Immunofluorescence reveals that BKα and β4 are substantially localized to the ER and that β4 is difficult to detect on the cell surface.

<p>(A) Immunocytochemistry against fixed, permeabilized HEK-293 cells for the FAP-BKα subunit (anti-BKα: green) and the ER-marker mRFP-KDEL (intrinsic fluorescence; red) in cells transfected with BKα alone. Inset shows a cell with overlapping expression of BKα and mRFP-KDEL with arrows pointing to regions containing only BKα. (B) Quantitation of normalized intensity versus normalized distance from the cell nucleus for anti-BKα (green) and mRFP (red) in transfected cells. (C) As in (A) but for cells expressing BKα (green), β4 (unlabeled; not visualized) and mRFP-KDEL (red). (D) Same as (B) but in cells expressing BKα, β4 and mRFP-KDEL. (E) Localization of the β4 subunit (anti-β4: green) and mRFP- KDEL (intrinsic fluorescence; red) in cells expressing BKα (unlabeled; not visualized), β4 and mRFP-KDEL. Scale bar = 20 µm or 5 µm for inset. (F) As in (B), but for β4 and mRFP-KDEL. (G) Localization of the β4 subunit (anti-β4: green), mRFP-KDEL (intrinsic fluorescence; red) and BKα (anti-α: blue) in transfected, fixed HEK-293 cells expressing BKα, β4, and mRFP-KDEL. (H) Expression pattern of the β4 subunit (anti-β4: green) and mRFP- KDEL (intrinsic fluorescence; red) under non-permeabilized immunohistochemistry conditions. Note that immunohistochemical detection of cell-surface BKα is difficult because of an intracellular epitope for BKα. Scale bar = 10 µm (G,H).</p

Genetic ablation of β4 results in larger BK channel currents in CA3 pyramidal neurons.

<p>(A) Fluorescent image of a living slice from a β4−/− knock-out mouse showing GFP-signal in hippocampus. Scale bar: 500 µm. (B) Mean total potassium current from β4 heterozygote (β4+/−) and β4 knock-out (β4−/−) mice. (C) Example paxilline-sensitive BK channel current from representative CA3 neurons in β4+/− and β4−/− mice. (D) Mean amplitude of paxilline-sensitive BK channel current in β4+/− and β4−/− mice.</p

An ER-retention/retrieval sequence at the C-terminus of β4 inhibits the surface expression of BKα and β4.

<p>(A) Schematic of the mutant β4 constructs showing the C-terminal amino acids. (B) Fluorescence from binding of cell-impermeable dye to cell-surface BKα channels in cells transfected with FAP-BKα (red) and GFP (green). (C) Same as (B) but in cells transfected with FAP-BKα and wild-type β4. (D) Same as (B) but in cells transfected with FAP-BKα and β4-Ala. (E) Same as (B) but in cells transfected with FAP-BKα and β4-polySer. Scale bar = 20 µm (B–E). (F) Mean intensity of surface fluorescence for β4 constructs normalized to fluorescence from cells transfected with FAP-BKα alone. (G) Histogram showing distribution of fluorescence intensity in cells transfected with FAP-BKα alone (red), FAP-BKα+β4-Ala (green) and FAP-BKα+β4-polySer (blue). Scale bar: 20 µm (B–E).</p

Mutation of the C-terminal ER-retention/retrieval sequence in β4 liberates β4 to the surface.

<p>(A) Expression pattern of the cotransfected FAP-BKα (unlabeled; not visualized), β4-Ala mutant (anti-β4 immunocytochemistry: green) and mRFP- KDEL (intrinsic fluorescence; red) in fixed, permeabilized HEK-293 cells. (B) Same as (A) but under non-permeabilized conditions. (C) Same as for (A) but for β4-polySer mutant under permeabilized conditions. (D) Same as (C) but under non-permeabilized conditions. Scale bar = 10 µm (A–D).</p

Co-expression of the β4 subunit reduces cell-surface trafficking of BK channels.

<p>(A) Membrane topology of the FAP-BKα and β4 proteins. The FAP tag is at the extracellular, N-terminus. The C-terminus of the BKα subunit is indicated. (B) Schematic of binding of cell-impermeable dye (pink) to the FAP results in significant increase in fluorescence (red). (C) Application of cell-impermeable dye labels only surface BKα channels in live HEK-293 cells co-transfected with FAP-BKα (red) and GFP (green). (D) Same as (C) but in cells co-transfected with β4, FAP-BKα, and GFP showing reduced surface expressed of the FAP-BKα. (E) Application of cell-permeable dye labels intracellular stores of channel in cells transfected with FAP-BKα (red) and GFP (green). (F) Same as (E) but in cells transfected with β4, FAP-BKα, and GFP. Scale bar = 20 µm (C–F). (G) Distribution of surface fluorescence intensity values after application of cell-impermeable dye in transfected cells for FAP-BKα (red bars) or FAP-BKα+β4 (black bars). (H–I) Proposed model for surface distribution of BK channels in the absence (H) or presence (I) of the β4 subunit.</p

β4-containing BK channels do not contribute to whole-cell BK channel currents.

