Taming unruly chloride channel inhibitors with rational design

Sepela, Rebecka J.; Sack, Jon T.

doi:10.1073/pnas.1805589115

Cited by 7 publications

(4 citation statements)

References 10 publications

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“…Pharmacological inhibitors for Cl − channels are notoriously promiscuous (Sepela and Sack, 2018 ). Therefore, we sought to evaluate the involvement of TMEM16A more specifically by employing CRISPR/Cas9.…”

Section: Resultsmentioning

confidence: 99%

Reduced Expression of TMEM16A Impairs Nitric Oxide-Dependent Cl− Transport in Retinal Amacrine Cells

Rodriguez

Zhong

Simpson

et al. 2022

Front. Cell. Neurosci.

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Postsynaptic cytosolic Cl− concentration determines whether GABAergic and glycinergic synapses are inhibitory or excitatory. We have shown that nitric oxide (NO) initiates the release of Cl− from acidic internal stores into the cytosol of retinal amacrine cells (ACs) thereby elevating cytosolic Cl−. In addition, we found that cystic fibrosis transmembrane conductance regulator (CFTR) expression and Ca2+ elevations are necessary for the transient effects of NO on cytosolic Cl− levels, but the mechanism remains to be elucidated. Here, we investigated the involvement of TMEM16A as a possible link between Ca2+ elevations and cytosolic Cl− release. TMEM16A is a Ca2+-activated Cl− channel that is functionally coupled with CFTR in epithelia. Both proteins are also expressed in neurons. Based on this and its Ca2+ dependence, we test the hypothesis that TMEM16A participates in the NO-dependent elevation in cytosolic Cl− in ACs. Chick retina ACs express TMEM16A as shown by Western blot analysis, single-cell PCR, and immunocytochemistry. Electrophysiology experiments demonstrate that TMEM16A functions in amacrine cells. Pharmacological inhibition of TMEM16A with T16inh-AO1 reduces the NO-dependent Cl− release as indicated by the diminished shift in the reversal potential of GABAA receptor-mediated currents. We confirmed the involvement of TMEM16A in the NO-dependent Cl− release using CRISPR/Cas9 knockdown of TMEM16A. Two different modalities targeting the gene for TMEM16A (ANO1) were tested in retinal amacrine cells: an all-in-one plasmid vector and crRNA/tracrRNA/Cas9 ribonucleoprotein. The all-in-one CRISPR/Cas9 modality did not change the expression of TMEM16A protein and produced no change in the response to NO. However, TMEM16A-specific crRNA/tracrRNA/Cas9 ribonucleoprotein effectively reduces both TMEM16A protein levels and the NO-dependent shift in the reversal potential of GABA-gated currents. These results show that TMEM16A plays a role in the NO-dependent Cl− release from retinal ACs.

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Section: Resultsmentioning

confidence: 99%

Reduced Expression of TMEM16A Impairs Nitric Oxide-Dependent Cl− Transport in Retinal Amacrine Cells

Rodriguez

Zhong

Simpson

et al. 2022

Front. Cell. Neurosci.

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“…To quantify CLC-2 run-up, we developed the following protocol: (1) on acquisition of whole-cell recording mode, an initial voltage-family of currents was recorded, as in Figure 6A ; (2) from a holding potential of 0 mV, a single 1-s test pulse to −100 mV was repeated every 5 seconds to monitor time-dependent changes in current amplitude over 5 minutes, after which a second voltage-family of currents was recorded; (3) 300 nM AK-42 was added to selectively block all CLC-2 current (Koster et al, 2020 ), and the steady state inhibition current was used for leak subtraction. In contrast to chloride-channel inhibitors historically used in electrophysiological experiments (Sepela and Sack, 2018 ), AK-42 is potent and highly selective for CLC-2 over other anion channels (Koster et al, 2020 ) The increase in WT currents is accompanied by an apparent decrease in channel rectification, towards the level displayed by Delta-N CLC-2. WT CLC-2 currents recorded in heterologous and native expression systems are generally highly inwardly rectifying.…”

Section: Functional Support For Pore Block By the N-terminal Hairpin ...mentioning

confidence: 99%

CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism

Xu,

Neelands,

Powers

et al. 2024

eLife

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CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different mammalian tissues and cell types. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating mechanisms among closely related CLC homologs has been a long-standing mystery, in part because few CLC channel structures are available, and those that exist exhibit high conformational similarity. Here, we report cryoEM structures of human CLC-2 at 2.46 – 2.76 Å, in the presence and absence of the potent and selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl−-permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct apo conformations of CLC-2 involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl−-permeation pathway from the intracellular side. This peptide is highly conserved among species variants of CLC-2 but is not present in any other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a “ball-and-chain” gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we show that loss of this short sequence increases the magnitude and decreases the rectification of CLC-2 currents expressed in mammalian cells. Furthermore, we show that with repetitive hyperpolarization WT CLC-2 currents increase in resemblance to the hairpin-deleted CLC-2 currents. These functional results combined with our structural data support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl−-permeation pathway.

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“…To quantify CLC-2 run-up, we developed the following protocol: (1) on acquisition of whole-cell recording mode, an initial voltage-family of currents was recorded, as in Figure 6A; (2) from a holding potential of 0 mV, a single 1-s test pulse to -100 mV was repeated every 5 seconds to monitor time-dependent changes in current amplitude over 5 minutes, after which a second voltage-family of currents was recorded; (3) 300 nM AK-42 was added to selectively block all CLC-2 current (Koster et al, 2020), and the steady state inhibition current was used for leak subtraction. In contrast to chloride-channel inhibitors historically used in electrophysiological experiments (Sepela and Sack, 2018), AK-42 is potent and highly selective for CLC-2 over other anion channels (Koster et al, 2020). Representative current traces and summary data for this set of experiments are shown in Figure 6F, G; individual I-V traces are shown in Figure 6 figure supplement 1.…”

Section: Functional Support For Pore Block By the N-terminal Hairpin ...mentioning

confidence: 99%

CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

Xu,

Neelands,

Powers

et al. 2023

Preprint

View full text Add to dashboard Cite

CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different mammalian tissues and cell types. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating mechanisms among closely related CLC homologs has been a long-standing mystery, in part because few CLC channel structures are available, and those that exist exhibit high conformational similarity. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 Å, in the presence and absence of the potent and selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl--permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct apo conformations of CLC-2 involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl--permeation pathway from the intracellular side. This peptide is highly conserved among species variants of CLC-2 but is not present in any other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a ball-and-chain gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we show that loss of this short sequence increases the magnitude and decreases the rectification of CLC-2 currents expressed in mammalian cells. Furthermore, we show that with repetitive hyperpolarization WT CLC-2 currents increase in resemblance to the hairpin-deleted CLC-2 currents. These functional results combined with our structural data support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl--permeation pathway.

show abstract

Taming unruly chloride channel inhibitors with rational design

Cited by 7 publications

References 10 publications

Reduced Expression of TMEM16A Impairs Nitric Oxide-Dependent Cl− Transport in Retinal Amacrine Cells

Reduced Expression of TMEM16A Impairs Nitric Oxide-Dependent Cl− Transport in Retinal Amacrine Cells

CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism

CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

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