Recombinant artificial forisomes provide ample quantities of smart biomaterials for use in technical devices

Forisomes are mechanoproteins that undergo ATP-independent contraction–expansion cycles triggered by divalent cations, pH changes, and electrical stimuli. Although native forisomes from Medicago truncatula comprise a number of subunits encoded by separate genes, here we show that at least two of those subunits (MtSEO1 and MtSEO4) can assemble into homomeric forisome bodies that are functionally similar to their native, multimeric counterparts. We expressed these subunits in plants and yeast, resulting in the purification of large quantities of artificial forisomes with unique characteristics depending on the expression platform. These artificial forisomes were able to contract and expand in vitro like native forisomes and could respond to electrical stimulation when immobilized between interdigital transducer electrodes. These results indicate that recombinant artificial forisomes with specific characteristics can be prepared in large amounts and used as components of microscale and nanoscale devices.     

Native and artificial forisomes: functions and applications

Forisomes are remarkable protein bodies found exclusively in the phloem of the Fabaceae. When the phloem is wounded, forisomes are converted from a condensed to a dispersed state in an ATP-independent reaction triggered by Ca²⁺, thereby plugging the sieve tubes and preventing the loss of photoassimilates. Potentially, forisomes are ideal biomaterials for technical devices because the conformational changes can be replicated in vitro and are fully reversible over a large number of cycles. However, the development of technical devices based on forisomes has been hampered by the laborious and time-consuming process of purifying native forisomes from plants. More recently, the problem has been overcome by the production of recombinant artificial forisomes. This is a milestone in the development of forisome-based devices, not only because large quantities of homogeneous forisomes can be produced on demand, but also because their properties can be tailored for particular applications. In this review, we discuss the physical and molecular properties of native and artificial forisomes, focusing on their current applications in technical devices and potential developments in the future.     

Biochemical and functional evidence for heteromeric assembly of P2X1 and P2X4 subunits

P2X receptors are ligand-gated ion channels activated by extracellular ATP. In expression systems, P2X subunits form homo- and heterotrimeric receptors. Heteromerization is also likely to occur in vivo as (i) most P2X subunits show overlapping distribution in different tissues and (ii) the functional properties of many native P2X receptors differ from those of heterologously expressed homomeric receptors. Here, we used the Xenopus laevis oocyte expression system to test for heteromerization of P2X1 and P2X4 subunits. Upon co-injection, P2X4 subunits were co-purified with hexahistidyl-tagged P2X1 subunits indicating heteromerization. Blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis of these P2X complexes excluded artificial aggregation and confirmed that both subunits were present in trimeric complexes of the same size. Two-electrode voltage-clamp experiments revealed functional P2X receptors with kinetic properties resembling homomeric P2X4 receptors and a pharmacological profile similar to homomeric P2X1 receptors. Thus, application of alpha,beta-methylene ATP evoked a slowly desensitizing current sensitive to the antagonists suramin and 2',3'-O-(2,4,6-trinitrophenyl)-ATP. This study provides for the first time biochemical and functional evidence for the formation of heteromeric P2X(1+4) receptors. These receptors may account for native P2X mediated responses that until now could not be correlated with previously described recombinant P2X receptors.     

Interactions of cyclic nucleotide-gated channel subunits and protein tyrosine kinase probed with genistein

The cGMP sensitivity of cyclic nucleotide-gated (CNG) channels can be modulated by changes in phosphorylation catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases. Previously, we used genistein, a PTK inhibitor, to probe the interaction between PTKs and homomeric channels comprised of alpha subunits (RETalpha) of rod photoreceptor CNG channels expressed in Xenopus oocytes. We showed that in addition to inhibiting phosphorylation, genistein triggers a noncatalytic interaction between PTKs and homomeric RETalpha channels that allosterically inhibits channel gating. Here, we show that native CNG channels from rods, cones, and olfactory receptor neurons also exhibit noncatalytic inhibition induced by genistein, suggesting that in each of these sensory cells, CNG channels are part of a regulatory complex that contains PTKs. Native CNG channels are heteromers, containing beta as well as alpha subunits. To determine the contributions of alpha and beta subunits to genistein inhibition, we compared the effect of genistein on native, homomeric (RETalpha and OLFalpha), and heteromeric (RETalpha+beta, OLFalpha+beta, and OLFalpha+RETbeta) CNG channels. We found that genistein only inhibits channels that contain either the RETalpha or the OLFbeta subunits. This finding, along with other observations about the maximal effect of genistein and the Hill coefficient of genistein inhibition, suggests that the RETalpha and OLFbeta subunits contain binding sites for the PTK, whereas RETbeta and OLFalpha subunits do not.     

