(B) Relative mRNA expression levels of Slo1, Slo3 and 1C4 subunits in WT (black) and 4 KO (white) hippocampi by RT-qPCR reveals no change in expression of all but the 4 subunit mRNA. NIHMS304455-supplement-Supp_Fig_S2.jpg (79K) GUID:?A45F63E3-4E09-4281-ADBC-D61BBF2589AD Abstract BK channels are large conductance calcium- and voltage-activated potassium channels critical for neuronal excitability. channels are large conductance calcium- and voltage-activated potassium channels critical for neuronal excitability. Some neurons express so called fast-gated, type I BK channels. Other neurons express BK channels assembled with the accessory 4 subunit conferring slow-gating of type II BK channels. However, it is not clear how protein phosphorylation modulates these two distinct BK channel types. Using 4 knockout mice, we compared fast- or slow-gated BK channels in response to changes in phosphorylation status of hippocampus dentate gyrus granule neurons. We utilized the selective PP2A/PP4 phosphatase inhibitor, Fostriecin, to study changes in Ethisterone action potential shape and firing properties of the neurons. In 4 knockout neurons, Fostriecin increases BK current, speeds BK channel activation, and reduces action potential amplitudes. Fostriecin increases spiking during early components of an action potential train. In contrast, Ethisterone inhibition of BK channels through 4 in wild type neurons or by BK channel inhibitor Paxilline opposes Fostriecin effects. Voltage clamp recordings of neurons reveal that Fostriecin increases both calcium and BK currents. However, Fostriecin does not activate BK alone channels in transfected HEK293 cells lacking calcium channels. In summary, these results suggest that the fast-gating, type I BK channels lacking 4 can increase neuronal excitability in response to reduced phosphatase activity and activation of calcium channels. By opposing BK channel activation; the 4 subunit plays an important role in moderating firing frequency regardless of changes in phosphorylation status. have not been studied to date. This is partly due to the relatively nonselective action of most traditional phosphatase inhibitors and their broad target specificity. We have overcame this hindrance by using a novel phosphatase inhibitor Fostriecin that has four orders of sensitivity higher for PP2A NOTCH1 and PP4 compared to other phosphatases (Lewy em et al. /em , 2002). In addition, we were able to discern the fast- and slow-gating BK types by comparing 4 knockout mice to their wild type counterparts (Brenner em et al. /em , 2005; Wang em et al. /em , 2009). Our approach using a novel PP2A/PP4 inhibitor with 4 knockout mice thus offers a unique way to study the role of phosphorylation status on BK channels with or without the 4 subunit. In this study we have investigated the role of 4 in BK channel response to changes in neuronal phosphorylation status. For the first time, we confirmed that this fast-gating BK channels in neurons are activated by PP2A/PP4 inhibition. Furthermore, we found that knockout of 4 sensitizes neurons to actions of PP2A/PP4 inhibitor Fostriecin. The consequences of Fostriecin are BK channel activation, truncation of action potential amplitude and increase spiking during early components of an action potential train. These results suggest a new role for 4 in normalizing BK channels response to increased phosphorylation status of neurons. METHODS Isolation of brain slices All animal procedures were reviewed and approved by the University of Texas Health Science Center at San Antonio Institutional Animal Care and Use Committee (IACUC). Brain slices were prepared from 4C7 weeks old animals. 4 knockout (KO) mice were generated as described previously (Brenner em et al. /em , 2005). Animals used for this study were inbred for 5 generations to C57BL/6J and compared to control wild-type (WT) C57BL/6J mice. In contrast to the original mixed 129svj/C57BL/6J background strain, the inbred C57BL/6J background fail to have spontaneous seizures. Therefore observed changes more likely represent direct effects of BK channels properties rather than indirect effects of seizures. Animals were fully anesthetized by Isoflurane (Butler Animal Health Supply, Dublin, OH, USA) prior to their sacrifice by decapitation. The whole brain was extracted from the skull in Ethisterone less than 1 minute and 15 seconds after decapitation and placed in ice-cold cutting solution made up of (in mM) 2 KCl, 2 MgSO4, 1.25 NaH2PO4, 1 CaCl2, 2 MgCl2, 26 NaHCO3, 10 D-dextrose, Ethisterone 0.4 Vitamin C, and 206 Sucrose). The brain was than attached to a cutting platform and sliced (while constantly bubbled with 95% O2/5% CO2 mixture) to 400 M thick coronal sections no later than 3C4 minutes after the brain extraction. The slicing was performed on a Leica VT1000S vibratome (Leica Microsystems, Bannockburn, IL, USA) in ice-cold cutting solution. Ethisterone The brain slices recovered in 30.