Fig 2: ANO1 and ANO2 expression levels in the cerebellum. (A) Membrane topology model for anoctamin Ca2+-activated Cl- channels based on the X-ray structure of a fungal TMEM16 protein [42]. The transmembrane domains 5 and 6 are thought to provide the pore-lining region in the homodimeric channel [95]. Five negatively charged amino-acid residues (E, D) and an asparagine residue (N) in transmembrane domains 6–8 serve as Ca2+-binding sites involved in channel gating [39–41]. Four alternatively spliced segments (a—d) determine the apparent Ca2+-sensitivity of the ANO1 channel [5]. ANO2 has two isoforms A and B and a regulatory motif at a position homologous to segment c in ANO1 [6]. (B) RT-PCR analysis from mouse olfactory epithelium (OE) and mouse cerebellum (CB) yield similarly strong ANO1 signals in cerebellum but weaker signals for ANO2. (C) Immunoblots obtained from lysates of cerebellum (CB) and main olfactory epithelium (OE) from wild-type and Ano2-/- mice show an ANO1-specific signal at ~120 kDa with the ANO1in antiserum. (D) Rabbit anti-ANO2ex serum stains ANO2-specific bands (asterisks) in immunoblots obtained from lysates of main olfactory epithelium (OE) and eye, as well as in membrane-protein preparations of main olfactory bulb (OB) and cerebellum (CB). ANO2 bands are not present in immunoblots from Ano2-/- mice.

Fig 3: Reversal of GABAergic postsynaptic currents by the ANO2 inhibitor. (A) At a Cl- concentration of 12 mM in the recording pipette, GABAergic postsynaptic currents were negative (Cl- efflux), indicating that postsynaptic ECl is less negative than Vhold. (B) Shortly after applying 5 μM ANO2 inhibitor, positive currents appear (circles) as some synapses experience a decline of postsynaptic [Cl-]i, while others still have high Cl- (asterisks). (C) During the continued presence of the ANO2 inhibitor, virtually all postsynaptic currents reverse to positive polarity (Cl- influx) indicating that GABAergic synapses experience an ECl more negative than Vhold. (D) The collected data from 12 Purkinje cells at [Cl-]i = 12 mM and Vhold = -60 mV demonstrate the polarity reversal of postsynaptic currents (PSCs) actuated by the ANO2 inhibitor. (E) Schematic representation of an hypothesis for the 12 mM [Cl-]i experiment. In the absence of the ANO2 inhibitor (upper scheme), the basal activity of ANO2 channels (green) 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 (blue). Together the Cl- pathways stabilize a slightly elevated level of [Cl-]i which results in a negative driving force (Vm—ECl < 0) for Cl- currents through GABAA receptors in GABAergic synapses (red). In this situation, Cl- currents are outwardly directed and cause negative postsynaptic currents. Application of the ANO2 inhibitor (lower scheme) 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 GABAA-receptor-mediated Cl- gradients in extended dendritic trees proposed by Jedlicka et al. (2011) [88].

Fig 4: Expression of ANO2 protein in Purkinje cells. (A) 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 (GL). (B) In the Purkinje cell, the ANO2 immunosignal (green) is visible in dendrites and the perinuclear region, but only weakly in the plasma membrane of the cell body. In contrast, ANO1 signals (red) label the entire Purkinje cell soma, but are not detectable in dendrites. (C) The ANO2 antiserum does not stain the cerebellar cortex of the Ano2-/- mouse. (D) ANO2 immunosignals in the glomeruli of the olfactory bulb serving as positive control for ANO2 in brain tissue; gl: glomerular layer, opl: outer plexiform layer. (E) Absence of ANO2 immunosignals from the olfactory bulb of the Ano2-/- mouse. Blue in B-E represents DAPI nuclear stain. All calibrations bars: 20 μm.

Fig 5: Identification of an inhibitor specific for cerebellar ANO2 channels. (A) Whole-cell recording from an HEK293 cell transfected with mouse cerebellar ANO2 isoform B at -70 mV. Chloride inward current was activated by diffusion of 7.5 μM Ca2+ from the pipette into the cell immediately after whole-cell breakthrough (arrow). (B) Results from current recordings without (black bars) and with (hatched bars) T16Ainh-A01 revealed that neither mouse ANO1ac nor ANO1abc were significantly inhibited by 5 μM of the compound, while the ANO2 isoform B showed a significantly reduced current density. At 25 μM T16Ainh-A01, significant inhibition was also observed with ANO1ac. Results were averaged from 19–30 cells for each condition. (C) When applied at the ANO2-specific concentration of 5 μM, T16Ainh-A01 blocked DDI: The number of detectable IPSCs was 48.9 ± 12.1 min-1 before and 53.4 ± 15.4 min-1 after CF stimulation. ANO2+/+ control data from Fig 4G are included for comparison. (D) IPSC traces from wildtype mice in the presence of 5 μM T16Ainh-A01 show no difference in IPSC shapes and amplitudes before (left traces) and after (right traces) CF-activation.