ß subunits of GABAA receptors form proton-gated chloride channels: Insights into the molecular basis.

Gamma-aminobutyric acid type A receptors (GABAARs) are ligand gated channels mediating inhibition in the central nervous system. Here, we identify a so far undescribed function of ß-subunit homomers as proton-gated anion channels. Mutation of a single H267A in ß3 subunits completely abolishes channel activation by protons. In molecular dynamic simulations of the ß3 crystal structure protonation of H267 increased the formation of hydrogen bonds between H267 and E270 of the adjacent subunit leading to a pore stabilising ring formation and accumulation of Cl- within the transmembrane pore. Conversion of these residues in proton insensitive ρ1 subunits transfers proton-dependent gating, thus highlighting the role of this interaction in proton sensitivity. Activation of chloride and bicarbonate currents at physiological pH changes (pH50 is in the range 6- 6.3) and kinetic studies suggest a physiological role in neuronal and non-neuronal tissues that express beta subunits, and thus as potential novel drug target.

A selectivity filter mutation provides insights into gating regulation of a K+ channel.

G-protein coupled inwardly rectifying potassium (GIRK) channels are key players in inhibitory neurotransmission in heart and brain. We conducted molecular dynamics simulations to investigate the effect of a selectivity filter (SF) mutation, G154S, on GIRK2 structure and function. We observe mutation-induced loss of selectivity, changes in ion occupancy and altered filter geometry. Unexpectedly, we reveal aberrant SF dynamics in the mutant to be correlated with motions in the binding site of the channel activator Gßγ. This coupling is corroborated by electrophysiological experiments, revealing that GIRK2wt activation by Gßγ reduces the affinity of Ba2+ block. We further present a functional characterization of the human GIRK2G154S mutant validating our computational findings. This study identifies an allosteric connection between the SF and a crucial activator binding site. This allosteric gating mechanism may also apply to other potassium channels that are modulated by accessory proteins.

Development of IKATP Ion Channel Blockers Targeting Sulfonylurea Resistant Mutant KIR6.2 Based Channels for Treating DEND Syndrome.

Introduction: DEND syndrome is a rare channelopathy characterized by a combination of developmental delay, epilepsy and severe neonatal diabetes. Gain of function mutations in the KCNJ11 gene, encoding the KIR6.2 subunit of the IKATP potassium channel, stand at the basis of most forms of DEND syndrome. In a previous search for existing drugs with the potential of targeting Cantú Syndrome, also resulting from increased IKATP, we found a set of candidate drugs that may also possess the potential to target DEND syndrome. In the current work, we combined Molecular Modelling including Molecular Dynamics simulations, with single cell patch clamp electrophysiology, in order to test the effect of selected drug candidates on the KIR6.2 WT and DEND mutant channels. Methods: Molecular dynamics simulations were performed to investigate potential drug binding sites. To conduct in vitro studies, KIR6.2 Q52R and L164P mutants were constructed. Inside/out patch clamp electrophysiology on transiently transfected HEK293T cells was performed for establishing drug-channel inhibition relationships. Results: Molecular Dynamics simulations provided insight in potential channel interaction and shed light on possible mechanisms of action of the tested drug candidates. Effective IKIR6.2/SUR2a inhibition was obtained with the pore-blocker betaxolol (IC50 values 27-37 µM). Levobetaxolol effectively inhibited WT and L164P (IC50 values 22 µM) and Q52R (IC50 55 µM) channels. Of the SUR binding prostaglandin series, travoprost was found to be the best blocker of WT and L164P channels (IC50 2-3 µM), while Q52R inhibition was 15-20% at 10 µM. Conclusion: Our combination of MD and inside-out electrophysiology provides the rationale for drug mediated IKATP inhibition, and will be the basis for 1) screening of additional existing drugs for repurposing to address DEND syndrome, and 2) rationalized medicinal chemistry to improve IKATP inhibitor efficacy and specificity.

RNA polymerase II-binding aptamers in human ACRO1 satellites disrupt transcription in cis.

Transcription elongation is a highly regulated process affected by many proteins, RNAs and the underlying DNA. Here we show that the nascent RNA can interfere with transcription in human cells, extending our previous findings from bacteria and yeast. We identified a variety of Pol II-binding aptamers (RAPs), prominent in repeat elements such as ACRO1 satellites, LINE1 retrotransposons and CA simple repeats, and also in several protein-coding genes. ACRO1 repeat, when translated in silico, exhibits ~50% identity with the Pol II CTD sequence. Taken together with a recent proposal that proteins in general tend to interact with RNAs similar to their cognate mRNAs, this suggests a mechanism for RAP binding. Using a reporter construct, we show that ACRO1 potently inhibits Pol II elongation in cis. We propose a novel mode of transcriptional regulation in humans, in which the nascent RNA binds Pol II to silence its own expression.

Computational Insights Into Voltage Dependence of Polyamine Block in a Strong Inwardly Rectifying K+ Channel.

Inwardly rectifying potassium (KIR) channels play important roles in controlling cellular excitability and K+ ion homeostasis. Under physiological conditions, KIR channels allow large K+ influx at potentials negative to the equilibrium potential of K+ but permit little outward current at potentials positive to the equilibrium potential of K+, due to voltage dependent block of outward K+ flux by cytoplasmic polyamines. These polycationic molecules enter the KIR channel pore from the intracellular side. They block K+ ion movement through the channel at depolarized potentials, thereby ensuring, for instance, the long plateau phase of the cardiac action potential. Key questions concerning how deeply these charged molecules migrate into the pore and how the steep voltage dependence arises remain unclear. Recent MD simulations on GIRK2 (=Kir3.2) crystal structures have provided unprecedented details concerning the conduction mechanism of a KIR channel. Here, we use MD simulations with applied field to provide detailed insights into voltage dependent block of putrescine, using the conductive state of the strong inwardly rectifying K+ channel GIRK2 as starting point. Our µs long simulations elucidate details about binding sites of putrescine in the pore and suggest that voltage-dependent rectification arises from a dual mechanism.