Inactivation is an intrinsic property of several voltage-dependent ion channels, closing the conduction pathway during membrane depolarization and dynamically regulating neuronal activity. BK K+ channels undergo N-type inactivation via their β2 subunit, but the physiological significance is not clear. Here, we report that inactivating BK currents predominate during the day in the suprachiasmatic nucleus, the brain's intrinsic clock circuit, reducing steady-state current levels. At night inactivation is diminished, resulting in larger BK currents. Loss of β2 eliminates inactivation, abolishing the diurnal variation in both BK current magnitude and SCN firing, and disrupting behavioural rhythmicity. Selective restoration of inactivation via the β2 N-terminal ‘ball-and-chain' domain rescues BK current levels and firing rate, unexpectedly contributing to the subthreshold membrane properties that shift SCN neurons into the daytime ‘upstate'. Our study reveals the clock employs inactivation gating as a biophysical switch to set the diurnal variation in suprachiasmatic nucleus excitability that underlies circadian rhythm.

Large-conductance calcium-activated potassium channels (BK) are potent negative regulators of excitability in neurons and muscle, and increasing BK current is a novel therapeutic strategy for neuroand cardioprotection, disorders of smooth muscle hyperactivity, and several psychiatric diseases. However, in some neurons, enhanced BK current is linked with seizures and paradoxical increases in excitability, potentially complicating the clinical use of agonists. The mechanisms that switch BK influence from inhibitory to excitatory are not well defined. Here we investigate this dichotomy using a gain-of-function subunit (BK R207Q ) to enhance BK currents. Heterologous expression of BK R207Q generated currents that activated at physiologically relevant voltages in lower intracellular Ca 2+ , activated faster, and deactivated slower than wild-type currents. We then used BK R207Q expression to broadly augment endogenous BK currents in vivo, generating a transgenic mouse from a circadian clock-controlled Period1 gene fragment (Tg-BK R207Q ). The specific impact on excitability was assessed in neurons of the suprachiasmatic nucleus (SCN) in the hypothalamus, a cell type where BK currents regulate spontaneous firing under distinct day and night conditions that are defined by different complements of ionic currents. In the SCN, Tg-BK R207Q expression converted the endogenous BK current to fast-activating, while maintaining similar current-voltage properties between day and night. Alteration of BK currents in Tg-BK R207Q SCN neurons increased firing at night but decreased firing during the day, demonstrating that BK currents generate bidirectional effects on neuronal firing under distinct conditions.V oltage-gated K + channels generally oppose excitability by producing hyperpolarizing current in response to membrane depolarization (1). One distinctive member of this family is the BK channel (K Ca 1.1), encoded by the Kcnma1 gene (2, 3). BK channels are widely expressed in excitable and nonexcitable cells (4), suggesting that they are highly versatile players in membrane signaling. In neurons and muscle, BK currents are activated by simultaneous membrane depolarization and an increase in intracellular Ca 2+ (Ca 2+ i ) (1, 5), shaping the falling phase of the action potential, the afterhyperpolarization (AHP), and some Ca 2+ transients that underlie neurotransmitter release and secretion (6-9). Deletion of Kcnma1 leads to a constellation of defects related to hyperexcitability (2, 10). For this reason, BK agonists have been pursued as novel targets for treating stroke, seizure, cardiac ischemia, urinary incontinence, asthma, erectile dysfunction, and hypertension (11,12). Linkage analysis and expression profiling have also implicated Kcnma1 in schizophrenia, autism, mental retardation, and alcoholism (13-17).Whereas selectively activating BK currents that suppress excitability would be therapeutically useful, a paradoxical excitatory role for BK has also been uncovered in several tissues. BK antagonists can reduce heart rate (...

