Modulation of nicotinic acetylcholine receptor function ...

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Modulation of nicotinic acetylcholine receptor function through the outer and middle rings of transmembrane domains Francisco J Barrantes Address UNESCO Chair of Biophysics & Molecular Neurobiology and Instituto de Investigaciones Bioquímicas Universidad Nacional del Sur-CONICET CC 857 F8000FWB Bahía Blanca Argentina Email: [email protected] Current Opinion in Drug Discovery & Development 2003 6(5):620-632  Current Drugs ISSN 1367-6733

The photoaffinity labeling of amino acid residues embedded in the lipid bilayer, in combination with site-directed mutagenesis and patch-clamp electrophysiology, are beginning to delineate a topographic map of lipid-sensitive residues in the transmembrane (TM) region of the nicotinic acetylcholine receptor (nAChR), and to dissect their contribution to channel gating, opening and closing mechanisms. Recent structural data reveal that the TM segments form three concentric layers around the ion channel. An inner ring, shaped by five M2 segments (one from each subunit) excluded from contact with the lipid, forms the walls of the channel. The middle ring is formed by M1 and M3 segments, which exhibit contact with lipids, and the M4 membraneembedded domains constitute an outer ring, distant from the channel and loosely separated from the middle ring. Although they are not part of the ion conduction pathway, the lipid-exposed middle and outer rings significantly modulate nAChR function. Sterols and steroids, in particular, are among the ligands that act on this functionally relevant moiety of the nAChR. A major challenge for the future is to elucidate the mechanism of propagation of information from the lipid-exposed TM rings to the rest of the receptor molecule. Keywords Cell-surface receptor, lipid-protein interactions, membrane proteins, nicotinic receptor, non-genomic steroid effects, steroids, structure-function correlations

Abbreviations ACh Chol FA LGIC nAChR NCB PC PS TM TMD wt

Acetylcholine Cholesterol Fatty acid Ligand-gated ion channel Nicotinic acetylcholine receptor Non-competitive blockers Phosphatidylcholine Phosphatidylserine Transmembrane Transmembrane domain Wild-type

Introduction The ligand-gated ion channel (LGIC) superfamily comprises several families of evolutionarily related proteins coded by, according to current knowledge, a few hundred genes. These LGICs are neurotransmitter receptors that function as transducers of various chemical signals. Structurally, they are transmembrane (TM) proteins mostly localized at the post-synaptic (and, in some cases, at the pre-synaptic) membrane of chemically excitable synapses. The nicotinic

acetylcholine receptor (nAChR) is one of the bestcharacterized proteins of this family. Within the LGIC superfamily, nAChRs and subtype 3 of the 5hydroxytryptamine (serotonin, 5-HT3) receptors are two families of cation-selective channels, whereas Gly and γaminobutyric acid type A and C (GABAA and GABAC) receptors are anion-selective channels. Members of the LGIC superfamily are also known as Cys-loop receptors, due to the fact that all the N-terminal extracellular halves of these receptors contain a pair of disulfide-bonded Cys residues that are separated by only 13 residues [1]. Glutamate and histidine receptors are also Cys-loop receptors, although their structures do not conform to the canonical LGIC superfamily. The basic mechanism of signal transduction is common to all members of the LGIC superfamily, and results from fast and simple steps: the neurotransmitter binds to the receptor, and this results in conformational transitions in the receptor protein, which in turn leads to changes in the ionic permeability of the post-synaptic membrane. In the specific case of the nAChR, acetylcholine (ACh) initiates, upon binding, a conformational change that triggers the transient opening of its intrinsic cation-specific channel across the post-synaptic membrane. At the molecular level, this is accomplished by the concerted action of an array of four different, but highly homologous nAChR subunits in the stoichiometry α2βεδ in adult skeletal muscle. To date, α1-, β1-, γ- and δ-subunits have been isolated from fetal skeletal muscle, BC3H1 muscle cells and electric fish electrocytes, and 12 different types of vertebrate neuronal nAChR subunits, α2 to α10 and β2 to β4, are found in the central nervous system. The permeability response of the post-synaptic membrane to the natural neurotransmitter ACh is modulated by a heterogeneous group of compounds named non-competitive blockers (NCB) of the nAChR [1]. There are at least two classes of NCBs: the high-affinity NCBs, such as lidocaine, which bind to a site presumably located at, or close to the lumen of the ion channel, and a more heterogeneous group of hydrophobic agents, including the sterols, that bind to sites whose precise location is currently unknown. The mechanism by which the latter type of lipophilic agents affects nAChR activity also remains unknown, although its importance is widely recognized and is being implicated in anesthesia and in the effects of alcohols. Sterols have been reported to affect the function of various types of ion channels [2-5], including the nAChR, and may be loosely classified as NCBs of the nAChR. The naturally occurring sterol cholesterol (Chol) modulates nAChR channel open probability in vitro in a concentration-dependent manner [6], and it is well known that sterols, and particularly Chol, are closely associated with the nAChR [6,7•,8-13]. This review focuses on the structural and functional relationships between the nAChR protein and its membrane microenvironment, and the lipid modulation of

Modulation of nicotinic acetylcholine receptor function Barrantes 621

receptor function exerted on the lipid-exposed TM moieties of the protein. The topological relationship between this nAChR region and the surrounding lipid will be discussed first. The cation-selective pore region itself, delineated by one of the TM segments (M2) and currently envisaged as a domain excluded from contact with the membrane lipid, is not considered in this article. Recent progress on the modulation exerted by lipids and sterols on nAChR function will then be analyzed, using data from the paradigm combination of patch-clamp electrophysiology and site-directed mutagenesis. This approach will help form a topographic map of lipid-sensitive residues in the TM region and provide data on their contribution to nAChR channel gating, opening and closing. The structural-functional cross-talk between the lipid-sensing domains of the nAChR and the rest of the molecule remains to be elucidated.

