Decremental Response to High-Frequency Trains of Acetylcholine Pulses but Unaltered Fractional Ca2+ Currents in a Panel of “Slow-Channel Syndrome” Nicotinic Receptor Mutants

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

2014

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On the basis of single-channel currents recorded from the muscle nicotinic acetylcholine receptor (AChR), we have recently hypothesized that the conformation adopted by the glutamate side chains at the first turn of the pore-lining α-helices is a key determinant of the rate of ion permeation. In this paper, we set out to test these ideas within a framework of atomic detail and stereochemical rigor by conducting all-atom molecular dynamics and Brownian dynamics simulations on an extensively validated model of the open-channel muscle AChR. Our simulations provided ample support to the notion that the different rotamers of these glutamates partition into two classes that differ markedly in their ability to catalyze ion conduction, and that the conformations of the four wild-type glutamates are such that two of them "fall" in each rotamer class. Moreover, the simulations allowed us to identify the mm (χ 1 ≅ -60°; χ 2 ≅ -60°) and tp (χ 1 ≅ 180°; χ 2 ≅ +60°) rotamers as the likely conduction-catalyzing conformations of the AChR's selectivity-filter glutamates. More generally, our work shows an example of how experimental benchmarks can guide molecular simulations into providing a type of structural and mechanistic insight that seems otherwise unattainable.nicotinic receptor | glutamate rotamers T he role an ion channel can play in the physiology of a cell is dictated by the rate at which ions permeate, the type of ions that permeate, the stimulus that gates the channel open, the rates of interconversion among all conductive and nonconductive conformations, and the channel's level of expression in the membrane. In this paper, we are concerned with the chemical determinants of the rate at which cations permeate through the muscle nicotinic acetylcholine receptor (AChR), an archetypal neurotransmitter-gated ion channel.Several "rings" of negatively charged residues decorate the walls of the ion-permeation pathway of the muscle nicotinic AChR. Of these, it is the ring of four glutamates and one glutamine in the first (N-terminal) turn of the pore-lining M2 transmembrane α-helices (the "intermediate ring of charge" at position -1′; Fig. 1A) that lowers the energetic barrier to cation permeation the most (1). Recently, on the basis of single-channel currents recorded from mutant AChRs, we proposed that only two of the four glutamates in the ring contribute to set the size of the unitary currents, and that these glutamates are deprotonated even at pH 6.0 (2). This is in stark contrast with the situation in voltage-dependent Ca 2+ channels (Ca V channels) and cyclicnucleotide-gated channels (CNG channels), for example, where all four selectivity-filter glutamates have been suggested to contribute (directly or indirectly) to the formation of one (in Ca V channels) or two (in CNG channels) proton-binding sites that are largely protonated at pH 6.0 (3, 4). Moreover, our results led us to propose that the difference between the muscle-AChR glutamates that catalyze cation permeation and those that do not is the conformation adopte...

Section: Resultsmentioning

confidence: 67%

Side-chain conformation at the selectivity filter shapes the permeation free-energy landscape of an ion channel

Harpole

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

2014

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“…Agonist‐concentration jumps were applied to outside‐out patches using a two‐barrelled glass‐capillary ‘θ‐tube’ (Hilgenberg, Malsfeld, Germany) through which two solutions flow, as described by Jonas (1995; see also Elenes et al . 2006, 2009).…”

Section: Methodsmentioning

confidence: 99%

Desensitization of neurotransmitter‐gated ion channels during high‐frequency stimulation: a comparative study of Cys‐loop, AMPA and purinergic receptors

