Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters

Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phosphoregulation in both N- and C-termini. We show that phosphomimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1D19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large Cterminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development

Mutagenic Mapping Suggests a Novel Binding Mode for Selective Agonists of M1 Muscarinic Acetylcholine Receptors

Point mutations and molecular modeling have been used to study the activation of the M1 muscarinic acetylcholine receptor (mAChR) by the functionally selective agonists 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42), and 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1), comparing them with N-desmethylclozapine (NDMC) and acetylcholine (ACh). Unlike NDMC and ACh, the activities of AC-42 and 77-LH-28-1 were undiminished by mutations of Tyr404 and Cys407 (transmembrane helix 7), although they were reduced by mutations of Tyr408. Signaling by AC-42, 77-LH-28-1, and NDMC was reduced by L102A and abolished by D105E, suggesting that all three may interact with transmembrane helix 3 at or near the binding site Asp105 to activate the M1 mAChR. In striking contrast to NDMC and ACh, the affinities of AC-42 and 77-LH-28-1 were increased 100-fold by W101A, and their signaling activities were abolished by Y82A. Tyr82 and Leu102 contact the indole ring of Trp101 in a structural model of the M1 mAChR. We suggest the hypothesis that the side chain of Trp101 undergoes conformational isomerization, opening a novel binding site for the aromatic side chain of the AC-42 analogs. This may allow the positively charged piperidine nitrogen of the ligands to access the neighboring Asp105 carboxylate to activate signaling following a vector within the binding site that is distinct from that of acetylcholine. NDMC does not seem to use this mechanism. Subtype-specific differences in the free energy of rotation of the side chain and indole ring of Trp101 might underlie the M1 selectivity of the AC-42 analogs. Tryptophan conformational isomerization may open up new avenues in selective muscarinic receptor drug design

Structure of the adenosine A 2A receptor in complex with ZM241385 and the xanthines XAC and caffeine

Methylxanthines, including caffeine and theophylline are among the most widely consumed stimulant drugs in the world. These effects are mediated primarily via blockade of adenosine receptors. Xanthine analogues with improved properties have been developed as potential treatments for diseases such as Parkinson’s disease. Here we report the structures of a thermostabilised adenosine A(2A) receptor in complex with the xanthines xanthine amine congener and caffeine, as well as the A(2A) selective inverse agonist ZM241385. The receptor is crystallised in the inactive state conformation as defined by the presence of a salt bridge known as the ionic lock. The complete third intracellular loop, responsible for G protein coupling, is visible consisting of extended helices 5 and 6. The structures provide new insight into the features which define the ligand binding pocket of the adenosine receptor for ligands of diverse chemotypes as well as the cytoplasmic regions which interact with signal transduction proteins

Phospho‐regulation, nucleotide binding and ion access control in potassium‐chloride cotransporters

Potassium‐coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho‐regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo‐EM structures of human KCC3b and KCC1, revealing structural determinants for phospho‐regulation in both N‐ and C‐termini. We show that phospho‐mimetic KCC3b is arrested in an inward‐facing state in which intracellular ion access is blocked by extensive contacts with the N‐terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho‐regulatory site in the KCC1 N‐terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP‐binding pocket in the large C‐terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development