Topology and structure of nAChR transmembrane segments Each nAChR subunit has four hydrophobic segments, M1 to M4, referred to as TM domains (TMDs), which are embedded in the membrane. The M2 segments form the walls of the ion channel itself. Figure 1A depicts the TM and the extracellular regions of the nAChR and their dimensions, based on recently available cryoelectron microscopy data [14•] and X-ray crystallography data on the water-soluble ACh-binding protein, respectively [15••]. The TM region consists of five all-helical bundles from the α(two copies), β-, γ- and δ-subunits. Each bundle is built from four anti-parallel helices, thus making a total of 20 TM helices (Figure 1). Helical bundles based on four antiparallel helices are a well-known super-secondary structure found, for example, in myohemerythrin. In a collaboration involving centers based in Argentina, Germany and UK, we have used such a structural motif in homology modeling studies of the LGIC [16,17]. Earlier studies [18] suggested that the tertiary structure of the TM region might resemble that of the B5 pentamer of the heat-labile enterotoxin [19]. This toxin is a homomeric pentamer with a central channel formed by five α-helices, one from each B-subunit. The remainder of the toxin is a β-sheet, with the exception of a small helix at the exterior of each subunit. A mixed αhelical/β-sheet molecular model of the nAChR TM region has been proposed [20]. The recently published electron microscopy data at 4-Å resolution, by Unwin and co-workers [14•], constitutes a major advance in the understanding of the structure of nAChR. I would like to propose the distinction of three concentric rings of nAChR TM segments: firstly, an inner ring exclusively formed by five M2 segments, corresponding to the walls of the nAChR ion pore, which have no contact with the membrane lipid. A middle ring, formed by ten helices corresponding to the M1 and M3 TM segments, can also be observed. This middle ring is separated from the inner five-member ring of M2s, and its outer face is exposed to lipids and to the outermost ring, consisting of five M4 segments (Figure 1B). This lateral perspective, together with the end-on view, supports the proposed subdivision into three concentric regions (Figure 1C).

The above structural data provides strong support to early photoaffinity labeling experiments designed to test which portions of the nAChR are in contact with the membrane lipid. These experiments take advantage of lipophilic photoreactive probes that favorably partition into the hydrophobic lipid phase and label the regions of the receptor that are in contact with the lipid phase [13]. Results from studies using probes such as 3-trifluoromethyl-3-(3125 125 [ I]iodophenyl)diazirine ([ I]TID) [21], the Chol analog 3α125 (4-azido-3-[ I]iodosalicylic)-cholest-5-ene (azido-Chol) [22], the steroid derivative promegestone [23] and a phosphatidylserine (PS) arylazido derivative [24] support the hypothesis that the TM segments of the outer (M4) and middle (M1 and M3) rings are in contact with membrane lipids. In Torpedo nAChR, these probes labeled amino acid residues in all four subunits [22,25•]. Moreover, photolabeling experiments provide evidence on the secondary structure of M4: the periodicity of the labeling in αM4 is typical of an αhelical secondary structure [21,25•,26]. The elegant series of affinity labeling studies by Blanton, Cohen and co-workers [21,25•,26] provides the strongest support to the hypothesis that not only M2, but also M3 and M4 are helical. These authors could not, however, assign the pattern obtained for M1 to a helix or a β-sheet. In addition, the loop connecting M2 and M3, a region that is not usually considered to be in the membrane, was also labeled. Fluorescent spectroscopy studies are also compatible with an α-helical structure in αM4 and γM4, whereas the evidence is more ambiguous in the case of αM1 and γM1 [27]. Other experimental approaches using Raman, circular dichroism/optical rotary dispersion (CD/ORD) or Fourier Transform Infra Red (FTIR) spectroscopy, molecular modeling studies and theoretical analysis of LGIC sequences using prediction algorithms have been used to analyze the structure of nAChR TM segments. No consensus has currently emerged from these studies, as the relative amount of α-helical or β-sheet structure is still controversial. Regardless of the tentative status and the several aspects of current models of nAChR TM topography that are still obscure, the experimental data demonstrate that extensive physical contact occurs between membrane lipid and a significant portion of the nAChR protein and, hence, that mutual interactions between lipids and the nAChR are operative within the bilayer. The nature of such interactions, and their possible consequences on receptor structure and function, are beginning to be unraveled by the combined application of various experimental approaches.

Structural basis of nAChR channel modulation by lipophilic compounds The study of the requisites for nAChR channel gating has provided information on the structural relationships between protein and lipid moieties in the nAChR-rich postsynaptic membrane. Early in vitro studies indicated that the nAChR ion translocation machinery requires negatively charged phospholipids and Chol [28-31]. These early findings implicitly pointed to the fact that nAChR channel function is sensitive to the lipid environment, which, in turn, suggested that the ability of the protein to 'sense' the lipid was likely to be located at the lipid-facing surface of the protein.

622 Current Opinion in Drug Discovery & Development 2003 Vol 6 No 5

Figure 1. Model of the nAChR deduced from cryoelectron microscopy data.

