Molecular biology: generation of transgenic mice (homologous recombination) and CRISPR gene-editing, RT-PCR, cloning, site-directed mutagenesis, transcript and protein expression, immunohistochemistry and in situ hybridization, microarray analysis of gene expression.
Electrophysiology: macroscopic currents, action potentials, single and multi-unit extracellular recordings, planar multi-electrode arrays, whole-cell recording, patch-clamp, acute brain slices, dissociated primary neuronal cultures, and organotypic cultures.
Systems physiology: telemetry (ECG, EEG, pressure, temperature), isometric tension recordings from smooth muscle, cardiovascular function, bladder cystometry, and behavior (circadian rhythm, locomotor coordination, baseline and paroxysmal dyskinesia).
Transgenic Mouse Lines
Global BK channel knockout (Slo–/–, Kcnma1–/–)
Available from Jax Labs (Stock #035902 B6.129(FVB)-Kcnma1tm1Rwa/J)
Citation: JBC 279:36746-36752 (2004)
PCR genotyping protocol & strain info
Transnetyx genotyping (Probes: Kcnma1 WT and Neomycin)
MTA
KCNMA1 channelopathy mouse models (CRISPR gene-edited)
GOF: Kcnma1N999S (Jax Stock #037787)and Kcnma1D434G (Jax Stock #037788) and LOF: Kcnma1H444Q (Jax Stock #037789)
Citation: eLife 2022;11:e77953.
Transnetyx genotyping (Probes Kcnma1-9, 1-8, and 1-7)
MTA
BK channel gain-of-function, Per1-driven expression (Tg-BKR207Q)
Citation: PNAS 109(46):18997-9002 (2012). Genbank Accession #JX429072.
Transnetyx genotyping (Probes: Kcnma1-2 Tg)
MTA
Conditional BK channel knockout, expressing tdTomato (Kcnma1fl)
Available from Jax Labs (Stock #035901 B6.Cg-Kcnma1tm1.1Alme/J)
Citation: Physiological Reports 3(11): e12612 (2015). Genbank Accession #
Transnetyx genotyping (Probes: Kcnma1-3 WT and tdRFP)
MTA
Beta2 subunit knockout, conditional, (Kcnmb2–/–), made by Chris Lingle (Wash U)
Citation: Nature Communications Mar 4;7:10837 (2016).
Original Citation for generation of mouse line: J Gen Physiol 144(4):275-95 (2014).
Available from Chris Lingle or Jax Labs (Stock #028416).
Transnetyx genotyping (Probes: Kcnmb2-1 WT and Kcnmb2-1 EX)
Genbank Links for BK Variants (Organized by Publication)
HUMAN BK CHANNEL cDNA sequences
BKVYR (MG279689) was used in the following publications:
Characterization of new human KCNMA1 Loss-of-Function Mutations. Biophysical Journal 118(3), 114A (2020).
Comparative Gain-of-Function Effects of KCNMA1-N999S Mutation on Human BK Channel Properties. J Neurophysiology 123:560 (2020).
Effects of single nucleotide polymorphisms in human KCNMA1 on BK current properties. Frontiers in Molecular Neuroscience. 12:285(2019).
BKQEERL (MG279688) used in: Effects of single nucleotide polymorphisms in human KCNMA1 on BK current properties. Frontiers in Molecular Neuroscience. 12:285(2019).
MOUSE BK CHANNEL cDNA sequences
BK channels regulate sinoatrial node firing rate and cardiac pacing in vivo. AJP- Heart 307(9):H1327-38 (2014).
– BKQEERL (KF530043), BKVYR (KF530042)
Phosphorylation of a constitutive serine inhibits BK channel variants containing the alternate exon ‘SRKR’. J General Physiology 142 (6):585-598 (2013).
– BKSRKR (JX462785); Mutations: BKSRKR-S642A, , BKSRKR-R640Q/R645Q
– BKSRKR with YFP tag (JX462786)
– mbr5:YFP (KF530038)
– C2:YFP (KF530044)
– C3:YFP (KF530036)
– C4:YFP, also called BKVYR (KF530042)
– BK0:YFP (KF530040); Mutations: BK0-S642D
Mis-expression of the BK K+ channel disrupts suprachiasmatic nucleus circuit rhythmicity and alters clock-controlled behavior. AJP- Cell Physiology 304(4):C299-C311 (2013).
Genetic activation of BK currents in vivo generates bi-directional effects on neuronal excitability. PNAS 109(46):18997-9002 (2012).
– Tg-BKR207Q (JX429072)
Others
MOUSE