Cation–chloride cotransporters (CCCs) catalyze electroneutral symport of Cl − with Na + and/or K + across membranes. CCCs are fundamental in cell volume homeostasis, transepithelia ion movement, maintenance of intracellular Cl − concentration, and neuronal excitability. Here, we present a cryoelectron microscopy structure of human K + –Cl − cotransporter (KCC)1 bound with the VU0463271 inhibitor in an outward-open state. In contrast to many other amino acid–polyamine–organocation transporter cousins, our first outward-open CCC structure reveals that opening the KCC1 extracellular ion permeation path does not involve hinge-bending motions of the transmembrane (TM) 1 and TM6 half-helices. Instead, rocking of TM3 and TM8, together with displacements of TM4, TM9, and a conserved intracellular loop 1 helix, underlie alternate opening and closing of extracellular and cytoplasmic vestibules. We show that KCC1 intriguingly exists in one of two distinct dimeric states via different intersubunit interfaces. Our studies provide a blueprint for understanding the mechanisms of CCCs and their inhibition by small molecule compounds.
Cation-coupled chloride cotransporters (CCCs) modulate the transport of sodium and/or potassium cations coupled with chloride anions across the cell membrane. CCCs thus help regulate intracellular ionic concentration and consequent cell volume homeostasis. This has been largely exploited in the past to develop diuretic drugs that act on CCCs expressed in the kidney. However, a growing wealth of evidence has demonstrated that CCCs are also critically involved in a great variety of other pathologies, motivating most recent drug discovery programs targeting CCCs. Here, we examine the structure-function relationship of CCCs. By linking recent high-resolution cryogenic electron microscopy (cryo-EM) data with older biochemical/functional studies on CCCs, we discuss the mechanistic insights and opportunities to design selective CCC modulators to treat diverse pathologies. Cation-coupled chloride cotransporters (CCCs): from structure to function and modulationCCCs are proteins (~130 kDa) of the subfamily of solute carrier transporters (see Glossary) 12 (SLC12) that modulate transport of sodium and/or potassium cations (Na + , K + ) and chloride anions (Cl -) across the cell membrane (Figure 1A). In humans, there are seven known electroneutral CCCs: the Na + -Clcotransporter NCC, the Na + -K + -Clcotransporters NKCC1 and NKCC2, and the K + -Clcotransporters KCC1, KCC2, KCC3, and KCC4 (Figure 1B) [1]. NCC and NKCC2 are expressed predominantly in the kidney [2]. NKCC1, KCC1, KCC3, and KCC4 are expressed throughout the body. KCC2 is expressed specifically in neurons [3]. CCCs are involved in physiological processes, including salt absorption and secretion, cell volume regulation, and intracellular Clconcentration setting [1,4]. As such, CCCs are primarily implicated in blood pressure regulation, cardiovascular and brain physiopathology, and diuresis, and are also associated with hearing and tumoral diseases (Figure 1C,D). Structure-function relationshipsCCCs mainly form homodimers, although multimeric states have been described in functional assays for N(K)CCs and KCCs [5][6][7]. Monomers for each of the CCC family members are characterized by a well-conserved transmembrane (TM) domain formed by 12 TM helices, one N-glycosylated extracellular (EC) domain, and, on the cytosolic side, the amino-terminal (NT) and the large carboxy-terminal (CT) domains (Figure 1A). All SLC12-CCC members share the same TM protein fold of the leucine transporter (LeuT [8], Box 1). The EC domain is characterized by a connecting loop between TM7 and TM8 in Na + -dependent CCCs, whereas Na + -independent CCCs have a longer loop between TM5 and TM6. The NT and EC domains are variable in amino acid length, much shorter than the CT domain, and poorly conserved among the CCC members. The EC domains contain glycosylation sites, whereas the intracellular domains contain phosphorylation sites to modulate CCC function (Figure 1A) [9]. Molecular heterogeneity is also provided by multiple CCC isoforms, generated by alternative splicing and alternative promote...
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