Chris Shelley

Properties of the human muscle nicotinic receptor, and of the slow-channel myasthenic syndrome mutant eL221F, inferred from maximum likelihood fits

The mechanisms that underlie activation of nicotinic receptors are investigated using human recombinant receptors, both wild type and receptors that contain the slow channel myasthenic syndrome mutation, εL221F. The method uses the program HJCFIT, which fits the rate constants in a specified mechanism directly to a sequence of observed open and shut times by maximising the likelihood of the sequence with exact correction for missed events. A mechanism with two different binding sites was used. The rate constants that apply to the diliganded receptor (opening, shutting and total dissociation rates) were estimated robustly, being insensitive to the exact assumptions made during fitting, as expected from simulation studies. They are sufficient to predict the main physiological properties of the receptors. The εL221F mutation causes an approximately 4‐fold reduction in dissociation rate from diliganded receptors, and a smaller increase in opening rate and mean open time. These are sufficient to explain the approximately 6‐fold slowing of decay of miniature synaptic currents seen in patients. The distinction between the two binding sites was less robust, the estimates of rate constants being dependent to some extent on assumptions, e.g. whether an extra short‐lived shut state was included or whether the EC50 was constrained. The results suggest that the two binding sites differ by roughly 10‐fold in the affinity of the shut receptor for ACh in the wild type, and that in the εL221F mutation the lower affinity is increased so the sites become more similar.

Recessive inheritance and variable penetrance of slow-channel congenital myasthenic syndromes

Background Slow-channel congenital myasthenic syndromes (SCCMS) typically show dominant inheritance. They are caused by missense mutations within the subunits of muscle nicotinic acetylcholine receptors (AChR) that result in prolonged ion channel activations. SCCMS mutations within the AChR &agr; subunit are located in various functional domains, whereas fully described mutations in AChR non-&agr; subunits have, thus far, been located only in the M2 channel-lining domain. The authors identified and characterized two &egr;-subunit mutations, located outside M2, that underlie SCCMS in three kinships. In two of the three kinships, the syndrome showed an atypical inheritance pattern. Methods These methods included clinical diagnosis, mutation detection, haplotype analysis, and functional expression studies using single-channel recordings of mutant AChR transiently transfected into HEK293 cells. Results The authors identified two SCCMS mutations in the AChR &egr; subunit, &egr;L78P and &egr;L221F. Both mutations prolonged ACh-induced ion channel activations. &egr;L78P is present in a consanguineous family and appears to be pathogenic only when present on both alleles, and &egr;L221F shows variable penetrance in one of the two families that were identified harboring this mutation. Conclusion SCCMS mutations may show a recessive inheritance pattern and variable penetrance. A diagnosis of SCCMS should not be ruled out in cases of CMS with an apparent recessive inheritance pattern.

Cornichons Modify Channel Properties of Recombinant and Glial AMPA Receptors

Ionotropic glutamate receptors, which underlie a majority of excitatory synaptic transmission in the CNS, associate with transmembrane proteins that modify their intracellular trafficking and channel gating. Significant advances have been made in our understanding of AMPA-type glutamate receptor (AMPAR) regulation by transmembrane AMPAR regulatory proteins. Less is known about the functional influence of cornichons—unrelated AMPAR-interacting proteins, identified by proteomic analysis. Here we confirm that cornichon homologs 2 and 3 (CNIH-2 and CNIH-3), but not CNIH-1, slow the deactivation and desensitization of both GluA2-containing calcium-impermeable and GluA2-lacking calcium-permeable (CP) AMPARs expressed in tsA201 cells. CNIH-2 and -3 also enhanced the glutamate sensitivity, single-channel conductance, and calcium permeability of CP-AMPARs while decreasing their block by intracellular polyamines. We examined the potential effects of CNIHs on native AMPARs by recording from rat optic nerve oligodendrocyte precursor cells (OPCs), known to express a significant population of CP-AMPARs. These glial cells exhibited surface labeling with an anti-CNIH-2/3 antibody. Two features of their AMPAR-mediated currents—the relative efficacy of the partial agonist kainate (IKA/IGlu ratio 0.4) and a greater than fivefold potentiation of kainate responses by cyclothiazide—suggest AMPAR association with CNIHs. Additionally, overexpression of CNIH-3 in OPCs markedly slowed AMPAR desensitization. Together, our experiments support the view that CNIHs are capable of altering key properties of AMPARs and suggest that they may do so in glia.

