DOI: 10.5061/dryad.3bk3j9kr0
title: Dataset: Disease-associated KCNMA1 variants decrease circadian clock robustness in channelopathy mouse models

description: KCNMA1 encodes the voltage- and calcium-activated K+ (BK) channel, which
 regulates suprachiasmatic nucleus (SCN) neuronal firing and circadian
 behavioral rhythms. Gain-of-function (GOF) and loss-of-function (LOF)
 alterations in BK channel activity disrupt circadian behavior, but the
 effect of human disease-associated KCNMA1 channelopathy variants has not
 been studied on clock function. Here, we assess circadian behavior in two
 GOF and one LOF mouse lines. Heterozygous Kcnma1N999S/WT and homozygous
 Kcnma1D434G/D434G mice are validated as GOF models of paroxysmal
 dyskinesia (PNKD3), but whether circadian rhythm is affected in this
 hypokinetic locomotor disorder is unknown. Conversely, homozygous LOF
 Kcnma1H444Q/H444Q mice do not demonstrate PNKD3. We assessed circadian
 behavior by locomotor wheel running activity. All three mouse models were
 rhythmic, but Kcnma1N999S/WT and Kcnma1D434G/D434G showed reduced
 circadian amplitude and decreased wheel activity, corroborating prior
 studies focused on acute motor coordination. In addition,
 Kcnma1D434G/D434G mice had a small decrease in period. However, the
 phase-shifting sensitivity for both GOF mouse lines was abnormal.
 Both Kcnma1N999S/WT and Kcnma1D434G/D434G mice displayed
 increased responses to light pulses and took fewer days to re-entrain to a
 new light:dark cycle. In contrast, the LOF Kcnma1H444Q/H444Q mice showed
 no difference in any of the circadian parameters tested. The enhanced
 sensitivity to phase-shifting stimuli in Kcnma1N999S/WT
 and Kcnma1D434G/D434G mice was similar to other Kcnma1 GOF mice.
 Together with previous studies, these results suggest that increasing BK
 channel activity decreases circadian clock robustness, without rhythm
 ablation.
description: Wheel activity was sampled every 1 min in ClockLab software
 (Actimetrics). Actograms were constructed by double-plotting consecutive
 days of activity over the recording period. Circadian period (τ), χ2
 amplitude and Fourier transform relative power (FFT rPSD for 0.04–0.042
 cycles/hr) were determined from the last 10 days in LD and the first 15
 days of wheel running activity in DD in ClockLab software. Activity period
 (α) was determined as the length of time an animal had consolidated
 activity using default settings with manual adjustment, with ρ defined as
 the portion of the cycle outside of α. For re-entrainment experiments,
 after 7 days of stable entrainment, the LD cycle was phase advanced by 6
 h. The response was calculated as the number of days to stable
 re-entrainment of at least 3 days. After keeping mice in DD for 7 days,
 phase shifts in response to a light pulse were calculated as the number of
 hours between the activity onset regression fits the day before and after
 a 30-min light pulse delivered in early subjective night (CT16). All bout
 parameters (bout duration, bouts/day, counts/bout) were calculated from
 the first 15 days in DD, where a bout of activity is defined as periods
 during which the activity does not fall below 30 counts/min for more than
 10 min. Data was excluded from mice that failed to record activity counts
 for two consecutive days. All animal experiments were conducted blinded to
 genotype during data collection and analysis from at least three
 independent cohorts for each transgenic line. Data was obtained from males
 and females, and no randomization was used to allocate animals into
 cohorts. WT controls were compared to transgenic littermates within each
 individual transgenic line.  Current-clamp recordings were made with a
 Multiclamp 700B amplifier and pCLAMP 11.2 software (Molecular Devices,
 Sunnyvale, CA). Data were acquired at a 50-kHz sampling rate. Recording
 window was at the peak (ZT4–8) of the circadian rhythm. All recordings
 were made with synaptic transmission intact to more closely approximate in
 vivo activity. Electrodes (4–8 MΩ resistance) were filled internal
 solution (in mM: 123 K-methane-sulfonate, 9 NaCl, 0.9 EGTA, 9 HEPES, 14
 Tris-phosphocreatine, 2 Mg-ATP, 0.3 Tris-GTP, and 2 Na2-ATP, pH adjusted
 to 7.3 with KOH). After gigaseal formation and whole cell break-in,
 membrane properties were elicited from a +50-mV voltage step from a
 holding potential (V<sub>h</sub>) of -70 mV. Access resistance
 was verified to be &lt;30 MΩ with less than 20% change at the end of
 the recording. Series resistance was compensated at 60–70%. For action
 potentials, data were acquired in 10 s sweeps. Silent cells were
 identified by injecting a 20-pA current to elicit an action potential.
 Frequency was calculated as the average of each sweep. Baseline potential
 was determined as the average inter-spike potential in the presence of
 spontaneous activity. Input resistance was calculated as the linear slope
 of the voltage from hyperpolarizing current injections (‒10 to ‒25 pA in 5
 pA increments). Instantaneous frequency histograms were constructed by
 normalizing the number of events in 0.5-Hz bins to the total number of
 events from all neurons (GraphPad Prism v9.3.0 (San Diego, CA)). All data
 were corrected for liquid junctional potential (10
 mV).
description: Statistics and graphs:  Graphpad Prism v9.3
 Wheel actogram data:  Actimetrics Clocklab running in MatLab
 (available from the authors upon request) Action
 potential recordings:  pClamp v11.2
description: ### Figure 1 The analysis and statistical tests presented in Figure 1
 panels C-F of wheel running records are contained within the GraphPad
 Prism file entitled 'Figure 1 analysis'. The individual
 circadian wheel running records in MATLAB file format for data analyzed in
 Figure 1 are available upon request from the authors. ### Figure 2 The
 analysis and statistical tests presented in Figure 2 panels B-E of wheel
 running records are contained within the GraphPad Prism file entitled
 'Figure 2 analysis'. The individual circadian wheel running
 records in MATLAB file format for data analyzed in Figure 2 are available
 upon request from the authors. ### Figure 3 The analysis and statistical
 tests presented in Figure 3 panels B and D of wheel running records are
 contained within the GraphPad Prism file entitled 'Figure 3
 analysis'. The individual circadian wheel running records in MATLAB
 file format for data analyzed in Figure 3 are available upon request from
 the authors. Figure 4 The analysis and statistical tests presented in
 Figure 4 panels A-C and F of electrophysiological recordings are contained
 within the GraphPad Prism file Figure 4 analysis file. Data for individual
 recordings are provided as ABF files with the File name structure:
 FigureNo\_Genotype\_MouseNo\_CellNo