Identification of a novel di-leucine motif mediating K+/Cl− cotransporter KCC2 constitutive endocytosis
Introduction
Fast inhibitory neurotransmission is mediated by two classes of ligand gated Cl− channels, the γ-amino-butyric acid type-A receptor (GABAAR) and the glycine receptor (GlyR) [1], [2]. In each case Cl− influx is triggered upon channel opening resulting in hyperpolarization of the postsynaptic membrane. This in turn, leads to a reduction in the likelihood of further neurotransmitter release [1], [2]. In the majority of neurons, Cl− influx and fast hyperpolarizing inhibition are critically dependent on low intracellular chloride concentrations ([Cl−]i). The neuron-specific K+–Cl− cotransporter, KCC2, has now been identified as an essential protein in establishing and maintaining this low [Cl−]i, by controlling Cl− extrusion [3].
During central nervous system (CNS) development, KCC2 gene expression is upregulated and underlies the transition of GABA and glycine responses from the immature depolarizing to the hyperpolarizing responses found in adults [3]. This transition occurs as the overall developmental increase in KCC2 activity leads to a reduction in [Cl−]i, which at resting membrane potentials promotes Cl− influx (hyperpolarization) rather than efflux upon ion channel opening [3]. Further, in mature neurons a reduction in KCC2 gene expression, via antisense oligonucleotide suppression, leads to an increase in [Cl−]i and a shift in GABAAR responses to immature depolarizing [3]. These observations have demonstrated that dynamic regulation of KCC2 gene expression can alter the direction of GABAAR and GlyR signaling. Consistent with this and its essential role in inhibition, KCC2 knockout mice die shortly after birth [4]. In addition, a loss of KCC2 expression is recognized as a contributing factor in the pathological conditions of chronic pain [5], nerve injury [6] and epilepsy [7], [8].
Besides the above-mentioned regulation of KCC2 gene expression, short-term modulation of the KCC2 protein has also been demonstrated. Several kinase activities can modulate KCC2 Cl− transport activity in both immature [9], [10], [11], [12] and mature [13] hippocampal neurons. The precise molecular mechanisms involved however, have yet to be eluded. In addition a rapid loss of KCC2 cell surface expression has been demonstrated under conditions of increased interictal activity [14] and oxidative stress [15]. Indicating that regulation of KCC2 membrane trafficking may be a crucial mechanism by which KCC2 function can be controlled.
KCC2 is a 12 transmembrane protein with both amino and carboxyl intracellular termini and belongs to the cation-chloride cotransporter (CCC) superfamily, which consists of one Na+–Cl− cotransporter (NCC), two Na+–K+–Cl− cotransporters (NKCCs), and four K+–Cl− cotransporters (KCCs). Different CCCs exert opposite Cl− transport activities, with NCC and NKCCs taking up Cl−, while KCCs extrude Cl− [16]. The functional unit of CCCs is most likely to be a dimer, as homo- and hetero-dimerization have now been demonstrated for NKCC [17], NCC [18] and KCC family proteins [19]. The molecular mechanisms regulating the membrane trafficking of any CCC family member however are presently unknown, albeit they play essential roles in controlling chloride homeostasis in multiple tissues.
The cellular mechanisms controlling the cell-surface expression of many transporters and their membrane internalization, in particular, have been shown to have a profound and dynamic effect on overall transporter activity [20], [21]. As one requirement for protein internalization is an interaction with the cellular endocytic machinery, the identification of the molecular motifs governing these interactions has in several cases revealed pivotal regulatory domains within these proteins [22], [23], [24]. How KCC2 membrane expression is controlled, specifically the cellular mechanism and the molecular motifs contributing to its membrane internalization are presently unknown. Therefore in the present study we set out to investigate the mechanisms controlling the membrane internalization of KCC2. Here we report our findings, using an array of endocytosis reporter systems and site-directed mutagenesis, of the cellular mechanisms and the molecular motif within KCC2 directing its constitutive internalization from the plasma membrane.
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Antibodies
The following antibodies were used, mouse monoclonal anti-HA (HA.11, Covance, Berkeley, CA, USA), rabbit polyclonal anti-KCC2 (Upstate, Lake Placid, NY, USA), mouse monoclonal anti-IL2 receptor alpha (Tac; kindly provided by Julie Donaldson, NIH, Bethesda, MD, USA), mouse monoclonal anti-alpha adaptin-clone 8 for immunoblotting (BD Biosciences, Mississauga, ON Canada) and anti-alpha adaptin-clone AP.6 for immunoprecipitation (Affinity BioReagents, Golden CO USA). Donkey anti-mouse and
Endogenous neuronal KCC2 interacts with the clathrin-mediated endocytic machinery
Clathrin-mediated endocytosis (CME) is a prominent mechanism by which plasma membrane proteins are internalized. It involves the recruitment of membrane proteins (cargo) by the adaptor protein-2 (AP-2), to clathrin-coated pits, which are subsequently pinched from the plasma membrane to form internalized endocytic vesicles [28]. To examine whether neuronal KCC2 interacts with the CME machinery, we tested whether the AP-2 complex is bound endogenously to neuronal KCC2 by co-immunoprecipitation.
Discussion
KCC2 is essential in the vast majority of CNS neurons for the development and maintenance of inhibitory neurotransmission [3], [4], [5], [6], [7]. In addition loss of KCC2 expression and function is associated with several neuropathological conditions including chronic pain [5], nerve injury [6] and epilepsy [7]. Given this seemingly crucial role for KCC2 in the mature nervous system, it was surprising how little was known of the cellular mechanisms controlling KCC2 stability and function, in
Acknowledgements
We thank Julie Donaldson, Yves Rouille, Robert lodge, Mark McNiven, Matthew Mulvey, Robert Harvey, Peter McPherson and Stephane Laporte for constructs and antibodies. This work was supported, in part by the Canadian Institute of Health Research (CIHR) operating grant awarded to, F. B. (MOP-62822) and J.P. (MOP-49590). F.B. and D.B. are CIHR-Canada Research Chairs-Tier II recipients.
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