A

B Ser Gly

60 Å

M4

269

275

M3

M1

M2

285

Val

254

Thr 251 Leu 10 Å

30 Å

M1-M2 Gly Outer

240

Middle

Inner

C

(A) Extracellular and TM regions of the nAChR. The view is normal to the nAChR main axis, showing the α-helical structure (dark gray structure) in relation to the membrane surfaces (broken lines) and the β-sheet structure (extracellular, light gray structure), comprising the ligand binding domain; the asterisk denotes open space at a subunit interface. It is interesting to note that some of the supposed hydrophobic TM residues extend beyond the membrane bilayer [14•]. The extracellular moiety is based on the crystallographic data of the water-soluble ACh-binding protein [15••]. (B) The helix bundle motif. A lateral view of the TM segments of the α-subunit, depicted as a 'classic' all-helical bundle, is presented [14•]. This pattern is the same as that adopted in early molecular modeling studies of the nAChR TM region [16,17]. This view emphasizes the three concentric layers, or rings, around the ion pore. On the right, the slightly skewed M2 segment, which constitutes the inner ring and is excluded from contact with the lipid, forms the walls of the channel. The middle ring is formed by M1 and M3 segments, and can contact with lipids. On the left, the M4 membrane-embedded domain constitutes the outer ring, distant from the channel and loosely joined to the middle ring by only one (M3-M4) cytoplasmic-facing loop. (C) End-on view of the nAChR TM region, generated with PyMOL (DeLano Scientific LLC) from the atomic coordinates of the structure, deposited in the Protein Data Bank. Three distinct concentric rings of TM segments are indicated in this view. (Figure 1A and 1B are reproduced and adapted, respectively, with permission from Nature Publishing Group and Miyazawa A, Fujiyoshi Y, Unwin N: Structure and gating mechanism of the acetylcholine receptor pore. Nature (2003) 424(6943):949-955.  2003 Nature Publishing Group.)


Fatty acids (FAs) [32-34], phospholipids containing shortchain FAs [35] and steroids [36-38] exert effects on the function of muscle-type nAChR. Unlike Chol ester, unsaturated FAs and dioleoylphosphatidylcholine decrease the generalized polarization (GP) of bulk and lipid-belt regions in the native nAChR membrane [39]. This observation is in agreement with fluorescence quenching studies with brominated lipids, which identified annular sites for phospholipids and non-annular sites for Chol, and confirmed both sites to be accessible to FAs [9,40]. By studying the fluorescence resonance energy transfer (FRET) between the intrinsic fluorescence of nAChR-rich membranes (donor) and Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) molecules (acceptors), it was demonstrated that FRET efficiency decreased upon addition of exogenous lipids, which displace Laurdan molecules from the nAChR microenvironment [39,41]. The maximum decrease in energy transfer efficiency (E) resulting from the addition of a FA (18:1) was ~ 60%, whereas Chol or phospholipid reduced E by 35 and 25%, respectively. Therefore, it appears that independent sites exist for phospholipid and sterol that are both accessible to FAs [39,41]. The M2-(M1-M3) interface as a putative alcohol and anesthetic site Alcohols and anesthetics might bind to sites between the outer surface of the pore-lining M2 inner ring and the middle M1-M3 ring, and involve the amino acid residue 257 αLeu [14•]. This residue, and homologous residues in other members of the LGIC, can be found in water-filled cavities. Laurdan GP, which reflects the molecular dynamics of water molecules, can be correlated in a variety of mammalian cells with the energetic changes in nAChR ion channel conductance measured by single-channel recordings [42]. Laurdan molecules expend energy during solvent (water) reorientation, as evidenced by a red shift in its emission spectrum. When temperature is increased, the Laurdan GP decreases, reflecting the increase in water diffusion into the membrane and the nAChR lipid microenvironment. Water diffusion is facilitated by the increased thermal-induced disorder in the bilayer lipid. The higher nAChR channel conductance observed upon increases in temperature is a manifestation of the augmented ion and water permeability in the nAChR channel region, as observed by using single-channel recordings with the patchclamp technique. nAChR channel kinetics depend on intrinsic properties of the nAChR protein and on the physical state of the lipid microenvironment in which the receptor is embedded. Thus, the purported water-filled cavities can occur in the nAChR-vicinal bilayer lipid.

M1 It has been proposed that the M1 domain is exposed to the ion conducting pathway and to the bilayer lipid, as revealed by substituted Cys accessibility studies and by labeling with membrane-partitioning hydrophobic reagents [21,43]. According to recent cryoelectron microscopy studies [14•], however, the M1 segment appears to form part of the intermediate shell or ring of TM segments, together with M3, leaving an inner ring exclusively formed by five copies of M2, each from a different nAChR subunit (Figure 1). However, mutations in the M1 segment have been demonstrated to affect nAChR function [44].