Linking Exponential Components to Kinetic States in Markov Models for Single-Channel Gating

Discrete state Markov models have proven useful for describing the gating of single ion channels. Such models predict that the dwell-time distributions of open and closed interval durations are described by mixtures of exponential components, with the number of exponential components equal to the number of states in the kinetic gating mechanism. Although the exponential components are readily calculated (Colquhoun and Hawkes, 1982, Phil. Trans. R. Soc. Lond. B. 300:1-59), there is little practical understanding of the relationship between components and states, as every rate constant in the gating mechanism contributes to each exponential component. We now resolve this problem for simple models. As a tutorial we first illustrate how the dwell-time distribution of all closed intervals arises from the sum of constituent distributions, each arising from a specific gating sequence. The contribution of constituent distributions to the exponential components is then determined, giving the relationship between components and states. Finally, the relationship between components and states is quantified by defining and calculating the linkage of components to states. The relationship between components and states is found to be both intuitive and paradoxical, depending on the ratios of the state lifetimes. Nevertheless, both the intuitive and paradoxical observations can be described within a consistent framework. The approach used here allows the exponential components to be interpreted in terms of underlying states for all possible values of the rate constants, something not previously possible.

TARP‐associated AMPA receptors display an increased maximum channel conductance and multiple kinetically distinct open states

•  Signalling of information in the nervous system relies on the activation of specific neurotransmitter receptors. •  Here we characterise some of the properties of GluA1 AMPA receptors, whose ion‐permeable channel is opened by the neurotransmitter glutamate. •  We found that the individual single‐channel openings exhibit several discrete conductance levels that persist in the presence of saturating glutamate concentrations, and that the presence of modulatory accessory subunits differentially influences the durations of these channel openings. •  Our data also indicate that there are at least two kinetically distinguishable stable open states for each conductance level. •  These observations place constraints on models of GluA1 function that can be used to relate receptor properties to synaptic function.

Coupling and cooperativity in voltage activation of a limited-state BK channel gating in saturating Ca2+

Voltage-dependent gating mechanisms of large conductance Ca2+ and voltage-activated (BK) channels were investigated using two-dimensional maximum likelihood analysis of single-channel open and closed intervals. To obtain sufficient data at negative as well as positive voltages, single-channel currents were recorded at saturating Ca2+ from BK channels mutated to remove the RCK1 Ca2+ and Mg2+ sensors. The saturating Ca2+ acting on the Ca2+ bowl sensors of the resulting BKB channels increased channel activity while driving the gating into a reduced number of states, simplifying the model. Five highly constrained idealized gating mechanisms based on extensions of the Monod-Wyman-Changeux model for allosteric proteins were examined. A 10-state model without coupling between the voltage sensors and the opening/closing transitions partially described the voltage dependence of Po but not the single-channel kinetics. With allowed coupling, the model gave improved descriptions of Po and approximated the single-channel kinetics; each activated voltage sensor increased the opening rate approximately an additional 23-fold while having little effect on the closing rate. Allowing cooperativity among voltage sensors further improved the description of the data: each activated voltage sensor increased the activation rate of the remaining voltage sensors approximately fourfold, with little effect on the deactivation rate. The coupling factor was decreased in models with cooperativity from ∼23 to ∼18. Whether the apparent cooperativity among voltage sensors arises from imposing highly idealized models or from actual cooperativity will require additional studies to resolve. For both cooperative and noncooperative models, allowing transitions to five additional brief (flicker) closed states further improved the description of the data. These observations show that the voltage-dependent single-channel kinetics of BKB channels can be approximated by highly idealized allosteric models in which voltage sensor movement increases Po mainly through an increase in channel opening rates, with limited effects on closing rates.

Phosphorylation of a constitutive serine inhibits BK channel variants containing the alternate exon “SRKR”

BK Ca2+-activated K+ currents exhibit diverse properties across tissues. The functional variation in voltage- and Ca2+-dependent gating underlying this diversity arises from multiple mechanisms, including alternate splicing of Kcnma1, the gene encoding the pore-forming (α) subunit of the BK channel, phosphorylation of α subunits, and inclusion of β subunits in channel complexes. To address the interplay of these mechanisms in the regulation of BK currents, two native splice variants, BK0 and BKSRKR, were cloned from a tissue that exhibits dynamic daily expression of BK channel, the central circadian pacemaker in the suprachiasmatic nucleus (SCN) of mouse hypothalamus. The BK0 and BKSRKR variants differed by the inclusion of a four–amino acid alternate exon at splice site 1 (SRKR), which showed increased expression during the day. The functional properties of the variants were investigated in HEK293 cells using standard voltage-clamp protocols. Compared with BK0, BKSRKR currents had a significantly right-shifted conductance–voltage (G-V) relationship across a range of Ca2+ concentrations, slower activation, and faster deactivation. These effects were dependent on the phosphorylation state of S642, a serine residue within the constitutive exon immediately preceding the SRKR insert. Coexpression of the neuronal β4 subunit slowed gating kinetics and shifted the G-V relationship in a Ca2+-dependent manner, enhancing the functional differences between the variants. Next, using native action potential (AP) command waveforms recorded from SCN to elicit BK currents, we found that these splice variant differences persist under dynamic activation conditions in physiological ionic concentrations. AP-induced currents from BKSRKR channels were significantly reduced compared with BK0, an effect that was maintained with coexpression of the β4 subunit but abolished by the mutation of S642. These results demonstrate a novel mechanism for reducing BK current activation under reconstituted physiological conditions, and further suggest that S642 is selectively phosphorylated in the presence of SRKR.