<p>(A) In situ hybridization showing regions of β4 expression in a mouse brain. Arrow points to high levels of expression in the CA3. (B) Hippocampal area CA3 shows robust β4 expression. Scale bar = 200 µm. (C) Bright field image showing a whole cell electrode in the CA3. Scale bar = 500 µm. (D) Fluorescent image of a CA3 pyramidal cell filled with Alexa fluor 568 after whole cell patch clamp recording. Scale bar = 50 µm. (E) Overlaid traces from a representative cell showing total potassium current and the residual potassium current after application of a BK channel blocker. (F) Schematic overlap between paxilline- and iberiotoxin-sensitive currents when β4-containing BK channels contribute to whole-cell current. (G) Schematic overlap between paxilline- and iberiotoxin-sensitive currents when β4 containing BK channels do not contribute to whole-cell current. (H) Example paxilline-sensitive BK channel current from a representative cell. (I) Example iberiotoxin-sensitive BK channel current from a representative cell. (J) Quantitation of mean paxilline (pax)- and iberiotoxin (ibtx)-sensitive currents from CA3 pyramidal cells.</p

Control and seizure animals express different ratios of type II and type I BK channels.

<p>(A-B) Type II channels have longer open dwell times and were resistant to IBTX. Type I channels had observably shorter open dwell times and were consistently blocked by IBTX. All channels were blocked by paxilline. (C) β4 KO mice only express Type I channels that are sensitive to IBTX and paxilline. (D) Type I BK channels were more predominant from seizure experienced mice (Control n = 15, Seizure n = 19). (E) Single channel current amplitude was similar in control, pilocarpine treated, and β4 KO mice. Single channel current was an average of 7 ± 1.5 pA at a 0 mV holding potential. (Control n = 8, Seizure n = 7, β4 KO n = 5) (F) The average number of BK channels per membrane patch was similar in control and seizure mice. (Control n = 15, Seizure n = 19). Panels A-E data was acquired at 0 mV potential with 60 μM buffered calcium internal solution.</p

Pilocarpine-induced seizures reduce <i>KCNMB4</i>-EGFP reporter expression.

<p>(A) Anti-EGFP (top panels) and anti-MAP2 (middle panels) immunofluorescence staining from heterozygous mice containing the EGFP cDNA that replaces the first coding exon of <i>KCNMB4</i> gene [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188064#pone.0188064.ref010" target="_blank">10</a>]. The pictures are oriented from left, medial (the dentate gyrus) to right, temporal (hilar mossy fibers extending to CA3 region). Bottom panels are merged of above images. (B) Average fluorescence intensity of EGFP normalized to MAP2 measured at the granule cell area. Control is 4.1 ± 0.20, N = 7, pilocarpine 3.4 ± 0.18, N = 6, P = 0.034.</p

cfos immunostaining marks neurons with reduced gene expression of <i>KCNMB4</i>.

<p>(A) Anti-EGFP (top panels) and anti-c-Fos (middle panels) immunofluorescence staining from heterozygous mice containing EGFP knockin into the <i>KCNMB4</i> locus [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188064#pone.0188064.ref010" target="_blank">10</a>]. Left panels are control mice, right panels are pilocarpine treated mice. The pictures are focused on the granule cell layer of the dentate gyrus. Bottom panels are merged images from panels above. (B) Average anti-EGFP immunofluorescence in c-Fos positive cells normalized to EGFP immunofluorescence in c-Fos negative, neighboring cells. Control cells show a reduced EGFP immunofluorescence in c-Fos positive cells (bar value) normalized to c-Fos negative cells in the section (0.91 ± 0.017, N = 12, P < 0.001). Pilocarpine-treated mice show no reduction in EGFP immunofluorescence in c-Fos positive cells relative to c-Fos negative cells. (0.98 ± 0.019, N = 15, P = 0.38).</p

Seizures do not change BK channel contribution to early spike frequency.

<p>(Top) Representative traces of first 20 ms of voltage response to a 200 pA constant current injection. The voltages traces are aligned at the first spike (pre-spike tracings are not shown) to display effects on instantaneous frequency. Before (black trace) and after BK channel block (paxilline, grey traces) in control (A) and pilocarpine treated (B) granule cells. (C) Average instantaneous firing frequency of 1^st action potential before and after paxilline from control and seizure experienced animals. (D) Average change in instantaneous firing frequency resulting from blocking BK channels in control and pilocarpine treated mice. (Control n = 9, seizure n = 8).</p