M3 Recent structural data place M3 at the intermediate shell of TMDs (Figure 1) [14•]. However, this membrane-embedded segment also contributes to the rates of opening and closing of the nAChR channel [45]. M4 On the basis of the classic four-helix model of the nAChR, it has been argued that the M4 segment (residues 409 to 426 in the Torpedo α-chain) is the most likely candidate among the TM portions to be in contact with the lipid bilayer. The arguments in favor of this hypothesis are that: (i) M4 is the least conserved among all TM segments, an argument early invoked in the case of the muscarinic AChR to postulate lipid contacts for analogous portions of this protein [46]; (ii) M4 is the most hydrophobic segment (it even has a face virtually devoid of hydrogen bonding groups [47]); (iii) photoreactive arylazido phosphatidylcholine (PC) analogs result in the labeling of all subunits, but predominantly αsubunits, from the lipid phase, and M4 is postulated to be the target of PC labeling [48]; and (iv) the PS photoaffinity label developed by Blanton and Wang [24,49] tags the αM4 segment at the two extremes of the polypeptide stretch, at the level of the phospholipid polar head regions, ie, at 408 429 residues His and Arg . As M4 emerges as the most likely lipid-contacting TM segment, and has a low degree of conservation, it has been proposed that it is located away from the pore region and, therefore, does not contribute to the ion permeation process. The 4-Å resolution cryoelectron microscopy data place the M4 segments at the outermost periphery of the TM region [14•], an independent TMD that is different from the intermediate M1-M3 ring and proposed to form the outer ring. The data also confirm the paucity of the hydrogenbonding capacity of M4 [47]. In early studies, it was hypothesized that deletion of two to four amino acids in Torpedo αM4, or its total replacement by foreign TM sequences resulted in no loss of nAChR channel activity [50]. These conclusions appeared to exclude, ab initio, any participation of M4 in the function of the nAChR. Other TM segments were intensively studied as possible candidates for functional regulation, or as structural constituents of the ion pore region. However, increases of up to 20% in ion conductance were observed in the resulting chimeras containing non-receptor M4-replacing segments [50]. The alleged low degree of conservation of the M4 domain is also open to debate [51]. Single-site mutations of residues presumably located at the nAChR/lipid interface have led to a different view on the participation of M4 in nAChR channel gating [52-58]. Systematic studies at the single-channel level have only recently become available [59•,60,62,63•]. In early studies, the mutagenesis of the Cys416 and Cys420 residues of the γ-subunit with the bulkier side group Phe led to partial inhibition of channel activity, and mutations with the Ser residue had little or no effect, leading to the conclusion that the 'HA' domain under study, a loop segment located between M3 and M4 (and containing the two Cys residues), was not functionally related to nAChR channel activity [58]. Conversely, Cys residues in TMDs


appeared to be important. Site-directed mutagenesis on Torpedo αCys418 provided the first strong suggestion that the M4 segment was indeed involved in channel activity: substitution of αCys418 with Trp caused an ~ 40-fold increase in ion permeability per cell surface area [53]. In contrast, 451 previous mutagenesis studies of γCys with Ser or Trp produced the opposite effect, decreasing conductance by ~ 50% [54]. It is interesting to note that mutation of this Cys residue with Ser or Trp would abolish FA acylation sites, which normally occur through the formation of a thioester linkage between the FA and the sulfhydryl group of Cys residues. In Torpedo californica nAChRs, γM4 Cys451, which is the only Cys in the TM segment, lies within the bilayer at the level of the interface between the phospholipid polar head 418 region and the cytoplasmic compartment, whereas αCys is located around the middle of M4. Further experiments reinforced the involvement of M4 in 418 nAChR channel kinetics [56]. Mutation of the two αCys 447 in M4 (one in each α-chain) or of βCys resulted in an ~ 30-fold increase in nAChR channel mean open time, without affecting single-channel conductance. Similarly, mutations in γLeu458 (equivalent to Torpedo αCys412) and γMet460 of the BC3H-1 mouse muscle nAChR lengthened the duration of single-channel events [52]. Furthermore, substitution of Gly421 of the Torpedo α-subunit by Phe or Trp produced a substantial increase in the duration of the open state of the channel [55]. Thus, lipid-exposed residues in the M4 segment appear to play an important and unexpected role in the modulation of the nAChR channel kinetics. The amino acid residues of the mouse nAChR presumed to be involved in protein-lipid contact were further studied by

using a combination of site-directed mutagenesis and singlechannel patch-clamp recording [51]. The first quadruplepoint mutant α-subunit was constructed through the substitution of Leu411, Met415, Cys418 and Thr422 with Ala. As determined by TID labeling pattern [25•], all these residues should be oriented toward the lipid moiety. The mutant nAChR resulted in channels characterized by a significantly reduced open state [51]. To determine which amino acid is responsible for this reduction, the quadruple mutant was dissected into four individual point mutations. Two residues 418 422 in mouse αM4, Cys and Thr , were demonstrated to significantly contribute to gating kinetics [59•]. nAChRs that contained αCys418Ala or αThr422Ala mutants formed channels characterized by a 3- and 5-fold reduction in the mean open time, respectively, suggesting that the increase in the closing rate was due to the mutations. Cys418 and Thr422 are highly conserved among species and subunits, and are likely to be exposed to the lipid milieu. Lasalde-Dominicci et al adopted a similar systematic approach, using Trp-scanning mutagenesis, to establish the participation of putative lipid-facing residues in αM4 [61,62] and in αM3 [60]. In a recent study on αThr422, several different mutants of this residue were produced, and the changes in the activation kinetics of the nAChR were evaluated [59•]. Aromatic and hydrophobic substitutions 422 422 422 422 (Thr Val, Thr Ala, Thr Tyr and Thr Trp) profoundly modified nAChR activation, increasing the closing rate by 6to 9-fold and decreasing the opening rate by 2- to 5-fold. The experimental data did not correspond with the classical activation model (Scheme 1). To account for the modified kinetics on mutant nAChRs, it was necessary to introduce a second double-liganded open state (ie, two sequentially connected open states), as depicted in Scheme 2.

Scheme 1. Classical activation model of nAChR.

AR*

β1 α 1

k+1 A+R

AR + A k-1

β2

k+2 A2R

k-2

α2

k+b A2R*

k-b

A2B

This scheme illustrates two agonists A binding to the receptor R in the resting state. k+1, k+2, association rates; k+b forward blocking rate constant; k-1, k-2, dissociation rates; k-b unblocking rate constant; β1 rate at which the receptor occupied by one agonist opens; β2 rate at which the receptor occupied by two agonists opens; α1 rate at which the receptor occupied by one agonist closes; α2 rate at which the receptor occupied by two agonists closes; A2B blocked state.

