19 research outputs found
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
Metabolic and thermal stimuli control K 2P
K(2P)2.1 (TREK-1) is a polymodal two-pore domain leak potassium channel that responds to external pH, GPCR-mediated phosphorylation signals, and temperature through the action of distinct sensors within the channel. How the various intracellular and extracellular sensory elements control channel function remains unresolved. Here, we show that the K(2P)2.1 (TREK-1) intracellular C-terminal tail (Ct), a major sensory element of the channel, perceives metabolic and thermal commands and relays them to the extracellular C-type gate through transmembrane helix M4 and pore helix 1. By decoupling Ct from the pore-forming core, we further demonstrate that Ct is the primary heat-sensing element of the channel, whereas, in contrast, the pore domain lacks robust temperature sensitivity. Together, our findings outline a mechanism for signal transduction within K(2P)2.1 (TREK-1) in which there is a clear crosstalk between the C-type gate and intracellular Ct domain. In addition, our findings support the general notion of the existence of modular temperature-sensing domains in temperature-sensitive ion channels. This marked distinction between gating and sensory elements suggests a general design principle that may underlie the function of a variety of temperature-sensitive channels
Tethered Protein Display Identifies a Novel Kir3.2 (GIRK2) Regulator from Protein Scaffold Libraries
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Tethered Protein Display Identifies a Novel Kir3.2 (GIRK2) Regulator from Protein Scaffold Libraries
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
Novel Insights into the Structural Basis of pH-Sensitivity in Inward Rectifier K<sup>+</sup> Channels Kir2.3
Graded contribution of the Gβγ binding domains to GIRK channel activation
G protein coupled inwardly rectifying K(+) channels (GIRK/Kir3.x) are mainly activated by a direct interaction with Gβγ subunits, released upon the activation of inhibitory neurotransmitter receptors. Although Gβγ binding domains on all four subunits have been found, the relative contribution of each of these binding sites to channel gating has not yet been defined. It is also not known whether GIRK channels open once all Gβγ sites are occupied, or whether gating is a graded process. We used a tandem tetrameric approach to enable the selective elimination of specific Gβγ binding domains in the tetrameric context. Here, we show that tandem tetramers are fully operational. Tetramers with only one wild-type channel subunit showed receptor-independent high constitutive activity. The presence of two or three wild-type subunits reconstituted receptor activation gradually. Furthermore, a tetramer with no GIRK1 Gβγ binding domain displayed slower kinetics of activation. The slowdown in activation was found to be independent of regulator of G protein signaling or receptor coupling, but this slowdown could be reversed once only one Gβγ binding domain of GIRK1 was added. These results suggest that partial activation can occur under low Gβγ occupancy and that full activation can be accomplished by the interaction with three Gβγ binding subunits
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
Inward rectifier potassium current (I K1) and Kir2 composition of the zebrafish (Danio rerio) heart.
Electrophysiological properties and molecular background of the zebrafish (Danio rerio) cardiac inward rectifier current (IK1) were examined. Ventricular myocytes of zebrafish have a robust (-6.7 ± 1.2 pA pF(-1) at -120 mV) strongly rectifying and Ba(2+)-sensitive (IC50 = 3.8 μM) IK1. Transcripts of six Kir2 channels (drKir2.1a, drKir2.1b, drKir2.2a, drKir2.2b, drKir2.3, and drKir2.4) were expressed in the zebrafish heart. drKir2.4 and drKir2.2a were the dominant isoforms in both the ventricle (92.9 ± 1.5 and 6.3 ± 1.5 %) and the atrium (28.9 ± 2.9 and 64.7 ± 3.0 %). The remaining four channels comprised together less than 1 and 7 % of the total transcripts in ventricle and atrium, respectively. The four main gene products (drKir2.1a, drKir2.2a, drKir2.2b, drKir2.4) were cloned, sequenced, and expressed in HEK cells for electrophysiological characterization. drKir2.1a was the most weakly rectifying (passed more outward current) and drKir2.2b the most strongly rectifying (passed less outward current) channel, whilst drKir2.2a and drKir2.4 were intermediate between the two. In regard to sensitivity to Ba(2+) block, drKir2.4 was the most sensitive (IC50 = 1.8 μM) and drKir2.1a the least sensitive channel (IC50 = 132 μM). These findings indicate that the Kir2 isoform composition of the zebrafish heart markedly differs from that of mammalian hearts. Furthermore orthologous Kir2 channels (Kir2.1 and Kir2.4) of zebrafish and mammals show striking differences in Ba(2+)-sensitivity. Structural and functional differences needs to be taken into account when zebrafish is used as a model for human cardiac electrophysiology, cardiac diseases, and in screening cardioactive substances