ANO1 and ANO2 expression levels in the cerebellum.

<p><b>(A)</b> Membrane topology model for anoctamin Ca²⁺-activated Cl^- channels based on the X-ray structure of a fungal TMEM16 protein [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142160#pone.0142160.ref042" target="_blank">42</a>]. The transmembrane domains 5 and 6 are thought to provide the pore-lining region in the homodimeric channel [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142160#pone.0142160.ref095" target="_blank">95</a>]. Five negatively charged amino-acid residues <i>(E</i>, <i>D)</i> and an asparagine residue <i>(N)</i> in transmembrane domains 6–8 serve as Ca²⁺-binding sites involved in channel gating [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142160#pone.0142160.ref039" target="_blank">39</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142160#pone.0142160.ref041" target="_blank">41</a>]. Four alternatively spliced segments <i>(a—d)</i> determine the apparent Ca²⁺-sensitivity of the ANO1 channel [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142160#pone.0142160.ref005" target="_blank">5</a>]. ANO2 has two isoforms <i>A</i> and <i>B</i> and a regulatory motif at a position homologous to segment <i>c</i> in ANO1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142160#pone.0142160.ref006" target="_blank">6</a>]. <b>(B)</b> RT-PCR analysis from mouse olfactory epithelium <i>(OE)</i> and mouse cerebellum <i>(CB)</i> yield similarly strong ANO1 signals in cerebellum but weaker signals for ANO2. <b>(C)</b> Immunoblots obtained from lysates of cerebellum <i>(CB)</i> and main olfactory epithelium <i>(OE)</i> from wild-type and Ano2^-/- mice show an ANO1-specific signal at ~120 kDa with the ANO1_in antiserum. <b>(D)</b> Rabbit anti-ANO2_ex serum stains ANO2-specific bands <i>(asterisks)</i> in immunoblots obtained from lysates of main olfactory epithelium <i>(OE)</i> and eye, as well as in membrane-protein preparations of main olfactory bulb <i>(OB)</i> and cerebellum <i>(CB)</i>. ANO2 bands are not present in immunoblots from Ano2^-/- mice.</p

Reversal of GABAergic postsynaptic currents by the ANO2 inhibitor.

<p><b>(A)</b> At a Cl^- concentration of 12 mM in the recording pipette, GABAergic postsynaptic currents were negative (Cl^- efflux), indicating that postsynaptic E_Cl is less negative than V_hold. <b>(B)</b> Shortly after applying 5 μM ANO2 inhibitor, positive currents appear <i>(circles)</i> as some synapses experience a decline of postsynaptic [Cl^-]_i, while others still have high Cl^-<i>(asterisks)</i>. <b>(C)</b> During the continued presence of the ANO2 inhibitor, virtually all postsynaptic currents reverse to positive polarity (Cl^- influx) indicating that GABAergic synapses experience an E_Cl more negative than V_hold. <b>(D)</b> The collected data from 12 Purkinje cells at [Cl^-]_i = 12 mM and V_hold = -60 mV demonstrate the polarity reversal of postsynaptic currents (PSCs) actuated by the ANO2 inhibitor. <b>(E)</b> Schematic representation of an hypothesis for the 12 mM [Cl^-]_i experiment. In the absence of the ANO2 inhibitor <i>(upper scheme)</i>, the basal activity of ANO2 channels <i>(green)</i> provides a Cl^- conductance in the dendritic membrane. ANO2 contributes to the Cl^—transport machinery, whose various pathways are represented by the K⁺/Cl^—cotransporter KCC2 <i>(blue)</i>. Together the Cl^- pathways stabilize a slightly elevated level of [Cl^-]_i which results in a negative driving force (V_m—E_Cl < 0) for Cl^- currents through GABA_A receptors in GABAergic synapses <i>(red)</i>. In this situation, Cl^- currents are outwardly directed and cause negative postsynaptic currents. Application of the ANO2 inhibitor <i>(lower scheme)</i> reduces the Cl^- conductance. This causes a polarity reversal of the Cl^- driving force, as the balance shifts towards Cl^- extrusion, causing local [Cl^-]_i to decrease. This hypothesis provides a qualitative concept for the role of ANO2 channels in the inversion of postsynaptic currents that is depicted in panels A to C. The proximity of Cl^—transport pathways and GABAergic synapses, as well as the occurrence of local Cl^-.gradients within dendritic segments, are inspired by the model for GABA_A-receptor-mediated Cl^- gradients in extended dendritic trees proposed by Jedlicka et al. (2011) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142160#pone.0142160.ref088" target="_blank">88</a>].</p

Expression of ANO1 in neurons of the cerebellar cortex.