A human congenital myasthenia-causing mutation (εL78P) of the muscle nicotinic acetylcholine receptor with unusual single channel properties

A mutation in the epsilon subunit of the human nicotinic acetylcholine receptor (ɛL78P) is known to cause a congenital slow channel myasthenic syndrome. We have investigated the changes in receptor function that result in the mutant receptor producing prolonged endplate currents, and consequent muscle damage. The rate constants for channel gating and for the binding and dissociation of acetylcholine were investigated by analysis of single ion channel recordings. A conventional mechanism with two non‐equivalent binding sites, and variations upon this mechanism, were fitted to data using a maximum likelihood method that uses the Hawkes‐Jalali‐Colquhoun (HJC) treatment of missed brief events. The mutant receptor produced prolonged activations, bursts of openings that cause a slow decay of simulated synaptic currents. The main reason for the longer bursts of openings seen with mutant receptor was a decrease in the rate of ACh dissociation from diliganded receptors, though the lifetime of individual openings was somewhat increased too. As well as producing long bursts, the mutant receptor also produced many very short openings, though these carry little current. The burst structure for the mutant receptor at low ACh concentration is unusual in that most long bursts appear to start in a very brief monoliganded open state that then usually binds another ACh molecule to produce a long diliganded activation. The first opening is so short that it will usually be missed (together with the shut time that follows it), so the true burst length is likely to be underestimated.

Structural Abnormalities of the AChR Caused by Mutations Underlying Congenital Myasthenic Syndromes

Abstract: The objective was to define the molecular mechanisms underlying congenital myasthenic syndromes (CMS) by studying mutations within genes encoding the acetylcholine receptor (AChR) and related proteins at the neuromuscular junction. It was found that mutations within muscle AChRs are the most common cause of CMS. The majority are located within the ε‐subunit gene and result in AChR deficiency.

