Single-layer graphene modulates neuronal communication and augments membrane ion currents

The use of graphenebased materials to engineer sophisticated biosensing interfaces that can adapt to the central nervous system requires a detailed understanding of how such materials behave in a biological context. Graphene's peculiar properties can cause various cellular changes, but the underlying mechanisms remain unclear. Here, we show that singlelayer graphene increases neuronal firing by altering membraneassociated functions in cultured cells. Graphene tunes the distribution of extracellular ions at the interface with neurons, a key regulator of neuronal excitability. The resulting biophysical changes in the membrane include stronger potassium ion currents, with a shift in the fraction of neuronal firing phenotypes from adapting to tonically firing. By using experimental and theoretical approaches, we hypothesize that the graphene\u2013ion interactions that are maximized when singlelayer graphene is deposited on electrically insulating substrates are crucial to these effects

Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block

BACKGROUND:Understanding the interactions between ion channels and blockers remains an important goal that has implications for delineating the basic mechanisms of ion channel function and for the discovery and development of ion channel directed drugs. METHODOLOGY/PRINCIPAL FINDINGS:We used genetic selection methods to probe the interaction of two ion channel blockers, barium and amantadine, with the miniature viral potassium channel Kcv. Selection for Kcv mutants that were resistant to either blocker identified a mutant bearing multiple changes that was resistant to both. Implementation of a PCR shuffling and backcrossing procedure uncovered that the blocker resistance could be attributed to a single change, T63S, at a position that is likely to form the binding site for the inner ion in the selectivity filter (site 4). A combination of electrophysiological and biochemical assays revealed a distinct difference in the ability of the mutant channel to interact with the blockers. Studies of the analogous mutation in the mammalian inward rectifier Kir2.1 show that the T-->S mutation affects barium block as well as the stability of the conductive state. Comparison of the effects of similar barium resistant mutations in Kcv and Kir2.1 shows that neighboring amino acids in the Kcv selectivity filter affect blocker binding. CONCLUSIONS/SIGNIFICANCE:The data support the idea that permeant ions have an integral role in stabilizing potassium channel structure, suggest that both barium and amantadine act at a similar site, and demonstrate how genetic selections can be used to map blocker binding sites and reveal mechanistic features

Novel insertion mutation in KCNJ5 channel produces constitutive aldosterone release from H295R cells

Primary aldosteronism accounts for 5%-10% of hypertension and in a third of cases is caused by autonomous aldosterone production by adenomas (APA). Somatic mutations in the potassium channel encoded by KCNJ5 have been detected in surgically removed APAs. To better understand the role of these mutations, we resequenced the KCNJ5 channel in a large Australian primary aldosteronism cohort. KCNJ5 mutations were detected in 37 APAs (45% of the cohort), including previously reported E145Q (n = 3), G151R (n = 20), and L168R (n = 13) mutations. In addition, we found a novel 12-bp in-frame insertion mutation (c.414-425dupGCTTTCCTGTTC, A139_F142dup) that duplicates the AFLF sequence in the pore helix upstream of the selectivity filter. Expressed in Xenopus oocytes, the A139_F142dup mutation depolarized the oocytes and produced a G-protein-sensitive Na(+) current with altered K(+) selectivity and loss of inward rectification but retained Ba(2+) sensitivity. Transfected into H295R cells, A139_F142dup increased basal aldosterone release 2.3-fold over the wild type. This was not increased further by incubation with angiotensin II. Although the A139_F142dup mutant trafficked to the plasma membrane of H295R cells, it showed reduced tetramer stability and surface expression compared with the wild-type channel. This study confirms the frequency of somatic KCNJ5 mutations in APAs and the novel mutation identified (A139_F142dup) extend the phenotypic range of the known KCNJ5 APA mutations. Being located in the pore helix, it is upstream of the previously reported mutations and shares some features in common with selectivity filter mutants but additionally demonstrates insensitivity to angiotensin II and decreased channel stability

Evolving potassium channels by means of yeast selection reveals structural elements important for selectivity

