Genetic activation of BK currents in vivo generates bidirectional effects on neuronal excitability.

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 neuro- and 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.

Mis-expression of the BK K(+) channel disrupts suprachiasmatic nucleus circuit rhythmicity and alters clock-controlled behavior.

In 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(+) current, encoded by the BK Ca(2+)-activated K(+) current channel (K(Ca)1.1 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.

BK channel inactivation gates daytime excitability in the circadian clock.

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 ß2 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 ß2 eliminates inactivation, abolishing the diurnal variation in both BK current magnitude and SCN firing, and disrupting behavioural rhythmicity. Selective restoration of inactivation via the ß2 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.

Evaluation of mouse urinary bladder smooth muscle for diurnal differences in contractile properties.

Most physiological systems show daily variations in functional output, entrained to the day-night cycle. Humans exhibit a daily rhythm in urinary voiding (micturition), and disruption of this rhythm (nocturia) has significant clinical impact. However, the underlying mechanisms are not well-understood. Recently, a circadian rhythm in micturition was demonstrated in rodents, correlated with functional changes in urodynamics, providing the opportunity to address this issue in an animal model. Smooth muscle cells from mouse bladder have been proposed to express a functional and autonomous circadian clock at the molecular level. In this study, we addressed whether a semi-intact preparation of mouse urinary bladder smooth muscle (UBSM) exhibited measurable differences in contractility between day and night. UBSM tissue strips were harvested at four time points over the diurnal cycle, and spontaneous (phasic) and nerve-evoked contractions were assessed using isometric tension recordings. During the active period (ZT12-24) when micturition frequency is higher in rodents, UBSM strips had no significant differences in maximal- (high K(+)) or nerve-evoked contractions compared to strips harvested from the resting period (ZT0-12). However, a diurnal rhythm in phasic contraction was observed, with higher amplitudes at ZT10. Consistent with the enhanced phasic amplitudes, expression of the BK K(+) channel, a key suppressor of UBSM excitability, was lower at ZT8. Higher expression of BK at ZT20 was correlated with an enhanced effect of the BK antagonist paxilline (PAX) on phasic amplitude, but PAX had no significant time-of-day dependent effect on phasic frequency or nerve-evoked contractions. Overall, these results identify a diurnal difference for one contractile parameter of bladder muscle. Taken together, the results suggest that autonomous clocks in UBSM make only a limited contribution to the integrated control of diurnal micturition patterns.

Phosphorylation of a constitutive serine inhibits BK channel variants containing the alternate exon "SRKR".

BK Ca(2+)-activated K(+) currents exhibit diverse properties across tissues. The functional variation in voltage- and Ca(2+)-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 ß 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 Ca(2+) 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 ß4 subunit slowed gating kinetics and shifted the G-V relationship in a Ca(2+)-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 ß4 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.