Homomeric and heteromeric assembly of KCNQ (Kv7) K+ channels assayed by total internal reflection fluorescence/fluorescence resonance energy transfer and patch clamp analysis

M-type K(+) channels, consisting of KCNQ1-5 (Kv7.1-7.5) subunits, form a variety of homomeric and heteromeric channels. Whereas all the subunits can assemble into homomeric channels, the ability of the subunits to assemble into heteromultimers is highly variable. KCNQ3 is widely thought to co-assemble with several other KCNQ subtypes, whereas KCNQ1 and KCNQ2 do not. However, the existence of other subunit assemblies is not well studied. To systematically explore the heteromeric assembly of KCNQ channels in individual living cells, we performed fluorescence resonance energy transfer (FRET) between cyan fluorescent protein- and yellow fluorescent protein-tagged KCNQ subunits expressed in Chinese hamster ovary cells under total internal reflection fluorescence microscopy in which excitation light only penetrates several hundred nanometers into the cell, thus isolating membrane events. We found significant FRET between homomeric subunits as expected from their functional expression in heterologous expression systems. Also as expected from previous work, robust FRET was observed between KCNQ2 and KCNQ3. KCNQ3 and KCNQ4 also showed substantial FRET as did KCNQ4 and KCNQ5. To determine functional assembly of KCNQ4/KCNQ5 heteromers, we performed two types of experiments. In the first, we constructed a mutant tetraethylammonium ion-sensitive KCNQ4 subunit and tested its assembly with KCNQ5 by patch clamp analysis of the tetraethylammonium ion sensitivity of the resulting current; however, those data were not conclusive. In the second, we co-expressed a KCNQ4 (G285S) pore mutant with KCNQ5 and found the former to act as a dominant negative, suggesting co-assembly of the two types of subunits. These data confirm that among the allowed assembly conformations are KCNQ3/4 and KCNQ4/5 heteromers.     

High zinc sensitivity and pore formation in an invertebrate P2X receptor

To investigate fast purinergic signaling in invertebrates, we examined the functional properties of a P2X receptor subunit cloned from the parasitic platyhelminth Schistosoma mansoni. This purinoceptor (SmP2X) displays unambiguous homology of primary sequence with vertebrate P2X subunits. SmP2X subunits assemble into homomeric ATP-gated channels that exhibit slow activation kinetics and are blocked by suramin and PPADS but not TNP-ATP. SmP2X mediates the uptake of the dye YO-PRO-1 through the formation of large pores and can be blocked by submicromolar concentrations of extracellular Zn2+ ions (IC50 = 0.4 microM). The unique receptor phenotype defined by SmP2X suggests that slow kinetics, modulation by zinc and the ability to form large pores are ancestral properties of P2X receptors. The high sensitivity of SmP2X to zinc further reveals a zinc regulation requirement for the parasite's physiology that could potentially be exploited for therapeutic purposes.     

Different structural requirements for functional ion pore transplantation suggest different gating mechanisms of NMDA and kainate receptors

Although considerable progress has been made in characterizing the physiological function of the high-affinity kainate (KA) receptor subunits KA1 and KA2, no homomeric ion channel function has been shown. An ion channel transplantation approach was employed in this study to directly test if homomerically expressed KA1 and KA2 pore domains are capable of conducting currents. Transplantation of the ion pore of KA1 or KA2 into GluR6 generated perfectly functional ion channels that allowed characterization of those electrophysiological and pharmacological properties that are determined exclusively by the ion pore of KA1 or KA2. This demonstrates for the first time that KA1 and KA2 ion pore domains are intrinsically capable of conducting ions even in homomeric pore assemblies. NMDA receptors, similar to KA1- or KA2-containing receptors, function only as heteromeric complexes. They are composed of NR1 and NR2 subunits, which both are non-functional when expressed homomerically. In contrast to NR1, the homomeric NR2B ion pore failed to translate ligand binding into pore opening when transplanted into GluR6. Similarly, heteromeric coexpression of the ion channel domains of both NR1 and NR2 inserted into GluR6 failed to produce functional channels. Therefore, we conclude that the mechanism underlying the ion channel opening in the obligatorily heterotetrameric NMDA receptors differs significantly from that in the facultatively heterotetrameric alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and KA receptors.     

