Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel

Anna K. Koster; Austin L. Reese; Yuri Kuryshev; Xianlan Wen; Keri A. McKiernan; Erin E. Gray; Caiyun Wu; John R. Huguenard; Merritt Maduke; J. Du Bois

doi:10.1073/pnas.2009977117

Edited by Richard W. Aldrich, The University of Texas at Austin, Austin, TX, and approved October 22, 2020 (received for review May 21, 2020)

Significance

The CLC-2 ion channel facilitates selective passage of Cl^– ions across cell membranes. In the central nervous system, CLC-2 is expressed in both neurons and glia and is proposed to regulate electrical excitability and ion homeostasis. CLC-2 has been implicated in various central nervous system disorders, including certain types of epilepsy and leukodystrophy. Establishing a causative role for CLC-2 in neuropathologies, however, has been limited by the absence of selective reagents that enable acute and specific channel modulation. Our studies have resulted in the identification of a highly potent, small-molecule inhibitor that enables specific block of CLC-2 Cl^– currents in hippocampal brain slices. This precise molecular tool should enable future efforts to identify and treat CLC-2–related disease.

Abstract

CLC-2 is a voltage-gated chloride channel that is widely expressed in mammalian tissues. In the central nervous system, CLC-2 appears in neurons and glia. Studies to define how this channel contributes to normal and pathophysiological function in the central nervous system raise questions that remain unresolved, in part due to the absence of precise pharmacological tools for modulating CLC-2 activity. Herein, we describe the development and optimization of AK-42, a specific small-molecule inhibitor of CLC-2 with nanomolar potency (IC₅₀ = 17 ± 1 nM). AK-42 displays unprecedented selectivity (>1,000-fold) over CLC-1, the closest CLC-2 homolog, and exhibits no off-target engagement against a panel of 61 common channels, receptors, and transporters expressed in brain tissue. Computational docking, validated by mutagenesis and kinetic studies, indicates that AK-42 binds to an extracellular vestibule above the channel pore. In electrophysiological recordings of mouse CA1 hippocampal pyramidal neurons, AK-42 acutely and reversibly inhibits CLC-2 currents; no effect on current is observed on brain slices taken from CLC-2 knockout mice. These results establish AK-42 as a powerful tool for investigating CLC-2 neurophysiology.

Footnotes

↵¹To whom correspondence may be addressed. Email: john.huguenard{at}stanford.edu, maduke{at}stanford.edu, or jdubois{at}stanford.edu.

Author contributions: A.K.K., A.L.R., Y.K., J.R.H., M.M., and J.D.B. designed research; A.K.K., A.L.R., Y.K., X.W., K.A.M., E.E.G., and C.W. performed research; A.K.K. and E.E.G. contributed new reagents/analytic tools; A.K.K., A.L.R., X.W., J.R.H., M.M., and J.D.B. analyzed data; and A.K.K., M.M., and J.D.B. wrote the paper.
Competing interest statement: A.K.K., J.D.B., and M.M. have filed for a patent “Compositions and Methods to Modulate Chloride Ion Channel Activity,” USSN 16/449,021 from the U.S. Patent & Trademark Office, January 16, 2020.
This article is a PNAS Direct Submission.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2009977117/-/DCSupplemental.

Data Availability.

All study data are included in the article and supporting information.

Published under the PNAS license.

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Submit

[1] 1.↵
T. J. Jentsch,
M. Pusch
, CLC chloride channels and transporters: Structure, function, physiology, and disease. Physiol. Rev. 98, 1493–1590 (2018).
OpenUrl CrossRef PubMed

[2] T. J. Jentsch,

[3] M. Pusch

[4] 2.↵
A. Thiemann,
S. Gründer,
M. Pusch,
T. J. Jentsch
, A chloride channel widely expressed in epithelial and non-epithelial cells. Nature 356, 57–60 (1992).
OpenUrl CrossRef PubMed

[5] A. Thiemann,

[6] S. Gründer,

[7] M. Pusch,

[8] T. J. Jentsch

[9] 3.↵
W. Walz
, Chloride/anion channels in glial cell membranes. Glia 40, 1–10 (2002).
OpenUrl CrossRef PubMed

