Research Article: Human ClC-6 Is a Late Endosomal Glycoprotein that Associates with Detergent-Resistant Lipid Domains

Date Published: May 30, 2007

Publisher: Public Library of Science

Author(s): Sofie Ignoul, Jeannine Simaels, Diane Hermans, Wim Annaert, Jan Eggermont, Jean Gruenberg. http://doi.org/10.1371/journal.pone.0000474

Abstract: The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb) that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7.

Partial Text: CLC proteins form an evolutionary conserved family of chloride channels and/or transporters that are expressed from bacteria to man [1]. The human genome contains 9 genes (CLCN1–7, CLCNKA, CLCNKB) that encode the pore-forming α-subunits (ClC-1 to -7, ClC-Ka and –Kb). In addition, auxiliary β-subunits that affect plasma membrane location or expression level of the α-subunit, have been described for ClC-Ka and –Kb (barttin) and ClC-7 (Ostm1) [2], [3]. More recently it has transpired that α-subunits can differ in terms of subcellular location (plasma membrane versus intracellular organelles) and mode of Cl− transport (Cl− channel versus Cl−/H+ antiporter) [4]–[7]. Consequently, the mammalian α-subunits can be classified in two subgroups, one functioning as plasma membrane Cl− channels (ClC-1, -2, -Ka and –Kb) and another as intracellular Cl−/H+ antiporters (ClC-3 to -7). In mammals antiporter function has only been formally shown for ClC-4 and ClC-5 [5], [6], but the presence of a conserved glutamate corresponding to E203 in the E. coli ClC-ec1 that is responsible for H+-coupling of Cl− transport [7], suggests a similar antiporter mode for ClC-3, ClC-6 and ClC-7. Some of the intracellular CLC’s have been located in specific subcellular organelles: ClC-7 resides in late endosomes, lysosomes and the osteoclast resorption lacuna [8], ClC-5 in endosomes in the proximal tubule of the kidney [9], [10] and ClC-3 in (late) endosomes and synaptic vesicles [11]. Intracellular CLC’s are thought to facilitate acidification of endosomal and lysosomal compartments by dissipating the lumen-positive membrane potential that arises from the electrogenic H+-transport by the V-type H+-ATPase [12]. Nevertheless, alternative functions have been proposed for intracellular CLC’s, such as fusion of intracellular organelles [5] or trafficking of the endocytic receptor proteins megalin and cubulin [13].

We have developed a polyclonal antibody against human ClC-6 which has enabled us to study the N-glycosylation profile and the subcellular distribution of hClC-6 both endogenously in the neuronal SH-SY5Y cell line and in transiently transfected COS-1 or Hela cells. Our data are consistent with hClC-6 being a multiply N-glycosylated membrane protein that is targeted to late endosomes in neuronal cells. In transfected COS-1 cells hClC-6 resides in a lipid raft microenvironment and the association with detergent resistant membrane fractions is critically dependent on the positively charged juxtamembranous KKGRR sequence (amino acids 71–75).

Source:

http://doi.org/10.1371/journal.pone.0000474