Protons activate the delta-subunit of the epithelial Na+ channel in humans

The amiloride-sensitive epithelial Na(+) channel (ENaC) controls Na(+) transport into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as alpha, beta, gamma, and delta. ENaCdelta can associate with beta and gamma subunits and generate a constitutive current that is 2 orders of magnitude larger than that of homomeric ENaCdelta. However, the distribution pattern of ENaCdelta is not consistent with that of the beta and gamma subunits. ENaCdelta is expressed mainly in the brain in contrast to beta and gamma subunits, which are expressed in non-neuronal tissues. To explain this discrepancy, we searched for novel functional properties of homomeric ENaCdelta and investigated the detailed tissue distribution in humans. When human ENaCdelta was expressed in Xenopus oocytes and Chinese hamster ovary cells, a reduction of extracellular pH activated this channel (half-maximal pH for an activation of 5.0), and the acid-induced current was abolished by amiloride. The most striking finding was that the desensitization of the acid-evoked current was much slower (by approximately 10% 120 s later), dissociating from the kinetics of acid-sensing ion channels in the degenerin/epithelial Na(+) channel family, which were rapidly desensitized during acidification. RNA dot-blot analyses showed that ENaCdelta mRNA was widely distributed throughout the brain and was also expressed in the heart, kidney, and pancreas in humans. Northern blotting confirmed that ENaCdelta was expressed in the cerebellum and the hippocampus. In conclusion, human ENaCdelta activity is regulated by protons, indicating that it may contribute to the pH sensation and/or pH regulation in the human brain.     

Heteromerization of TRP channel subunits: extending functional diversity

Transient receptor potential (TRP) channels are widely found throughout the animal kingdom. By serving as cellular sensors for a wide spectrum of physical and chemical stimuli, they play crucial physiological roles ranging from sensory transduction to cell cycle modulation. TRP channels are tetrameric protein complexes. While most TRP subunits can form functional homomeric channels, heteromerization of TRP channel subunits of either the same subfamily or different subfamilies has been widely observed. Heteromeric TRP channels exhibit many novel properties compared to their homomeric counterparts, indicating that co-assembly of TRP channel subunits has an important contribution to the diversity of TRP channel functions.     

pH Dependency and desensitization kinetics of heterologously expressed combinations of acid-sensing ion channel subunits

The exact subunit combinations of functional native acid-sensing ion channels (ASICs) have not been established yet, but both homomeric and heteromeric channels are likely to exist. To determine the ability of different subunits to assemble into heteromeric channels, a number of ASIC1a-, ASIC1b-, ASIC2a-, ASIC2b-, and ASIC3-containing homo- and heteromeric channels were studied by whole-cell patch clamp recordings with respect to pH sensitivity, desensitization kinetics, and level of sustained current normalized to peak current. Analyzing and comparing data for these three features demonstrated unique heteromeric channels in a number of co-expression experiments. Formation of heteromeric ASIC1a+2a and ASIC1b+2a channels was foremost supported by the desensitization characteristics that were independent of proton concentration, a feature none of the respective homomeric channels has. Several lines of evidence supported formation of ASIC1a+3, ASIC1b+3, and ASIC2a+3 heteromeric channels. The most compelling was the desensitization characteristics, which, besides being proton-independent, were faster than those of any of the respective homomeric channels. ASIC2b, which homomerically expressed is not activated by protons per se, did not appear to form unique heteromeric combinations with other subunits and in fact appeared to suppress the function of ASIC1b. Co-expression of three subunits such as ASIC1a+2a+3 and ASIC1b+2a+3 resulted in data that could best be explained by coexistence of multiple channel populations within the same cell. This observation seems to be in good agreement with the fact that ASIC-expressing sensory neurons display a variety of acid-evoked currents.     

