The voltage-dependent chloride channel ClC-2 is a member of the CLC channel/transporter superfamily. A functional ClC-2 channel comprises a dimeric structure, with each monomer containing 18 intracellular or transmembrane α-helices (helices A to R). ClC-2 is ubiquitously expressed and has been detected in astrocytes and oligodendrocytes, where ClC-2 colocalizes with the adhesion molecule GlialCAM at astrocyte-astrocyte junctions. Mutations in human genes encoding ClC-2 or GlialCAM have been linked to the white matter disease leukodystrophy. A disease-causing mutation significantly increasing the side-chain volume of an alanine (A500V) in the transmembrane helix O of human ClC-2 channel is known to instigate substantial reduction of ClC-2 protein level. Notably, this alanine corresponds to one of the several highly conserved helix O alanine residues present in virtually all members of the CLC channel/transporter superfamily. We thus individually converted some other highly conserved alanine residues in helix O into valine to evaluate the effect of these modifications on protein homeostasis of ClC-2 channel. Our biochemical analyses indicate that all the helix O alanine mutations markedly attenuated total protein level and plasma membrane expression of ClC-2 channel, as well as promoting ClC-2 interaction with an E3 ubiquitin ligase contributing to protein quality control of the chloride channel in the endoplasmic reticulum (ER). These observations suggest that the side-chain volume of highly conserved helix O alanine residues may serve as an essential factor for ER quality control of human ClC-2 channel.