Papke

González-Gutiérrez

The Journal of Physiology

2011

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Non-technical summary During fast synaptic transmission, series of brief pulses of highly concentrated neurotransmitter impinge repetitively on the postsynaptic membrane. The number of neurotransmitter-gated ion channels (NGICs) that open in response to each neurotransmitter pulse may increase or decrease along such trains of stimuli to an extent that can affect the transmission of action potentials. This 'short-term' plasticity results from transient changes (lasting from milliseconds to minutes) in the properties of the presynaptic terminal, the postsynaptic terminal, or both. In this paper, we studied eight representative members of all three known superfamilies of NGICs to determine the extent to which short-term plasticity can occur at the postsynaptic-receptor level. We found that the responsiveness of all tested channels declines appreciably along trains of brief neurotransmitter pulses delivered at physiologically relevant frequencies. We suggest that the role of receptor desensitization in the synaptic control of action-potential transmission may be more general than previously thought.Abstract Changes in synaptic strength allow synapses to regulate the flow of information in the neural circuits in which they operate. In particular, changes lasting from milliseconds to minutes ('short-term changes') underlie a variety of computational operations and, ultimately, behaviours. Most studies thus far have attributed the short-term type of plasticity to activity-dependent changes in the dynamics of neurotransmitter release (a presynaptic mechanism) while largely dismissing the role of the loss of responsiveness of postsynaptic receptor channels to neurotransmitter owing to entry into desensitization. To better define the response of the different neurotransmitter-gated ion channels (NGICs) to repetitive stimulation without interference from presynaptic variables, we studied eight representative members of all three known superfamilies of NGICs in fast-perfused outside-out patches of membrane. We found that the responsiveness of all tested channels (two nicotinic acetylcholine receptors, two glycine receptors, one GABA receptor, two AMPA-type glutamate receptors and one purinergic receptor) declines along trains of brief neurotransmitter pulses delivered at physiologically relevant frequencies to an extent that suggests that the role of desensitization in the synaptic control of action-potential transmission may be more general than previously thought. Furthermore, our results indicate that a sizable fraction (and, for some NGICs, most) of this desensitization occurs during the neurotransmitter-free interpulse intervals. Clearly, an incomplete clearance of neurotransmitter from the synaptic cleft between vesicle-fusion events need not be invoked to account for NGIC desensitization upon repetitive stimulation. Abbreviations GlyR, glycine receptor; NGIC, neurotransmitter-gated ion channel; P2X 2 R, purinergic ionotropic type 2 receptor.

“…For outside-out recordings, the ligand was applied to the external aspect of outside-out patches as rapid jumps (solution-exchange time 10–90% <150 µs). These step changes in the concentration of ligand were achieved by the rapid switching of two solutions (differing only in the presence or absence of ligand) flowing from either barrel of a piece of theta-type capillary glass mounted on a piezo-electric device25,26 (Burleigh-LSS-3100; Exfo). For all outside-out recordings, the patch-pipette solution consisted of (in mM) 110 KF, 40 KCl, 1 CaCl 2 , 11 EGTA and 10 Hepes/KOH, pH 7.4, whereas the agonist-free solution flowing through one of the barrels of the theta-type tubing consisted of (in mM) 142 KCl, 5.4 NaCl, 1.8 CaCl 2 , 1.7 MgCl 2 and 10 Hepes/KOH, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

Bridging the Gap between Structural Models of Nicotinic Receptor Superfamily Ion Channels and Their Corresponding Functional States

González-Gutiérrez

Journal of Molecular Biology

2010

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Aromatic-aromatic interactions are a most prominent feature of the crystal structure of ELIC (PDB code 2VL0), a bacterial member of the nicotinic-receptor superfamily of ion channels where five pore-facing phenylalanines come together to form a structure akin to a narrow iris that occludes the transmembrane pore. To identify the functional state of the channel this structure represents, we engineered phenylalanines at various pore-facing positions of the muscle acetylcholine receptor (one position at a time), including the position that aligns with the native phenylalanine 246 of ELIC, and assessed the consequences of such mutations using electrophysiological and toxin-binding assays. From our experiments, we conclude that the interaction among the side chains of pore-facing phenylalanines, rather than the accumulation of their independent effects, leads to the formation of a non-conductive conformation that is unresponsive to the application of acetylcholine and is highly stable even in the absence of ligand. Moreover, electrophysiological recordings from a GLIC channel (another bacterial member of the superfamily) engineered to have a ring of phenylalanines at the corresponding pore-facing position suggest that this novel refractory state is distinct from the well-known desensitized state. It seems reasonable to propose, then, that it is in this peculiar non-conductive conformation that the ELIC channel was crystallized. It seems also reasonable to propose that, in the absence of rings of porefacing aromatic side chains, such stable conformation may never be attained by the acetylcholine receptor. Incidentally, we also noticed that the response of the proton-gated, wild-type GLIC channel to a fast change in pH from 7.4 to 4.5 (on the extracellular side) is only transient, with the evoked current fading completely in a matter of seconds. This raises the possibility that the crystal structures of GLIC obtained at pH 4.0 (PDB code 3EHZ) and 4.6 (PDB code 3EAM) correspond to the to the (well-known) desensitized state.