Scheme 2. Activation model of mutant nAChR.

k+1 A+R

AR + A k-1

β2

k+2 A2R k-2

β3 A2R*

α2

k+b A2R**

α3

A2B k-b

k+1, k+2, association rates; k+b blocking rate constant; k-1, k-2, dissociation rates; k-b unblocking rate constant; β2 rate at which the receptor occupied by two agonists opens; β3 rate at which the second double-liganded open state receptor opens; α2 rate at which the receptor occupied by two agonists closes; α3 rate at which the second double-liganded open state receptor closes; A2B blocked state. (Reproduced with permission from Rockefeller University Press and Bouzat C, Barrantes FJ, Sine S: Nicotinic receptor fourth transmembrane domain. Hydrogen bonding by conserved threonine contributes to channel gating kinetics. J Gen Physiol (2000) 115(5):663-672.  2000 Rockefeller University Press.)


Conversely, substitution of αThr422 with Ser only slightly affected the kinetics. With these results, it was surmised that a hydroxyl group correctly positioned at position 422 in αM4 plays a key role in ensuring rapid and efficient activation of the nAChR. In addition, it could be deduced that M4 contributes to nAChR activation by governing opening and closing transitions [59•]. The way in which the different side chains at position 422 affect the relative free energy for nAChR gating, opening 422 and closing were explored (Table 1) [59•]. When αThr was replaced by aromatic or aliphatic amino acids, changes of ~ 9 kJ/mol were observed in the channel gating energetics, and the energy barrier for opening increased. Conversely, only a 422 slight change occurred when αThr was substituted with a small residue, such as Ser. In addition, the barrier for the closing transition decreased in all mutants, changing by > 4.19 kJ/mol (Table 1). The analysis of the energetics of mutant αThr422 nAChRs revealed small contributions to the steps governing ACh binding to the resting state, but significant contributions to the steps governing opening and closing. The presence of a hydroxyl group at position 422 appears to be an absolute requirement for adequate nAChR channel gating. Table 1. Energetics of channel gating, opening and closing in αThr422 nAChR mutants. Mutation

Gating

∆(∆G) (kJ/mol) Opening

Closing

422

2.51 5.86 8.37 8.37 9.63

0 1.26 2.93 2.93 3.35

-2.51 -4.19 -5.02 -5.02 -5.86

Thr Ser 422 Thr Val Thr422Ala 422 Thr Trp 422 Thr Tyr

Gating: ∆(∆G) is the change between mutant and wild-type (wt) nAChRs in the energy of channel gating, calculated with the equation ∆(∆G)= -RT ln(θ2m/θ2wt). Opening and closing: ∆(∆G) is the change between mutant and wt nAChRs in the energy that the channel must overcome to make the transition from the bi-liganded closed state to the main open state (opening), and from this open to a closed state (closing). The differences in free energies are calculated with the equation ∆(∆G) = -RT ln(β2m/β2wt) and ∆(∆G) = -RT ln(α2m/α2wt) for opening and closing, respectively. R gas constant; T absolute temperature; θ2 initial channel opening equilibrium constant, ie, the ratio of the opening (β2) to closing (α2) rate constants corresponding to the major open state (β2/α2); m mutant; wt wild-type. (Reproduced with permission from Rockefeller University Press and Bouzat C, Barrantes FJ, Sine S: Nicotinic receptor fourth transmembrane domain. Hydrogen bonding by conserved threonine contributes to channel gating kinetics. J Gen Physiol (2000) 115(5):663-672.  2000 Rockefeller University Press.)

To summarize the findings from this series of studies designed to elucidate the structural contribution of putative lipid-exposed residues in the nAChR TMDs, the data suggest that M4, and possibly M3 residues, are involved in muscle-type nAChR channel kinetics (which presumably influences lipid-protein interactions), and mutations in γCys451, αThr422, αCys418, βCys447, αGly412 αVal425 modify nAChR kinetics [51,53-58,59•,60-62,63•,64]. Recent structural

data indicate that the lipid-exposed M1-M3 and M4 constitute outer and intermediate rings, respectively, distant from the ion channel lumen and from the walls of the channel, which are shaped by five M2 segments (all of which are excluded from contact with the lipid) [14•]. Although the lipid-exposed TM segments of the outer and middle rings are not part of the ion conduction pathway (inner ring), they appear to modulate nAChR function to a significant extent.

Defining lipid sites in structural and functional terms Several hypotheses have been considered to account for the general effects of sterols and lipids on the function of the nAChR ion channel. These include: (i) indirect effects exerted on the physical properties of the bilayer, such as membrane fluidity [65,66] (membrane curvature or lateral pressure have also been studied in this context [67]); and (ii) direct effects, mediated by the interactions of sterols with the TM portion of the nAChR protein [6,7•,9,10,22,30,39,41,49,61,68-72]. The most conspicuous indirect physical effect of Chol on the membrane bilayer is the restriction of phospholipid motional regimes, resulting in lipid ordering [73,74]. In the case of the nAChR-rich membrane, experimental approaches using Laurdan extrinsic fluorescence and FRET led to the characterization of the polarity and molecular dynamics of lipids in the nAChR membrane. The lipids were demonstrated to be in a liquid-ordered phase [75]. The nAChR-vicinal lipid is also in a liquid-ordered phase, although it exhibits a higher degree of order than the bulk bilayer lipid. Bulk membrane 'fluidity' is also diminished by increasing Chol concentrations [76,77], as is the polarity and the degree of water penetration of the protein-vicinal lipid microenvironment of the nAChR [39]. As expected, Chol also augments membrane stiffness [78]. Support for the direct effects of sterols on the nAChR stems from early observations on the natural abundance of Chol [79,80] and the protein-induced restriction in mobility of spin-labeled androstanol [7•] in native nAChR-rich membranes. Biophysical research suggested the presence of sterol-sensitive regions in the nAChR. It was subsequently demonstrated that the nAChR protein preferentially associates with other spin-labeled sterols [8]. Spin-labeled cholestane also preferentially associates with the nAChRvicinal lipids [81]. Further support was demonstrated by the displacement of a photoactivatable analog of Chol from the nAChR by unlabeled Chol in a desensitization-dependent manner [68]. Brominated sterol was used to define annular (~ 45 molecules per nAChR monomer) and non-annular (~ 5 to 10 per nAChR) sites, the latter having ~ 20-fold higher affinity for Chol [9]. Another sterol, androstanol, was demonstrated to exhibit a higher selectivity relative to PC, with an affinity constant 4-fold higher than that of PC [8]. Subsequent electron spin resonance (ESR) studies demonstrated the preference of nAChRs for a PS analog over other lipids [82]. After proteolytic removal of the extramembranous portions of the membrane-bound receptor, binding sites for spinlabeled androstane and stearic acid disappeared, while binding sites for phospholipid and sphingomyelin analogs remained. The occurrence of androstane sites in extracellular nAChR domains has been challenged by subsequent


