation is shifted nearly Ϫ65 mV relative to channel activation (V 0.5INACT ϭ Ϫ85 mV, V 0.5ACT ϭ Ϫ20 mV). A single mutation (S631A) in the P-loop of the outer pore causes a ϩ100 mV shift in the voltage dependence of inactivation, but no shift in activation compared with wild-type (WT) channels (6, 8). Furthermore, external cations have differential effects on the voltage dependence of activation and inactivation (9 -11). Together, these findings led to the suggestion that distinct voltage-sensing mechanisms underlie activation and inactivation gating in hERG channels.The highly charged S4 transmembrane helix functions as the primary voltage sensor in voltage-gated channels. Neutralization of basic S4 residues shifts the vol...