Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs.

Michelle S Reid,Stephen Graf Brohawn,David M Kern

doi:10.7554/elife.52505

Abstract

Cation-chloride-cotransporters (CCCs) catalyze transport of Cl- with K+ and/or Na+across cellular membranes. CCCs play roles in cellular volume regulation, neural development and function, audition, regulation of blood pressure, and renal function. CCCs are targets of clinically important drugs including loop diuretics and their disruption has been implicated in pathophysiology including epilepsy, hearing loss, and the genetic disorders Andermann, Gitelman, and Bartter syndromes. Here we present the structure of a CCC, the Mus musculus K+-Cl- cotransporter (KCC) KCC4, in lipid nanodiscs determined by cryo-EM. The structure, captured in an inside-open conformation, reveals the architecture of KCCs including an extracellular domain poised to regulate transport activity through an outer gate. We identify binding sites for substrate K+ and Cl- ions, demonstrate the importance of key coordinating residues for transporter activity, and provide a structural explanation for varied substrate specificity and ion transport ratio among CCCs. These results provide mechanistic insight into the function and regulation of a physiologically important transporter family.

Highlights

CCCs in mammals include the potassium-chloride cotransporters KCC1-4, the sodium-potassiumchloride cotransporters NKCC1-2, the sodium-chloride cotransporter NCC, and CCC8-9 (Figure 2— figure supplement 1; Arroyo et al, 2013; Marcoux et al, 2017; Gamba, 2005)
No significant difference in activity was observed between N- and C-terminally GFP-tagged mouse KCC4 (Figure 1B,C), in contrast to a previous report for KCC2 (Agez et al, 2017), and C-terminally tagged KCC4 was used for subsequent study
Three pairs of mutations designed to disrupt C-terminal domain (CTD)-CTD interactions in KCC4 (L678R, V680R; L670R, A673R; and S780R, L783R) resulted in a similar, but incomplete, reduction in KCC4 activity (Figure 3C,D,F). This reduction is comparable to that observed in a truncated KCC4 construct missing the entire C-terminal region (KCC4DC, which includes amino acids 1–658) (Figure 3F). These results suggest that monomeric KCC4 is active and that dimerization through the CTDs in a manner analogous to NKCC1 increases transport activity

Summary

Introduction

CCCs in mammals include the potassium-chloride cotransporters KCC1-4, the sodium-potassiumchloride cotransporters NKCC1-2, the sodium-chloride cotransporter NCC, and CCC8-9 (Figure 2— figure supplement 1; Arroyo et al, 2013; Marcoux et al, 2017; Gamba, 2005). KCC4 is expressed in tissues including the heart, nervous system, kidney, and inner ear and mice lacking KCC4 display progressive deafness and renal tubular acidosis (Marcoux et al, 2017; Mount et al, 1999; Karadsheh et al, 2004; Boettger et al, 2002). Hearing loss in these animals is due to disrupted K+ recycling by Dieter’s cells in the cochlea and hair cell excitotoxicity, while renal tubular. The structure reveals unique features of KCCs and, together with functional characterization of structure-based mutants, provides insight into the basis for ion binding, transport, and regulation of KCC4 activity

Results

Discussion

Materials and methods

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