research [10]. Corbin et al reported the results of photoaffinity labeling studies with the nAChR and azidoChol, which also do not support the occurrence of extracellular sites on nAChR for Chol [22]. These authors suggested that proteolytic cleavage of extracellular loops connecting the membrane-protected TM segments would result in destabilization of these segments. Considering the degree of flexibility of the M4 segment [14•], such a destabilization would not necessarily have an impact, as the segment might behave independently from the rest of the bundle. A photoaffinity label derivative of the steroid promegestone was incorporated into all subunits of the T californica nAChR, in the presence and in the absence of the agonist [23]. No [3H]promegestone incorporation was detected in the M2 segment, and residues labeled in the M4 segments were identical to those tagged by other probes reported to be in contact with the lipid bilayer [25•,26]. Promegestone was further demonstrated to be an NCB of the nAChR, allosterically affecting the receptor by interacting with residues situated at the lipid-protein interface. The photoactivatable Chol analog azido-Chol was demonstrated to tag residues Cys412, Val413, Phe414 and Met415 in the T californica αM4 segment [22]. These residues are closer to the cytoplasmic-facing hemilayer of the membrane. The identification of the protein-vicinal lipid-belt region in the immediate perimeter of the nAChR protein was first accomplished by ESR [7•,83,84]. The mobility of the lipid surrounding the nAChR (the lipid-belt region) is reduced relative to the mobility of the bulk membrane lipid, giving rise to a two-component ESR spectrum from which the number and selectivity of the lipids at the lipid-protein interface can be quantified. Spin-labeled sterols, phosphatidic acid and FAs were subsequently demonstrated to preferentially associate with the nAChR [8]. The lipidfacing surface of the nAChR contains grooves and protrusions to which the boundary lipid conforms (Figure 2). Bands of charged amino acid residues girdle the TM region at the membrane-water interfaces, whereas the hydrophobic core of the membrane-embedded region is virtually devoid of charges at the protein-lipid interface. Approximately 20 lipid molecules fit in the first-shell lipid 'annulus', ie, in the perimeter surrounding the nAChR in one hemilayer. The total number of lipid molecules per nAChR monomer is, therefore, ~ 40, which is in agreement with experimental data [8,16,17]. It should be emphasized that this is a static view, and that the exchange rate of nAChRboundary lipid and bulk lipid is extremely fast, being in the 6 7 order of 10 to 10 /s [8,16,17]. Recent research elucidated the association constants of spinlabeled lipids (relative to PC) in nAChR-rich membranes from Torpedo marmorata. The lipids studied, in decreasing order of association constant, were: cardiolipin (5.1), stearic acid (4.9), phosphatidylinositol (4.7), PS (2.7), phosphatidylglycerol (1.7), GD1b (1.0), GM1 (1.0), GM2 (1.0), GM3 (1.0; GD and GM are bovine brain gangliosides), PC (1.0) and phosphatidyl-

ethanolamine (0.5). Little or no selectivity for mono- or di-sialogangliosides was found over that for PC [84].

Modulation of nAChR channel function by sterols, alcohols and local anesthetics Non-genomic steroid effects on nAChR Although the predominant action of corticoid hormones is exerted on the synthesis of a restricted number of specific mRNA species in target cells, direct action of steroids on ion channels has also been reported [85,86]. Natural corticosteroids are lipophilic steroids synthesized by the adrenal cortex from Chol. Neuronal nAChRs were initially reported as targets of steroids in chromaffin cells [87], and the activity of neuronal nAChR expressed in oocytes or in the quail ciliary ganglion was affected by natural steroids [88,89]. In the peripheral nervous system, steroids modulate nAChR function in cultured myoblasts [90]. These actions are rapid, and are mediated by the non-genomic effects of steroid hormones. The non-genomic actions of steroids have recently been reviewed [91•,92•,93•]. The natural steroid Chol is an abundant component of the post-synaptic membrane [94], and in some cases, Chol affects the activity of ionic channels [95]. Chol plays a major role in nAChR function [12,13,94,96,97], due to the fact that: (i) increases in Chol content (up to a certain concentration) augment nAChR-mediated ion influx [28,29,98,99]; (ii) Chol interacts with high affinity with the nAChR [8,100]; (iii) Chol stabilizes the nAChR structure in reconstituted vesicles [69,101,102]; (iv) sterols preserve the agonist-induced affinity state transitions of the nAChR [28,29]; (v) steroids exhibit selectivity for the boundary lipid surrounding the nAChR [7•,8,82]; and (vi) it is necessary to add Chol to nAChR preparations reconstituted in pure phospholipid to increase the thermal stability of the protein induced by cholinergic ligands [40,68,69,102]. When nAChRs are reconstituted into lipid bilayers lacking Chol, agonists can no longer stimulate cation flux [6]. Rankin et al also found that the ability of the nAChR to reach the open state after activation varies with the Chol concentration in the bilayer, whereas the rate of transition from the open state to the fast-desensitized state is unaffected. The synthetic glucocorticoid dexamethasone (DEXA) was demonstrated to affect nAChR function by: (i) dosedependently shortening the channel mean open time (at 2 mM of the drug, the mean open time was ~ 1/5 of that of the control values); (ii) grouping the single-channel openings into bursts; and (iii) causing the appearance of extremely rapid opening and closing transitions (ie, a flickering substructure) within the bursts of openings [36]. These acute effects can be described by the linear blocking model (Scheme 3). If only one open-blocked state is considered, the mean open time would decrease from 1/α to 1/(α + f[B]), where α is the rate at which the channel closes from the open state, f is the forward rate constant for channel blocking and B is the blocking ligand. From the data gathered in one study, DEXA was calculated to have an f value of 7.3 × 105/M/s [37].


