Browsing School, Graduate by Subject "Large-Conductance Calcium-Activated Potassium Channels"
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Calcium and BK Potassium Channel Regulation of Circadian Rhythms in the Suprachiasmatic NucleusMammalian circadian rhythms are driven by a network of neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN exhibits daily (24-hour) rhythms in spontaneous action potential (AP) firing rate that encodes a time-of-day signal that coordinates the timing of circadian physiological and behavioral processes. Large-conductance Ca2+-activated K+ (BK) channels have a major role in driving the diurnal patterns of spontaneous firing in SCN neurons. BK K+ currents are larger at night, correlating with reduced neuronal excitability. The diurnal variation in BK current in the SCN is required for setting the day-night difference in firing frequency. BK currents undergo multi-level regulation by genetic and posttranslational mechanisms as well as functional coupling to Ca2+ channels. Intracellular Ca2+ (Ca2+i) is required for BK channel activation and previous studies have shown BK current is predominantly coupled to two types of Ca2+ sources in the SCN: L-type Ca2+ channels (LTCCs), and ryanodine receptors (RyRs). Circadian rhythms in Ca2+i have also been identified in SCN neurons. However, the Ca2+ channels involved in generating both AP and Ca2+i rhythms have not been clearly identified. First, to determine which Ca2+ channels are involved in AP rhythms, this study measured the impact of Ca2+ channel agonists and antagonists on the circadian parameters of spontaneous AP activity from organotypic SCN slice cultures grown on multi-electrode arrays. Next, to determine which Ca2+ channels are involved in Ca2+i rhythms, this study tested the effects of the same Ca2+ channel pharmacology on the circadian parameters of Ca2+i measured from SCN slice cultures transfected with a fluorescent Ca2+ sensor. Lastly, this investigated a potential mechanism by which LTCCs contribute to firing rate in SCN neurons by examining their ability to activate BK channels under controlled conditions. This study provides insight into the roles of specific Ca2+ sources in neural coding of the circadian time signal in the SCN.
Mechanisms of Diurnal Regulation of the BK current in the Brain's Central ClockThe large conductance Ca2+- and voltage-activated potassium (BK) channel is activated via two important cellular signaling mechanisms: increases in intracellular Ca2+ and membrane depolarization. Though the BK channel is encoded via a single gene, Kcnma1, currents produced by this channel exhibit a large degree of phenotypic variability across different cell types, indicating regulation of the BK channel can influence BK current properties. Furthermore, regulation of current properties, as opposed to changing expression of the channel, may act as a mechanism through which excitability can be modulated in various neurons. In order to determine whether regulation of BK current properties can influence neuronal excitability, we utilized the suprachiasmatic nucleus (SCN), as a model system. The SCN is the brain's central circadian oscillator, encoding time-of-day information through changes in neuronal excitability. Increased excitability is found during the day, promoting faster firing rates, while at night excitability is dampened, slowing firing rate. This diurnal change in firing rate is due in part to an increase in BK channel protein found in the SCN at night. While regulation of expression of the BK channel gene can account for part of the diurnal difference in excitability of SCN neurons, we hypothesized that daily regulation of BK current properties in the SCN via β-subunit association, alternative splicing, and control of Ca2+ sources limits a role for BK currents in setting firing frequency during the day. Using patch-clamp electrophysiolgy we demonstrate that BK current properties in the SCN are diurnally regulated, with smaller BK currents during the day found to be due to BK channel inactivation, Ca2+ source regulation and alternative splicing, rather than simply a decrease in BK channel expression. We also found loss of these mechanisms produces an increase in BK current and results in a decrease in firing rate during the day in SCN neurons. These results support the hypothesis that BK current regulation is just as important as regulation of BK protein expression in setting a diurnal difference in firing rate in the SCN. Furthermore, these results provide a framework through which modulation of current properties may impact firing in other pacemaking neurons.