Potassium channels are widely distributed. To serve their physiological functions, such as neuronal signaling, control of insulin release, and regulation of heart rate and blood flow, it is essential that K(+) channels allow K(+) but not the smaller and more abundant Na(+) ions to go through. The narrowest part of the channel pore, the selectivity filter formed by backbone carbonyls of the GYG-containing K(+) channel signature sequence, approximates the hydration shell of K(+) ions. However, the K(+) channel signature sequence is not sufficient for K(+) selectivity. To identify structural elements important for K(+) selectivity, we randomly mutagenized the G protein-coupled inwardly rectifying potassium channel 3.2 (GIRK2) bearing the S177W mutation on the second transmembrane segment. This mutation confers constitutive channel activity but abolishes K(+) selectivity and hence the channel's ability to complement the K(+) transport deficiency of Δtrk1Δtrk2 mutant yeast. S177W-containing GIRK2 mutants that support yeast growth in low-K(+) medium contain multiple suppressors, each partially restoring K(+) selectivity to S177W-containing double mutants. These suppressors include mutations in the first transmembrane segment and the pore helix, likely exerting long-range actions to restore K(+) selectivity, as well as a mutation of a second transmembrane segment residue facing the cytoplasmic half of the pore, below the selectivity filter. Some of these suppressors also affected channel gating (channel open time and opening frequency determined in single-channel analyses), revealing intriguing interplay between ion permeation and channel gating

Optical regulation of class C GPCRs by photoswitchable orthogonal remotely tethered ligands

G protein-coupled receptors (GPCRs) respond to a wide range of extracellular cues to initiate complex downstream signaling cascades that control myriad aspects of cell function. Despite a long-standing appreciation of their importance to both basic physiology and disease treatment, it remains a major challenge to understand the dynamic activation patterns of GPCRs and the mechanisms by which they modulate biological processes at the molecular, cellular, and tissue levels. Unfortunately, classical methods of pharmacology and genetic knockout are often unable to provide the requisite precision needed to probe such questions. This is an especially pressing challenge for the class C GPCR family which includes receptors for the major excitatory and inhibitory neurotransmitters, glutamate and GABA, which signal in a rapid, spatially-delimited manner and contain many different subtypes whose roles are difficult to disentangle. The desire to manipulate class C GPCRs with spatiotemporal precision, genetic targeting, and subtype specificity has led to the development of a variety of photopharmacological tools. Of particular promise are the photoswitchable orthogonal remotely tethered ligands (“PORTLs”) which attach to self-labeling tags that are genetically encoded into full length, wild-type metabotropic glutamate receptors (mGluRs) and allow the receptor to be liganded and un-liganded in response to different wavelengths of illumination. While powerful for studying class C GPCRs, a number of detailed considerations must be made when working with these tools. The protocol included here should provide a basis for the development, characterization, optimization, and application of PORTLs for a wide range of GPCRs

Tethered Protein Display Identifies a Novel Kir3.2 (GIRK2) Regulator from Protein Scaffold Libraries

[Image: see text] Use of randomized peptide libraries to evolve molecules with new functions provides a means for developing novel regulators of protein activity. Despite the demonstrated power of such approaches for soluble targets, application of this strategy to membrane systems, such as ion channels, remains challenging. Here, we have combined libraries of a tethered protein scaffold with functional selection in yeast to develop a novel activator of the G-protein-coupled mammalian inwardly rectifying potassium channel Kir3.2 (GIRK2). We show that the novel regulator, denoted N5, increases Kir3.2 (GIRK2) basal activity by inhibiting clearance of the channel from the cellular surface rather than affecting the core biophysical properties of the channel. These studies establish the tethered protein display strategy as a means to create new channel modulators and highlight the power of approaches that couple randomized libraries with direct selections for functional effects. Our results further underscore the possibility for the development of modulators that influence channel function by altering cell surface expression densities rather than by direct action on channel biophysical parameters. The use of tethered library selection strategies coupled with functional selection bypasses the need for a purified target and is likely to be applicable to a range of membrane protein systems