Figure 2. Topology of the lipid-facing surface of the nAChR TM region.

A

B

C

(A) The surface potential rendering (generated by WebLab Viewer Pro (Molecular Simulations Inc)) emphasizes the apolar nature of the protein surface exposed to the hydrophobic phospholipid acyl chains, devoid of charged amino acids, and the charged residues at the extracellular and cytoplasmic extremes of the TM region. (B) End-on view of lipid molecules docked on the nAChR outermost surface. One helix bundle is surface-rendered at the lipid-protein interface; the four other bundles are shown in ball-and-stick representation. Four phosphatidic acid molecules, which exhibit preferential affinity for the nAChR microenvironment [75,88], and a diacylglycerol molecule, as found in the crystal structure of a bacterial potassium channel [113], are tightly packed on one of the lipid-exposed faces of the protein. Approximately 20 lipid molecules fit in the perimeter of the nAChR in one hemilayer, bringing the total boundary lipid per nAChR molecule to ~ 40 lipid molecules, which is in close agreement with experimental data [8,16,17]. (C) Lateral view. One helix bundle is surface-rendered at the lipid-protein interface. The same set of lipid molecules shown in (B) is now seen to closely cover the nAChR outermost surface in the extracellular-facing hemilayer of the membrane. Charged residues appear in darker gray. The four other bundles are shown in ball-and-stick representation. Atomic coordinates of the nAChR [14•] were obtained from the Protein Data Bank.


Scheme 3. A linear blocking model of the nAChR channel.

C

β α

f [B] O

b

OB

C closed state; O open state; OB open-blocked state; β rate of channel opening from the closed state; α rate of channel closing from the open state; f forward rate constant for channel blocking; b backward rate constant for channel blocking; B blocking ligand.

Comparison with general anesthetic and alcohol effects Other lipophilic compounds, such as the general anesthetic benzyl alcohol [103], and the volatile general anesthetics isoflurane [104] and enflurane [105], also induce nAChR channel flickering. Conversely, the local anesthetics chlorpromazine and phencyclidine [106] reduce channelopen time without causing flickering. The affinity of these drugs for nAChR should be sufficiently high to allow open/blocked channels to close before drug dissociation. The value of 7.3 × 105/M/s for f derived for the DEXAmodified channel is similar to that found for other nAChR channel blockers such as alcohols (ranging from pentanol to nonanol). For the simple sequential blocking model (Scheme 3), the burst duration (defined as a series of opening events corresponding to the same nAChR molecule) is expected to increase as a function of blocker concentration, due to the fact that blocking events prolong the time channels spend in the open state before closing. In DEXA-modified channels, the burst duration decreased as a function of ligand concentration, a finding that is inconsistent with the idea that DEXA simply acts as an open-channel blocker. Thus, DEXA effects are not compatible with a classic open-channel blocking mechanism. Similar results were observed with isoflurane [104] and nalcohols [107]. In the case of DEXA, it is possible that the nAChR is able to undergo a conformational transition from an open-blocked (OB) to a closed-blocked (CB) state while interacting with - and being blocked by - the steroid, thus making the extended blocking model (Scheme 4) a more appropriate description of the action of the drug. Scheme 4. The extended blocking model of the nAChR channel.

C

β α

f [B] O b

OB

α' β'

CB

C closed state; O open state; OB open-blocked state; CB closedblocked state; β rate of channel opening from the closed state; α rate of channel closing from the open state; f forward rate constant for channel blocking; b backward rate constant for channel blocking; B blocking ligand; α' rate of channel closing from the open-blocked state; β' rate of channel opening from the closedblocked state.

