Journal: PLoS ONE
Article Title: Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex
doi: 10.1371/journal.pone.0142160
Figure Lengend Snippet: (A) 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) 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 E Cl more negative than V hold . (D) 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. (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 (V m —E Cl < 0) for Cl - currents through GABA A 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 GABA A -receptor-mediated Cl - gradients in extended dendritic trees proposed by Jedlicka et al. (2011) .
Article Snippet: Antibodies used for immunoblotting were guinea-pig anti-ANO1 (dilution 1:400; C-terminus encoding amino acids 962–1040; marked "ANO1 in " in the text) [ ], and a rabbit ANO2 antiserum directed against the extracellular loop that connects TMDs 5 and 6 in ANO2 (Alomone Labs, ACL012, dilution 1:1000, marked “ANO2 ex ” in the text).
Techniques: Concentration Assay, Transferring, Activity Assay
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![(A) Membrane topology model for anoctamin Ca 2+ -activated Cl - channels based on the X-ray structure of a fungal TMEM16 protein . The transmembrane domains 5 and 6 are thought to provide the pore-lining region in the homodimeric channel . Five negatively charged amino-acid residues (E , D) and an asparagine residue (N) in transmembrane domains 6–8 serve as Ca 2+ -binding sites involved in channel gating [ – ]. Four alternatively spliced segments (a—d) determine the apparent Ca 2+ -sensitivity of the ANO1 channel . ANO2 has two isoforms A and B and a regulatory motif at a position homologous to segment c in ANO1 . (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 ANO1 in antiserum. (D) Rabbit anti-ANO2 ex 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.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1602/pmc04641602/pmc04641602__pone.0142160.g001.jpg)
![(A) 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) 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 E Cl more negative than V hold . (D) 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. (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 (V m —E Cl < 0) for Cl - currents through GABA A 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 GABA A -receptor-mediated Cl - gradients in extended dendritic trees proposed by Jedlicka et al. (2011) .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1602/pmc04641602/pmc04641602__pone.0142160.g006.jpg)