<p><b>(A)</b> Schematic representation of the main cell types that constitute the microcircuits of the cerebellar cortex. Inhibitory interneurons <i>(red)</i>: basket cells <i>(BC)</i>, Golgi cells <i>(GC)</i> and stellate cells (SC); excitatory input to Purkinje cells <i>(PC) (green)</i>: climbing fibers <i>(CF)</i>, granule cells <i>(GrC)</i>, mossy fibers <i>(MF)</i> and parallel fibers <i>(PF)</i>. <b>(B)</b> Diaminobenzidine-labelled ANO1_in antiserum produced a continuous staining pattern in the Purkinje cell layer <i>(PCL)</i> as well as a scattered stain in the granule cell layer <i>(GL)</i> and the molecular layer <i>(ML)</i> of the mouse cerebellum. <b>(C)</b> Low-magnification image showing ANO1 immunofluorescence <i>(green)</i> and DAPI nuclear stain <i>(blue)</i>. Only few ANO1-positive cells are located among the many cells of the <i>GL</i>, most ANO-1 cells can be seen in the <i>PCL</i> and <i>ML</i>. <b>(D)</b> Detail of the <i>GL</i>, demonstrating co-localization of ANO1 and GAD^Cre, a marker for GABAergic neurons. <b>(E)</b> Co-localization of ANO1 with GAD^Cre cells in the <i>ML</i>. <b>(F)</b> Purkinje cell somata stained with the ANO1_ex antiserum and <i>NeuroTrace</i>^® to illustrate that all Purkinje cells are ANO1-positive. <b>(G)</b> Small, ANO1-negative granule-cell nuclei <i>(blue)</i> and somewhat larger ANO1-positive Golgi cells <i>(GoC)</i> in the <i>GL</i>. <b>(H)</b> In the molecular layer, ANO-1 positive cells are <i>SC</i> and <i>BC</i> inhibitory interneurons. <b>(I)</b> Preadsorption control for the ANO1_ex antiserum on cerebellar cortex. Scale bars: <i>B</i>, <i>C</i>: 200 μm, <i>D</i>,<i>E</i>: 50 μm, <i>F</i>: 20 μm, <i>G</i>: 50 μm, <i>H</i>: 20 μm, <i>I</i>: 50 μM. Blue represents DAPI nuclear stain.</p

Involvement of ANO2 in depolarization-induced depression of inhibition.

<p><b>(A)</b> A cerebellar Purkinje cell loaded with the fluorescent dye Alexa Fluor 568. Scale bar: 10 μm. <b>(B)</b> Spontaneous postsynaptic currents in a Purkinje cell with E_Cl near 0 mV and V_hold = -69 mV. Overlay of 764 current traces showing similar time courses but differing amplitudes, probably reflecting distinct positions of GABAergic synapses on the Purkinje cell dendritic tree. <b>(C)</b> Postsynaptic currents were completely blocked by 50 μM picrotoxin, an inhibitor of GABA_A-receptor chloride channels. <b>(D)</b> Protocol for activation of climbing fibers: Ten 0.1-ms current pulses were applied to the area near the proximal dendrite of a Purkinje cell while recording the whole-cell current of that cell at -70 mV. CF-activation produced characteristic complex spikes, as shown in the inset. <b>(E)</b><i>Upper traces</i>: GABAergic inhibitory postsynaptic currents recorded from a Purkinje cell at V_hold = -48 mV and with 5 mM Cl^- in the pipette solution. The positive polarity of IPSCs indicates Cl^- influx. <i>Lower traces</i>: postsynaptic currents, recorded immediately after the climbing-fiber stimulation, displayed decreased amplitudes. <b>(F)</b> IPSCs recorded from a Purkinje cell of an Ano2^-/—mouse before <i>(upper traces)</i> and immediately after <i>(lower traces)</i> CF-activation. <b>(G)</b> The number of detectable IPSC signals decreased by ~47% through climbing-fiber stimulation (before CF: 30.7 ± 6.5 min^-1; after CF: 14.6 ± 3.4 min^-1; 8 cells; <i>ctrl</i>). In slices from Ano2^-/- mice, more IPSCs were detected (54.5 ± 18.5 min^-1; 4 cells), and the activation of climbing fibers had no effect (52.5 ± 16.2 min^-1; 4 cells).</p

PCR primer pairs used to characterize cerebellar ANO1 and ANO2.

<p>PCR primer pairs used to characterize cerebellar ANO1 and ANO2.</p

Expression of ANO2 protein in Purkinje cells.

<p><b>(A)</b> ANO2 immunosignals from the cerebellar cortex are discernible in the dendrites of Purkinje cells. The signals are weak but stronger than the background signals emanating from the granule cell layer <i>(GL)</i>. <b>(B)</b> In the Purkinje cell, the ANO2 immunosignal <i>(green)</i> is visible in dendrites and the perinuclear region, but only weakly in the plasma membrane of the cell body. In contrast, ANO1 signals <i>(red)</i> label the entire Purkinje cell soma, but are not detectable in dendrites. <b>(C)</b> The ANO2 antiserum does not stain the cerebellar cortex of the Ano2^-/- mouse. <b>(D)</b> ANO2 immunosignals in the glomeruli of the olfactory bulb serving as positive control for ANO2 in brain tissue; <i>gl</i>: glomerular layer, <i>opl</i>: outer plexiform layer. <b>(E)</b> Absence of ANO2 immunosignals from the olfactory bulb of the Ano2^-/- mouse. Blue in <i>B-E</i> represents DAPI nuclear stain. All calibrations bars: 20 μm.</p