myasthenia gravis by virtue of their immunosuppressive action. In this disease, anti-nAChR antibodies cause accelerated destruction and functional blockage of the peripheral nAChR at the neuromuscular junction [108]. The results from the studies [36-38] described below support the observation that the clinical symptoms of myasthenia gravis often deteriorate during the initial phase of glucocorticoid treatment, when high doses are used. The impairment of the nAChR channel function by DEXA provides an explanation for the worsening of the myasthenic symptoms before the immunosuppressive effects of the steroid become apparent. Exposure of BC3H1 cells to another synthetic glucocorticoid, hydrocortisone (HC), induced a dose-dependent reduction in the channel-open time and burst duration, and an increase in the channel-closed time, with no changes in channel amplitude [36]. At high doses of HC, the nAChR channel lifetime was ~ 6-fold shorter than that of the control. Similar effects were observed with 11-desoxycortisone, suggesting that the oxygen atom of the hydroxyl group at the 11-position is not required for channel modification. Only a minor difference was observed between the effect of DEXA and that of HC or 11-desoxycortisone (the latter two compounds produced a slight increase in the number of opening events and in the length of the briefer channelclosed time). More hydrophilic steroids could preferentially concentrate at the level of the polar head region of the bilayer. Moreover, the nature of the perturbation of the lipid-protein interface might vary with the chemical structure of the steroid. A certain degree of vectoriality was observed in the steroid action on nAChR [36]. Possible explanations for this phenomenon are: (i) the asymmetry of the nAChR protein itself in the plane normal to the lipid bilayer; or (ii) an asymmetric distribution of lipids between the two leaflets of the membrane; or (iii) a combination of both. The expression of wild-type (wt) and a mutated nAChR associated with a 'slow channel syndrome' in HEK293 cells was used to study the mechanisms of action of glucocorticoids on this mutated nAChR [38]. The corticoid HC affected the gating kinetics of both types of nAChRs in a similar manner, producing briefer openings with normal amplitudes. The reduction in the channel-open time was slightly voltage-dependent, suggesting that HC binds to a site located inside the membrane that senses the electric field, and facilitates binding at more negative potentials. In competition studies with the open-channel blocker QX-222, HC induced an early termination of the burst, suggesting that the two ligands act at different sites. These results support the existence of sites sensed by the membrane field, that are different from those of open-channel blockers, and which are probably located at the lipid-protein interface. From this site(s), glucocorticoids and other hydrophobic non-competitive inhibitors could allosterically mediate channel blockage.

Narrowing down the search for steroid sites in the nAChR TM region It is possible to speculate on the implications of the singlechannel observations for the clinical effects of DEXA in vivo. Glucocorticoids, in combination with ACh esterase inhibitors, are used for the therapeutic treatment of

Different steroids affect nAChR channel kinetics in a qualitatively similar manner, albeit to different extents, where the effect is dependent on the inverse relationship between the lipophilicity and the inhibitory power of the


steroid [109]. This observation has two implications: (i) these compounds are unlikely to mediate their action on the nAChR via a global and unspecific alteration of the hydrophobic core of the membrane bilayer; and (ii) their effects depend on their accessibility to sites likely located at superficial regions of the nAChR-lipid interface, probably close to the phospholipid polar head region [96]. The depletion of ~ 40% of endogenous Chol from Xenopus oocytes did not significantly change the macroscopic response of the wt nAChR to ACh, whereas in the 418 αCys Trp mutant, the same Chol depletion caused a marked gain-in-function response [61]. Conversely, increasing the Chol content caused ~ 50% inhibition of the macroscopic response of the Torpedo wt nAChR expressed in oocytes, while the αCys418Trp mutation demonstrated an 418 81% inhibition. Unlike wt nAChR, the αCys Trp mutant recovered from the inhibition caused by Chol enrichment, suggesting that the interaction of the sterol with this lipidexposed residue is significantly different from that of the wt nAChR [61]. In a photoaffinity study using a promegestin probe, 125 [ I]promegestone labeling was observed in some Torpedo nAChR residues, in particular αCys412, that had been 125 previously observed to be labeled with [ I]TID [21,25]. Due to the steroidal nature of the probe, these results suggest that 412 Cys could be involved in the steroid-nAChR interaction 412 [23]. Based on the photoaffinity labeling of αCys , this position was mutated to different residues to explore the effects of side-chain volume, hydrophobicity and charge on steroid-nAChR interactions. All mutants demonstrated channel kinetics that were indistinguishable from those of the wt nAChR, in the absence and in the presence of HC (200 and 400 µM), in single-channel recordings at different ACh concentrations. An Ala-substituted quadruple mutant of 411 415 four putative lipid-exposed residues in αM4 (Leu , Met , 418 422 Cys and Thr ) exhibited less inhibition by HC than that observed in wt AChR. When the quadruple mutant was dissected into four individual Ala-substituted receptors, the Thr422 mutant nAChR behaved like the quadruple mutant, whereas the other three receptors were indistinguishable 422 from the wt nAChR. Therefore, Thr , a residue close to the extracellular-facing membrane hemilayer in αM4, directly influences the changes in HC sensitivity, and might be 422 involved in the steroid-nAChR interaction site [64]. Thr is relatively close to the C-terminal portion of the α-subunit, where site-directed mutagenesis studies have located the Ala-Gly-Met-Ile consensus sequence. This sequence is required for the potentiation exerted by the estrogenic steroid 17β-estradiol on the human neuronal α4 nAChR subunit [110,111]. Unlike rat α4β2 nAChR, human neuronal α4β2 nAChR, the predominant form in human brain, is potentiated by 17β-estradiol [112].

Conclusions The structural and functional studies reviewed in this article add to increasing evidence of the occurrence of lipid-sensing regions in the nAChR TMDs. Further refinement of this mapping strategy is likely to disclose details of these lipidsensitive TM regions and dissection, with increasing

resolution, of the specific residues involved in nAChR channel gating, opening and closing. The potential for pharmacological manipulation of channel activity and for the design of site-directed pharmacophores will improve as we learn more about these loci. The outer ring, formed by M4 segments, and the middle ring formed by M1 and M3 TM segments, appear to be the candidate regions to find such loci in the nAChR membrane-embedded domain.

Acknowledgments Thanks are due to Dr Nigel Unwin (Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK) for providing Figures 1A and 1B. The experimental work quoted in this review was supported by grants from the Universidad Nacional del Sur, the Agencia Nacional de Promoción Científica, Argentina, and FIRCA 1-RO3TW01225-01 to FJB.

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