[10] W. Walz

[11] 4.↵
C. S. Wilson,
A. A. Mongin
, The signaling role for chloride in the bidirectional communication between neurons and astrocytes. Neurosci. Lett. 689, 33–44 (2019).
OpenUrl CrossRef PubMed

[12] C. S. Wilson,

[13] A. A. Mongin

[14] 5.↵
T. Stauber,
T. J. Jentsch
, Chloride in vesicular trafficking and function. Annu. Rev. Physiol. 75, 453–477 (2013).
OpenUrl CrossRef PubMed

[15] T. Stauber,

[16] T. J. Jentsch

[17] 6.↵
N. Rahmati,
F. E. Hoebeek,
S. Peter,
C. I. De Zeeuw
, Chloride homeostasis in neurons with special emphasis on the olivocerebellar system: Differential roles for transporters and channels. Front. Cell. Neurosci. 12, 101 (2018).
OpenUrl

[18] N. Rahmati,

[19] F. E. Hoebeek,

[20] S. Peter,

[21] C. I. De Zeeuw

[22] 7.↵
M. Watanabe,
A. Fukuda
, Development and regulation of chloride homeostasis in the central nervous system. Front. Cell. Neurosci. 9, 371 (2015).
OpenUrl PubMed

[23] M. Watanabe,

[24] A. Fukuda

[25] 8.↵
D. R. Poroca,
R. M. Pelis,
V. M. Chappe
, ClC channels and transporters: Structure, physiological functions, and implications in human chloride channelopathies. Front. Pharmacol. 8, 151 (2017).
OpenUrl

[26] D. R. Poroca,

[27] R. M. Pelis,

[28] V. M. Chappe

[29] 9.↵
M. M. Bi et al
., Chloride channelopathies of ClC-2. Int. J. Mol. Sci. 15, 218–249 (2013).
OpenUrl

[30] M. M. Bi et al

[31] 10.↵
F. L. Fernandes-Rosa et al
., A gain-of-function mutation in the CLCN2 chloride channel gene causes primary aldosteronism. Nat. Genet. 50, 355–361 (2018).
OpenUrl CrossRef

[32] F. L. Fernandes-Rosa et al

[33] 11.↵
U. I. Scholl et al
., CLCN2 chloride channel mutations in familial hyperaldosteronism type II. Nat. Genet. 50, 349–354 (2018).
OpenUrl CrossRef

[34] U. I. Scholl et al

[35] 12.↵
J. Schewe et al
., Elevated aldosterone and blood pressure in a mouse model of familial hyperaldosteronism with ClC-2 mutation. Nat. Commun. 10, 5155 (2019).
OpenUrl

[36] J. Schewe et al

[37] 13.↵
C. Göppner et al
., Pathogenesis of hypertension in a mouse model for human CLCN2 related hyperaldosteronism. Nat. Commun. 10, 4678 (2019).
OpenUrl

[38] C. Göppner et al

[39] 14.↵
C. Depienne et al
., Brain white matter oedema due to ClC-2 chloride channel deficiency: An observational analytical study. Lancet Neurol. 12, 659–668 (2013).
OpenUrl CrossRef PubMed

[40] C. Depienne et al

[41] 15.↵
D. Di Bella et al
., Subclinical leukodystrophy and infertility in a man with a novel homozygous CLCN2 mutation. Neurology 83, 1217–1218 (2014).
OpenUrl CrossRef PubMed

[42] D. Di Bella et al

[43] 16.↵
H. A. Hanagasi et al
., Secondary paroxysmal kinesigenic dyskinesia associated with CLCN2 gene mutation. Parkinsonism Relat. Disord. 21, 544–546 (2015).
OpenUrl

[44] H. A. Hanagasi et al

[45] 17.↵
H. Gaitán-Peñas et al
., Leukoencephalopathy-causing CLCN2 mutations are associated with impaired Cl^- channel function and trafficking. J. Physiol. 595, 6993–7008 (2017).
OpenUrl CrossRef

[46] H. Gaitán-Peñas et al

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