SOLVING ION CHANNEL KINETICS WITH THE QuB SOFTWARE

Nicolai, Christopher; Sachs, Frederick

doi:10.1142/s1793048013300053

Cited by 131 publications

(117 citation statements)

References 62 publications

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“…Accompanying the traces are the activation times (10-90%) and deactivation time constants from either a single, or weighted sum of two exponential fits. The simulations were generated in QuB (Nicolai and Sachs, 2013) for 1000 receptors in response to a 1 ms application of agonist using 'flip' mechanisms. The mechanisms and rate constants employed in these simulations were obtained as follows: α1 homomeric GlyRs -scheme 5 from (Burzomato et al, 2004); α2 homomeric GlyRs -scheme 2 from (Krashia et al, 2011); α3…”

Section: Discussionmentioning

confidence: 99%

Functional reconstitution of glycinergic synapses incorporating defined glycine receptor subunit combinations

et al. 2015

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Glycine receptor (GlyR) chloride channels mediate fast inhibitory neurotransmission in the spinal cord and brainstem. Four GlyR subunits (α1-3, β) have been identified in humans, and their differential anatomical distributions underlie a diversity of synaptic isoforms with unique physiological and pharmacological properties. To improve our understanding of these properties, we induced the formation of recombinant synapses between cultured spinal neurons and HEK293 cells expressing GlyR subunits of interest plus the synapse-promoting molecule, neuroligin-2A. In the heterosynapses thus formed, recombinant α1β and α3β GlyRs mediated fast decaying inhibitory postsynaptic currents (IPSCs) whereas α2β GlyRs mediated slow decaying IPSCs. These results are consistent with the fragmentary information available from native synapses and single channel kinetic studies. As β subunit incorporation is considered essential for localizing GlyRs at the synapse, we were surprised that α1-3 homomers supported robust IPSCs with β subunit incorporation accelerating IPSC rise and decay times in α2β and α3β heteromers only. Finally, heterosynapses incorporating α1(D80A)β and α1(A52S)β GlyRs exhibited accelerated IPSC decay rates closely resembling those recorded in native synapses from mutant mice homozygous for these mutations, providing an additional validation of our technique. Glycinergic heterosynapses should prove useful for evaluating the effects of drugs, hereditary disease mutations or other interventions on defined GlyR subunit combinations under realistic synaptic activation conditions.

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Section: Discussionmentioning

confidence: 99%

Functional reconstitution of glycinergic synapses incorporating defined glycine receptor subunit combinations

et al. 2015

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“…Intensity trajectories were subjected to hidden Markov modeling (HMM) using QuB 22 in order to identify the number of binding and/or dissociation events ( N b+d ) and mean dwell times in the bound ( τ on ) and unbound ( τ off ) states for each candidate molecule. Based on control measurements in absence of target, a threshold of N b+d = 15 (6 standard deviations above background) was used to identify target molecules.…”

Section: Methodsmentioning

confidence: 99%

Kinetic fingerprinting to identify and count single nucleic acids

et al. 2015

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MicroRNAs (miRNAs) have emerged as promising diagnostic biomarkers. We introduce a kinetic fingerprinting approach called Single Molecule Recognition through Equilibrium Poisson Sampling (SiMREPS) for the amplification-free counting of single unlabeled miRNA molecules, which circumvents thermodynamic limits of specificity and virtually eliminates false positives. We demonstrate high-confidence single-molecule detection of synthetic and endogenous miRNAs in both buffer and minimally treated biological liquids, as well as >500-fold discrimination between single nucleotide polymorphisms.

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“…Electrophysiological recordings were performed on transiently transfected HEK cells using cell-attached patch-clamp and were analyzed using QUB software (33). The QuikChange site-directed mutagenesis kit was used to mutate AChR subunit cDNAs.…”

Section: Methodsmentioning

confidence: 99%

Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

Nayak

Bruhova

Chakraborty

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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A muscle acetylcholine receptor (AChR) has two neurotransmitter binding sites located in the extracellular domain, at αδ and either αe (adult) or αγ (fetal) subunit interfaces. We used single-channel electrophysiology to measure the effects of mutations of five conserved aromatic residues at each site with regard to their contribution to the difference in free energy of agonist binding to active versus resting receptors (ΔG B1 ). The two binding sites behave independently in both adult and fetal AChRs. For four different agonists, including ACh and choline, ΔG B1 is ∼−2 kcal/mol more favorable at αγ compared with at αe and αδ. Only three of the aromatics contribute significantly to ΔG B1 at the adult sites (αY190, αY198, and αW149), but all five do so at αγ (as well as αY93 and γW55). γW55 makes a particularly large contribution only at αγ that is coupled energetically to those contributions of some of the α-subunit aromatics. The hydroxyl and benzene groups of loop C residues αY190 and αY198 behave similarly with regard to ΔG B1 at all three kinds of site. ACh binding energies estimated from molecular dynamics simulations are consistent with experimental values from electrophysiology and suggest that the αγ site is more compact, better organized, and less dynamic than αe and αδ. We speculate that the different sensitivities of the fetal αγ site versus the adult αe and αδ sites to choline and ACh are important for the proper maturation and function of the neuromuscular synapse.allosteric protein | ion channel | ligand binding sites | single-channel electrophysiology | synaptic maturation R eceptors at synapses respond to specific chemical signals in the extracellular environment because the active conformation of the protein has a higher affinity for the ligand compared with the resting conformation (1, 2). The active vs. resting difference in binding free energy increases the relative stability of the active state and, hence, the probability of a cellular response. In this report, we describe and distinguish sources of ligandbinding free energy in three kinds of agonist site present in mouse muscle nicotinic acetylcholine receptors (AChRs). Our goal was to use single-channel electrophysiology to assess the relative contribution of significant functional groups to the overall free energy generated by the affinity change at each type of site.At cholinergic synapses, the main chemical signals are ACh released from nerve terminals and choline, which is an ACh precursor, hydrolysis product, and stable component of serum (3). The muscle AChR has central pore surrounded by five subunits of composition α 2 βδe in adult-type and α 2 βδγ in fetal-type (Fig. 1A) (4). The fetal, γ, subunit is essential for proper synapse maturation, and the adult, e, subunit is necessary for proper function of mature synapses (5-7). Each AChR pentamer has two agonist binding sites in the extracellular domain, at αδ and either αe (adult) or αγ (fetal) subunit interfaces.The change in agonist affinity occurs within the global, resting↔active...

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SOLVING ION CHANNEL KINETICS WITH THE QuB SOFTWARE

Cited by 131 publications

References 62 publications

Functional reconstitution of glycinergic synapses incorporating defined glycine receptor subunit combinations

Functional reconstitution of glycinergic synapses incorporating defined glycine receptor subunit combinations

Kinetic fingerprinting to identify and count single nucleic acids

Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

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