High zinc sensitivity and pore formation in an invertebrate P2X receptor

To investigate fast purinergic signaling in invertebrates, we examined the functional properties of a P2X receptor subunit cloned from the parasitic platyhelminth Schistosoma mansoni. This purinoceptor (SmP2X) displays unambiguous homology of primary sequence with vertebrate P2X subunits. SmP2X subunits assemble into homomeric ATP-gated channels that exhibit slow activation kinetics and are blocked by suramin and PPADS but not TNP-ATP. SmP2X mediates the uptake of the dye YO-PRO-1 through the formation of large pores and can be blocked by submicromolar concentrations of extracellular Zn²⁺ ions (IC₅₀=0.4 μM). The unique receptor phenotype defined by SmP2X suggests that slow kinetics, modulation by zinc and the ability to form large pores are ancestral properties of P2X receptors. The high sensitivity of SmP2X to zinc further reveals a zinc regulation requirement for the parasite's physiology that could potentially be exploited for therapeutic purposes.     

Calcium-energized motor protein forisome controls damage in phloem: potential applications as biomimetic “smart” material

Forisomes are ATP independent, mechanically active proteins from the Fabaceae family (also called Leguminosae). These proteins are located in sieve tubes of phloem and function to prevent loss of nutrient-rich photoassimilates, upon mechanical injury/wounding. Forisomes are SEO (sieve element occlusion) gene family proteins that have recently been shown to be involved in wound sealing mechanism. Recent findings suggest that forisomes could act as an ideal model to study self assembly mechanism for the development of nanotechnological devices like microinstruments, the microfluidic system frequently used in space exploration missions. Technology enabling improvement in micro instruments has been identified as a key technology by NASA in future space exploration missions. Forisomes are designated as biomimetic smart materials which are calcium-energized motor proteins. Since forisomes are biomolecules from plant systems it can be doctored through genetic engineering. In contrast, “smart” materials which are not derived from plants are difficult to modify in their properties. Current levels of understanding about forisomes conformational shifts with respect to calcium ions and pH changes requires supplement of future advances with relation to its 3D structure to understand self assembly processes. In plant systems it forms blood clots in the form of occlusions to prevent nutrient fluid leakage and thus proves to be a unique damage control system of phloem tissue.     

Response kinetics and pharmacological properties of heteromeric receptors formed by coassembly of GABA rho- and gamma 2-subunits

Two of the gamma-aminobutyric acid (GABA) receptors, GABAA and GABAC, are ligand-gated chloride channels expressed by neurons in the retina and throughout the central nervous system. The different subunit composition of these two classes of GABA receptor result in very different physiological and pharmacological properties. Although little is known at the molecular level as to the subunit composition of any native GABA receptor, it is thought that GABAC receptors are homomeric assemblies of rho-subunits. However, we found that the kinetic and pharmacological properties of homomeric receptors formed by each of the rho-subunits cloned from perch retina did not resemble those of the GABAC receptors on perch bipolar cells. Because both GABAA and GABAC receptors are present on retinal bipolar cells, we attempted to determine whether subunits of these two receptor classes are capable of interacting with each other. We report here that, when coexpressed in Xenopus oocytes, heteromeric (rho 1B gamma 2) receptors formed by coassembly of the rho 1B-subunit with the gamma 2-subunit of the GABAA receptor displayed response properties very similar to those obtained with current recordings from bipolar cells. In addition to being unresponsive to bicuculline and diazepam, the time-constant of deactivation, and the sensitivities to GABA, picrotoxin and zinc closely approximated the values obtained from the native GABAC receptors on bipolar cells. These results provide the first direct evidence of interaction between GABA rho and GABAA receptor subunits. It seems highly likely that coassembly of GABAA and rho-subunits contributes to the molecular organization of GABAC receptors in the retina and perhaps throughout the nervous system.     

