Structural basis for inhibition of the Cation-chloride cotransporter NKCC1 by the diuretic drug bumetanide

Abstract

Cation-chloride cotransporters (CCCs) NKCC1 and NKCC2 catalyze electroneutral symport of 1 Na⁺, 1 K⁺, and 2 Cl^- across cell membranes. NKCC1 mediates trans-epithelial Cl^- secretion and regulates excitability of some neurons and NKCC2 is critical to renal salt reabsorption. Both transporters are inhibited by the so-called loop diuretics including bumetanide, and these drugs are a mainstay for treating edema and hypertension. Here, our single-particle electron cryo-microscopy structures supported by functional studies reveal an outward-facing conformation of NKCC1, showing bumetanide wedged into a pocket in the extracellular ion translocation pathway. Based on these and the previously published inward-facing structures, we define the translocation pathway and the conformational changes necessary for ion translocation. We also identify an NKCC1 dimer with separated transmembrane domains and extensive transmembrane and C-terminal domain interactions. We further define an N-terminal phosphoregulatory domain that interacts with the C-terminal domain, suggesting a mechanism whereby (de)phosphorylation regulates NKCC1 by tuning the strength of this domain association.

Fig. 1. Bumetanide binds to the extracellular entryway of NKCC1.

Overall architecture of human NKCC1 bound with bumetanide shown in map (a) and ribbon diagram (b). c A single NKCC1 transmembrane domain highlights bumetanide (stick), K⁺ (purple sphere), and Cl⁻ (green sphere). Blue meshes are experimental cryo-EM densities. d Bumetanide binding pocket highlights key interacting residues along the extracellular ion pathway. e A 2D representation of bumetanide interactions with residues along the extracellular ion permeation path. Each eyelash indicates a hydrophobic interaction. f Superimposition of experimental NKCC1/bumetanide structure and a MD simulation model taken at 1000 ns shows almost identical binding pose of bumetanide. The K⁺ ions shown as spheres also maintain its position during simulations.

Fig. 2. An N-terminal phosphoregulatory segment associates with the C-terminal domain.

a An N-terminal segment shown as density map interacts with a swapped C-terminal domain shown as ribbon. b An enlarged view highlights residues involved in association of the N- and C-terminal domains. Polar contacts are indicated as dashed lines. c Cl⁻ transport rates in insect cells of wildtype NKCC1 and mutants designed to disrupt the N- and C-terminal domain interface. Each circle represents one kinetic measurement of a single sample in a 96 well plate. Unpaired one-tailed Student’s t tests are used for statistical analyses (n = 6; data are presented as mean values ± SD).

Fig. 3. Conformational changes upon isomerization between outward-open and inward-open states in NKCC1.

a A “slab” view of the electrostatic potential map of the outward-open NKCC1_iii/bumetanide structure, highlighting an extracellular vestibule if bumetanide is removed. b Superimposition of inward- (khaki) and outward-open (dodger blue) NKCC1 structures. Movements of helices are indicated as red arrows. c Left, gating interactions at the extracellular ion entryway and cytoplasmic exit in the outward-open NKCC1 structure. Right, two zoomed views highlight the broken salt bridge (R307-E389) seen in inward-open structures, and a newly formed ionic interaction (D510-K624) that closes the intracellular exit. d Superimposition of outward-open structures of NKCC1/bumetanide and KCC1/VU0463271 highlights a similar outward-open state and an analogous receptor site for inhibitors at the extracellular vestibule.

Fig. 4. A transmembrane and C-terminal domain interface regulates NKCC1 activity.

a A NKCC1 dimer structure highlights coupling among the N-terminal phosphoregulatory segment, extreme C-terminal tail, and ICL1. b The interface between the transmembrane (dodger blue) and a swapped C-terminal (khaki) domains is observed only in the herein described form of NKCC1_iii/bumetanide dimer. Polar interactions are indicated as dashed lines. c Cl⁻ transport rates in insect cells of wildtype human NKCC1 and mutants designed to disrupt the transmembrane and C-terminal domain interface. Each circle represents one kinetic measurement of a single sample in a 96 well plate. Unpaired one-tailed Student’s t tests are used for statistical analyses (n = 6; data are presented as mean values ± SD).

Fig. 5. NKCC1_iii/bumetanide adopts a dimeric architecture.

a Left, superimposition of human and zebrafish NKCC1 dimers based on their C-terminal domains shows that the two transmembrane units in human NKCC1 separate and interact extensively with its C-terminal domains. Right, superimposition of a single human and zebrafish NKCC1 subunit based on their transmembrane domains shows displacement of the scissor helix and C-terminal domain as the loop that connects the TM12 and scissor helices assumes different conformations in these structures. b In the NKCC1_iii/bumetanide structure, the loop connecting the TM12 and scissor helices is stabilized by interactions with the transmembrane unit. Hydrophilic interactions are depicted as dashed lines. c A hypothetical model depicts NKCC1 regulation by (de)phosphorylation. Kinases and phosphatases possibly oppositely tune the strength of association between the cytosolic N- and C-terminal domains with weak and strong association indicted by faded and bright N-terminal regulatory segment, respectively. It remains uncertain whether phosphorylation leads to disengagement of the two transmembrane units or triggers dissociation of the two interdigitating C-terminal domains as indicated by the two oppositely directed red arrows.