-expression of the BK K ϩ channel disrupts suprachiasmatic nucleus circuit rhythmicity and alters clock-controlled behavior. Am J Physiol Cell Physiol 304: C299 -C311, 2013. First published November 21, 2012; doi:10.1152/ajpcell.00302.2012In mammals, almost all aspects of circadian rhythmicity are attributed to activity in a discrete neural circuit of the hypothalamus, the suprachiasmatic nucleus (SCN). A 24-h rhythm in spontaneous firing is the fundamental neural intermediary to circadian behavior, but the ionic mechanisms that pattern circuit rhythmicity, and the integrated impact on behavior, are not well studied. Here, we demonstrate that daily modulation of a major component of the nighttime-phased suppressive K ϩ current, encoded by the BK Ca 2ϩ -activated K ϩ current channel (KCa1.1 or Kcnma1), is a critical arbiter of circadian rhythmicity in the SCN circuit. Aberrant induction of BK current during the day in transgenic mice using a Per1 promoter (Tg-BK R207Q ) reduced SCN firing or silenced neurons, decreasing the circadian amplitude of the ensemble circuit rhythm. Changes in cellular and circuit excitability in Tg-BK R207Q SCNs were correlated with elongated behavioral active periods and enhanced responses to phase-shifting stimuli. Unexpectedly, despite the severe reduction in circuit amplitude, circadian behavioral amplitudes in Tg-BK R207Q mice were relatively normal. These data demonstrate that downregulation of the BK current during the day is essential for the high amplitude neural activity pattern in the SCN that restricts locomotor activity to the appropriate phase and maintains the clock's robustness against perturbation. However, a residually rhythmic subset prevails over the ensemble circuit to drive the fundamental circadian behavioral rhythm. circadian rhythm; potassium channel; action potential; Kcnma1; BK channel A HIGHLY TRACTABLE SYSTEM to address neural coding of behavior is the generation of daily (circadian) rhythmicity. Lesion and transplantation studies have shown that the key aspects of circadian rhythmicity are mediated by a relatively discrete locus in the brain, the suprachiasmatic nucleus (SCN) in the hypothalamus (32,46,55,62). The bilateral SCN circuit is comprised of ϳ20,000 interconnected neurons that generate a synchronized network oscillation in spontaneous action potential firing that underlies circadian behavior (1,4,5,44,58,67,72).

BK Ca2+-activated K+ currents exhibit diverse properties across tissues. The functional variation in voltage- and Ca2+-dependent gating underlying this diversity arises from multiple mechanisms, including alternate splicing of Kcnma1, the gene encoding the pore-forming (α) subunit of the BK channel, phosphorylation of α subunits, and inclusion of β subunits in channel complexes. To address the interplay of these mechanisms in the regulation of BK currents, two native splice variants, BK0 and BKSRKR, were cloned from a tissue that exhibits dynamic daily expression of BK channel, the central circadian pacemaker in the suprachiasmatic nucleus (SCN) of mouse hypothalamus. The BK0 and BKSRKR variants differed by the inclusion of a four–amino acid alternate exon at splice site 1 (SRKR), which showed increased expression during the day. The functional properties of the variants were investigated in HEK293 cells using standard voltage-clamp protocols. Compared with BK0, BKSRKR currents had a significantly right-shifted conductance–voltage (G-V) relationship across a range of Ca2+ concentrations, slower activation, and faster deactivation. These effects were dependent on the phosphorylation state of S642, a serine residue within the constitutive exon immediately preceding the SRKR insert. Coexpression of the neuronal β4 subunit slowed gating kinetics and shifted the G-V relationship in a Ca2+-dependent manner, enhancing the functional differences between the variants. Next, using native action potential (AP) command waveforms recorded from SCN to elicit BK currents, we found that these splice variant differences persist under dynamic activation conditions in physiological ionic concentrations. AP-induced currents from BKSRKR channels were significantly reduced compared with BK0, an effect that was maintained with coexpression of the β4 subunit but abolished by the mutation of S642. These results demonstrate a novel mechanism for reducing BK current activation under reconstituted physiological conditions, and further suggest that S642 is selectively phosphorylated in the presence of SRKR.