Co-expression of the 5-HT3B serotonin receptor subunit alters the biophysics of the 5-HT3 receptor

Homomeric complexes of 5-HT(3A) receptor subunits form a ligand-gated ion channel. This assembly does not fully reproduce the biophysical and pharmacological properties of native 5-HT(3) receptors which might contain the recently cloned 5-HT(3B) receptor subunit. In the present study, heteromeric assemblies containing human 5-HT(3A) and 5-HT(3B) subunits were expressed in HEK 293 cells to detail the functional diversity of 5-HT(3) receptors. We designed patch-clamp experiments with homomeric (5-HT(3A)) and heteromeric (5-HT(3AB)) receptors to emphasize the kinetics of channel activation and desensitization. Co-expression of the 5-HT(3B) receptor subunit reduced the sensitivity for 5-HT (5-HT(3A) receptor: EC(50) 3 micro M, Hill coefficient 1.8; 5-HT(3AB) receptor: EC(50) 25 micro M, Hill coefficient 0.9) and markedly altered receptor desensitization. Kinetic modeling suggested that homomeric receptors, but not heteromeric receptors, desensitize via an agonist-induced open-channel block. Furthermore, heteromeric 5-HT(3AB) receptor assemblies recovered much faster from desensitization than homomeric 5-HT(3A) receptor assemblies. Unexpectedly, the specific 5-HT(3) receptor agonist mCPBG induced an open-channel block at both homomeric and heteromeric receptors. Because receptor desensitization and resensitization massively affect amplitude, duration, and frequency of synaptic signaling, these findings are evidence in favor of a pivotal role of subunit composition of 5-HT(3) receptors in serotonergic transmission.     

Trimeric architecture of homomeric P2X2 and heteromeric P2X1+2 receptor subtypes

Of the three major classes of ligand-gated ion channels, nicotinic receptors and ionotropic glutamate receptors are known to be organized as pentamers and tetramers, respectively. The architecture of the third class, P2X receptors, is under debate, although evidence for a trimeric assembly is accumulating. Here we provide biochemical evidence that in addition to the rapidly desensitising P2X1 and P2X3 receptors, the slowly desensitising subtypes P2X2, P2X4, and P2X5 are trimers of identical subunits. Similar (heteromeric) P2X subunits also formed trimers, as shown for co-expressed P2X1 and P2X2 subunits, which assembled efficiently to a P2X1+2 receptor that was exported to the plasma membrane. In contrast, P2X6 subunits, which are incapable of forming functional homomeric channels in Xenopus oocytes, were retained in the ER as apparent tetramers and high molecular mass aggregates. Altogether, we conclude from these data that a trimeric architecture is the structural hallmark of functional homomeric and heteromeric P2X receptors.     

Molecular biology of mammalian amino acid receptors

The amino acid receptor proteins are ubiquitous transducers of most excitatory and inhibitory synaptic transmission in the brain. In July 1987 two reports appeared describing the molecular cloning of a pair of subunits of the GABAA receptor (7) and one subunit of the glycine receptor (13). These papers sparked wide interest and led quickly to the concept of a ligand-gated receptor-ion channel superfamily that includes nicotinic acetylcholine receptors as well as certain amino acid receptors. The identification of additional subunits of each receptor followed; with the recent cloning of a kainate receptor subunit (14), only the NMDA receptor remains elusive. Several disciplines have been brought to bear on these receptor clones, including in situ hybridization and functional expression in Xenopus laevis oocytes and mammalian cell lines. In this review we compare cloning strategies that have been used for amino acid receptors and discuss structural similarities among the receptor subunits. Two findings that have arisen from molecular cloning and expression of these receptors receive special attention. First, the molecular heterogeneity of GABAA receptors is larger than expected from pharmacological studies of native receptors. Second, although the native receptors are thought to be heterooligomers, much like the model proposed for the nicotinic receptors, some individual amino acid receptor subunits can form functional receptor channels, presumably in a homomeric configuration. This review focuses, therefore, on what we have learned from cloning efforts about amino acid receptors and what might lie ahead in this field.     

Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit.

Functionally diverse GluR channels of the AMPA subtype are generated by the assembly of GluR-A, -B, -C, and -D subunits into homo- and heteromeric channels. The GluR-B subunit is dominant in determining functional properties of heteromeric AMPA receptors. This subunit exists in developmentally distinct edited and unedited forms, GluR-B(R) and GluR-B(Q), which differ in a single amino acid in transmembrane segment TM2 (Q/R site). Homomeric GluR-B(R) channels expressed in 293 cells display a low divalent permeability, whereas homomeric GluR-B(Q) and GluR-D channels exhibit a high divalent permeability. Mutational analysis revealed that both the positive charge and the size of the amino acid side chain located at the Q/R site control the divalent permeability of homomeric channels. Coexpression of Q/R site arginine- and glutamine-containing subunits generates cells with varying divalent permeabilities depending on the amounts of expression vectors used for cell transfection. Intermediate divalent permeabilities were traced to the presence of both divalent permeant homomeric and impermeant heteromeric channels. It is suggested that the positive charge contributed by the arginine of the edited GluR-B(R) subunit determines low divalent permeability in heteromeric GluR channels and that changes in GluR-B(R) expression regulate the AMPA receptor-dependent divalent permeability of a cell.     