Desensitization and models of receptor‐channel activation

Models of receptor activation have been widely used to describe fundamental properties of ion channels. Designing a kinetic scheme that could account for more than just simple receptor activation started with Bernard Katz and Stephen Thesleffs (1957) paper, which appeared more than fifty years ago in The Journal of Physiology. In this classic study, Katz and Thesleff attempted to quantify, and provide a simple physical explanation for, the phenomenon of acetylcholine (ACh) receptor desensitization at the frog endplate. In so doing, they extended del Castillo & Katzs (1957)‘working hypothesis’ that had appeared just a few months earlier, which proposed a two state model of receptor activation – with states that corresponded to the inactive and active forms of the bound (liganded) acetylcholine receptor (AChR). Del Castillo & Katzs scheme (Fig. 1) itself advanced earlier mathematical descriptions of drug and receptor binding and activation, such as those of Hill (1909), Clark (1933) and Gaddum (1937), by modifying Michaelis and Mentens (1913) concept that enzymatic conversion of substrate proceeds as a two step process – with the intermediate (fast) step involving the formation of an ‘inactive’ complex. The simple idea of a two state scheme for receptor activation not only provided an important and realistic distinction between ‘occupation (binding)’ and ‘activation (isomerisation)’ for transmitter-gated channels, but also allowed Katz and Thesleff to address the observation that many receptor mediated responses decline in the continued presence of an agonist. Figure 1 A scheme to explain the activation and desensitization of the ACh receptor In their experiments Katz & Thesleff (1957) used electrically controlled ionophoretic application of ACh from a double-barrelled glass micropipette positioned close to the endplate region of a frogs muscle fibre. One of the barrels provided a steady conditioning dose of ACh that elicited a desensitizing response, while the other was used to apply brief test pulses to monitor the development and recovery phase of desensitization (Fig. 2). From their data, Katz and Thesleff confirmed that the time course of onset of desensitization depended on dose, as described earlier by Paul Fatt (1950), and determined that this onset could occur more slowly than recovery from desensitization. Importantly, they established that the time course of recovery was relatively fast and independent of the conditioning dose or degree of desensitization (typically, with a time constant in the order of 4 s). Initially, Katz and Thesleff derived equations to describe the rate of desensitization onset and recovery, by considering the hypothetical ‘sequential’ and ‘simultaneous’ reaction schemes shown in Fig. 3. However, the resulting equations inferred that desensitization onset must always be faster than the recovery – a prediction that directly contradicted their experimental findings. Thus they were able to safely reject the following two reaction schemes (Fig. 3) as plausible models of receptor desensitization. Figure 3 Sequential and simultaneous reaction schemes considered and rejected Figure 2 Intracellular voltage recording of acetylcholine responses at the frog endplate They suggested, instead, a type of reaction scheme in which recovery from desensitization was slowed by the presence of agonist – namely a cyclical reaction. In such a scheme, the receptor would exist in two forms – normal (A) and desensitized (B) – both of which bind rapidly and reversibly with the agonist. However, the bound form of the active receptor is converted irreversibly to its desensitized bound form, and the unbound desensitized receptor returns irreversibly to its reactive form. Their experimental results fitted this model if the affinity of the agonist for the desensitized receptor (B) was much higher than for the normal receptor (A). Alternatively, they proposed a cyclical scheme in which reaction steps were reversible. A feature of such a scheme is that even in the absence of agonist a proportion of receptors will exist in their desensitized (B) state, and because of the high affinity of this form, will preferentially bind agonist. By proposing such cyclical schemes (Fig. 4), they introduced the concept that agonist can bind to and dissociate from multiple receptor conformations, albeit with different rates. This idea was extended over the following decade by the Monod–Wyman–Changeux (Monod et al. 1965) and Koshland–Nemethy–Filmer (Koshland et al. 1966) models of protein conformational change. Figure 4 The two cyclical schemes proposed by Katz and Thesleff The formalisation of the idea that the interaction of a neurotransmitter with receptor involves more than simply binding represents an important step in the history of receptor-channel theory, and hence in our views of fundamental mechanisms that underlie synaptic transmission. It paved the way for ever more complex models of channel gating. Desensitized states are routinely included in many receptor models and in the case of muscle acetylcholine receptors it is known that there are at least four or five distinct desensitized states (Elenes & Auerbach, 2002). Although, for the muscle nicotinic receptor, desensitization is expected to have minimal physiological effect, it plays an important role in the normal activity of many other ligand-gated receptor channels (Jones & Westbrook, 1996), and in many voltage-gated ion channels, in the form of ‘inactivation’ (Hille, 2001). Perhaps the height of complexity of ion channel models is found in the BK potassium channel, with its multiple Ca2+-binding sites and multiple voltage sensors, where models have been proposed that contain 1250 different states (Magleby, 2003). Importantly the schemes introduced by Katz and colleagues, with a channel-gating step that is separate from ligand binding, provided a physically realistic account for the reduced potency of partial agonists. Indeed the ‘curare-like’ inhibition elicited by coapplication of the full agonist ACh with the partial agonist choline was correctly attributed to competition between acetylcholine and choline for the same ligand-binding sites on the receptor. The concept of an initial binding step also helps us to visualise the mode of action of competitive antagonists; the antagonist occupies the agonist binding site, but its ability to open the channel (efficacy) is essentially zero. On this basis, competitive antagonists and partial agonists appear to form part of a continuum. Indeed, this view has recently been emphasised by the observation that 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a classic competitive glutamate antagonist at AMPA receptor channels, is converted to a partial agonist when AMPA receptors are expressed in the presence of auxiliary subunits that modify the channel gating (Menuz et al. 2007). Intriguingly, there appear to be two different mechanisms of partial agonism at ligand-gated ion channels. Some partial agonists at kainate receptors (Frydenvang et al. 2009), glycine-binding subunits of NMDA receptors (Inanobe et al. 2005), and pentameric ligand-gated receptors (Lape et al. 2008) seem to bind in ways very similar to full agonists, but the subsequent reaction steps have reduced efficacy. In contrast at AMPA-type glutamate receptors (Jin et al. 2003), and the glutamate-binding subunits of NMDA receptors (Erreger et al. 2005), different partial agonists promote differing extents of binding domain closure around the ligand. The fundamental advance offered by Katz and Thesleffs approach was the ability to distinguish between kinetic models. Discrimination between models is usually based on the models predicted properties, such as time constants of a particular process (e.g. desensitization, deactivation, rise-time), dwell-time distributions of single channel open and shut states, the likelihood of a particular reaction scheme generating observed single channel data, and the systematic ranking of proposed schemes. Katz and colleagues’ use of kinetic schemes gave a physical interpretation of the states involved in transmitter–receptor interaction long before three dimensional structures for receptors were available, or the molecular identity of receptors determined. It is now clear that at least some of these proposed states can be structurally identified as stable intermediates of a dynamic protein structure (Unwin, 2003). However, functional studies have identified many more stable states than those currently solved by structural methods, and the integration of kinetic and structural data therefore remains a major challenge in the field of receptor function.