Stoichiometry studies reveal functional properties of KDC1 in plant shaker potassium channels

Functional heteromeric plant Shaker potassium channels can be formed by the assembly of subunits from different tissues, as well as from diverse plant species. KDC1 (K(+) Daucus carota 1) produces inward-rectifying currents in Xenopus oocytes when coexpressed with KAT1 and other subunits appertaining to different plant Shaker subfamilies. Owing to the presence of KDC1, resulting heteromeric channels display slower activation kinetics, a shift of the activation threshold toward more negative membrane potentials and current potentiation upon the addition of external zinc. Despite available information on heteromerization of plant Shaker channels, very little is known to date on the properties of the various stoichiometric configurations formed by different subunits. To investigate the functional properties of heteromeric nKDC1/mKAT1 configurations, we realized a series of dimeric constructs combining KDC1 and KAT1 alpha-subunits. We found that homomeric channels, formed by monomeric or dimeric alpha-subunit constructs, show identical biophysical characteristics. Coinjections of diverse tandem constructs, instead, displayed significantly different currents proving that KDC1 has high affinity for KAT1 and participates in the formation of functional channels with at most two KDC1 subunits, whereas three KDC1 subunits prevented the formation of functional channels. This article brings a contribution to the understanding of the molecular mechanisms regulating plant Shaker channel functionality by association of modulatory subunits.     

Detection of constitutive homomeric associations of the integrins Mac-1 subunits by fluorescence resonance energy transfer in living cells

Integrins alpha(M)beta(2) plays important role on leukocytes, such as adhesion, migration, phagocytosis, and apoptosis. It was hypothesized that homomeric associations of integrin subunits provide a driving force for integrins activation, and simultaneously inducing the formation of integrins clusters. However, experimental reports on homomeric associations between integrin subunits are still controversial. Here, we proved the homomeric associations of the isolated Mac-1 subunits in living cells using three-channel fluorescence resonance energy transfer (FRET) microscopy and FRET spectra methods. We found that the extent of homomeric associations between beta(2) subunits is higher than alpha(M) subunits. Furthermore, FRET imaging indicated that the extent of homomeric associations of the Mac-1 subunits is higher along the plasma membrane than in the cytoplasm. Finally, we suggested that homomeric associations of the transmembrane domains or/and cytoplasmic domains may provide the driving force for the formation of constitutive homomeric associations between alpha(M) or beta(2) subunits.     

alpha-bungarotoxin receptors contain alpha7 subunits in two different disulfide-bonded conformations.

Neuronal nicotinic alpha7 subunits assemble into cell-surface complexes that neither function nor bind alpha-bungarotoxin when expressed in tsA201 cells. Functional alpha-bungarotoxin receptors are expressed if the membrane-spanning and cytoplasmic domains of the alpha7 subunit are replaced by the homologous regions of the serotonin-3 receptor subunit. Bgt-binding surface receptors assembled from chimeric alpha7/serotonin-3 subunits contain subunits in two different conformations as shown by differences in redox state and other features of the subunits. In contrast, alpha7 subunit complexes in the same cell line contain subunits in a single conformation. The appearance of a second alpha7/serotonin-3 subunit conformation coincides with the formation of alpha-bungarotoxin-binding sites and intrasubunit disulfide bonding, apparently within the alpha7 domain of the alpha7/serotonin-3 chimera. In cell lines of neuronal origin that produce functional alpha7 receptors, alpha7 subunits undergo a conformational change similar to alpha7/serotonin-3 subunits. alpha7 subunits, thus, can fold and assemble by two different pathways. Subunits in a single conformation assemble into nonfunctional receptors, or subunits expressed in specialized cells undergo additional processing to produce functional, alpha-bungarotoxin-binding receptors with two alpha7 conformations. Our results suggest that alpha7 subunit diversity can be achieved postranslationally and is required for functional homomeric receptors.