The subunit number and stoichiometry of membrane-bound proteins are difficult to determine without disrupting their membrane environment. Here we describe a single-molecule technique for counting subunits of proteins in live cell membranes by observing bleaching steps of GFP fused to a protein of interest. After testing the method with proteins of known stoichiometry expressed in Xenopus laevis oocytes, we resolved the composition of NMDA receptors composed of NR1 and NR3 subunits.Many membrane proteins form multimers before they achieve a functional state and are transported to the plasma membrane of the cell. The stoichiometry of a complex is precisely regulated and is fundamental to its functional properties. Subunit stoichiometry is usually assessed via bulk biochemical and macroscopic functional analyses. For example, the multimeric state of K + channels in the squid giant axon was originally predicted from the shape of the current trace during opening of the channels in voltage clamp 1 . A kinetic analysis of inactivation 2 and an analysis of currents in mixed subunit channels 3 indicated that the channels are probably composed of four similar or identical subunits, which was later confirmed by crystallography 4 . Sometimes macroscopic functional recordings do not distinguish stoichiometry precisely. For example, CNG channels for years had been assumed to be composed of a 2:2 stoichiometry of CNGA1 and CNGB1 subunits 5 , but has been shown to actually be composed of a 3:1 CNGA1 to CNGB1 stoichiometry by biochemical analysis 6 and using fluorescence energy resonance transfer between fluorescent proteins fused to CNGA1 and CNGB1 ( ref. 7 ).The well-studied glutamate-gated NMDA receptors are tetramers containing two NR1 and two NR2 subunits. This stoichiometry had been deduced from macroscopic functional analysis, in which coexpression of wild-type and mutant subunits resulted in a triphasic response to agonists that could be explained by mixture of receptors containing zero, one or two mutant NR1 or NR2 subunits 8 , and from single-channel recordings that showed distinct behaviors consistent with the possible mixed stoichiometries 9 , and confirmed by crystallography 10 . Less is known about NMDA receptors containing the more recently discovered NR3 subunit, which is thought to be involved in synaptic development 11 . Initially it was thought that NR3 coassembles with NR1 and NR2 to form glutamate-gated receptors with unique properties 11 . More recently, and quite surprisingly, the NR3 subunit ligand binding domain had been found to bind glycine and not glutamate, and

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate chemical communication between neurons at synapses. A variant iGluR subfamily, the Ionotropic Receptors (IRs), was recently proposed to detect environmental volatile chemicals in olfactory cilia. Here we elucidate how these peripheral chemosensors have evolved mechanistically from their iGluR ancestors. Using a Drosophila model, we demonstrate that IRs act in combinations of up to three subunits, comprising individual odor-specific receptors and one or two broadly expressed co-receptors. Heteromeric IR complex formation is necessary and sufficient for trafficking to cilia and mediating odor-evoked electrophysiological responses in vivo and in vitro. IRs display heterogeneous ion conduction specificities related to their variable pore sequences, and divergent ligand-binding domains function in odor recognition and cilia localization. Our results provide insights into the conserved and distinct architecture of these olfactory and synaptic ion channels and offer perspectives into use of IRs as genetically encoded chemical sensors.

In voltage-gated channels, ions flow through a single pore located at the interface between membrane-spanning pore domains from each of four subunits, and the gates of the pore are controlled by four peripheral voltage-sensing domains. In a striking exception, the newly discovered voltage-gated Hv1 proton channels lack a homologous pore domain, leaving the location of the pore unknown. Also unknown are the number of subunits and the mechanism of gating. We find that Hv1 is a dimer and that each subunit contains its own pore and gate, which is controlled by its own voltage sensor. Our experiments show that the cytosolic domain of the channel is necessary and sufficient for dimerization and that the transmembrane part of the channel is functional also when monomerized. The results suggest a mechanism of gating whereby the voltage sensor and gate are one and the same.

The KCNQ1 voltage-gated potassium channel and its auxiliary subunit KCNE1 play a crucial role in the regulation of the heartbeat. The stoichiometry of KCNQ1 and KCNE1 complex has been debated, with some results suggesting that the four KCNQ1 subunits that form the channel associate with two KCNE1 subunits (a 4∶2 stoichiometry), while others have suggested that the stoichiometry may not be fixed. We applied a single molecule fluorescence bleaching method to count subunits in many individual complexes and found that the stoichiometry of the KCNQ1 − KCNE1 complex is flexible, with up to four KCNE1 subunits associating with the four KCNQ1 subunits of the channel (a 4∶4 stoichiometry). The proportion of the various stoichiometries was found to depend on the relative expression densities of KCNQ1 and KCNE1. Strikingly, both the voltage-dependence and kinetics of gating were found to depend on the relative densities of KCNQ1 and KCNE1, suggesting the heart rhythm may be regulated by the relative expression of the auxiliary subunit and the resulting stoichiometry of the channel complex.channel that is expressed in a wide variety of tissues, including human heart, pancreas, kidney, lung, inner ear, and intestine (1-3). Like other Kv channels, each KCNQ1 subunit has six transmembrane segments (S1-S6), with S1-S4 segments serving as a voltage-sensor domain, S5-S6 segments forming a pore domain and four KCNQ1 subunits forming the ion channel (4-7). One of the most prominent features of the KCNQ1 channel is that its gating is dramatically affected by the single transmembrane domain proteins encoded by the KCNE gene family. KCNE1, which is coexpressed with KCNQ1 in the heart and inner ear, drastically slows the activation and deactivation kinetics of the KCNQ1 channel and enhances current amplitude (1,8,9). Another KCNE family member, KCNE3, makes the KCNQ1 channel constitutively open in the intestine (3). The remaining members of the KCNE family, KCNE2, 4, and 5 reduce KCNQ1 current amplitude or modulate the gating (10-12).Although there is little structural information about the KCNQ1 − KCNE1 complex (7, 13), KCNE1 has been shown to directly bind to the pore region of KCNQ1 (14). In addition, a functional interaction between KCNE proteins and the voltagesensor domain of KCNQ1 has been suggested by several reports, and the interaction surfaces have been modeled and mapped by cross linking (15)(16)(17)(18)(19)(20). The interaction studies suggest that KCNE1 resides between two adjacent voltage-sensor domains, at the junction with the pore region (15)(16)(17)(18)(19)(20). This kind of packing would seem to be compatible with a 4∶4 stoichiometry between KCNQ1 and the KCNE subunits. However, several studies concluded that only two KCNE1 subunits bind to four KCNQ1 subunits (4∶2 stoichiometry) (21-23). In contrast, a study of KCNE1 − KCNQ1 fusion proteins suggested the existence of multiple stoichiometries (24).In order to directly observe the number of KCNE1 subunits in the KCNQ1 − KCNE1 complex, we employed a single molecule im...

Voltage-sensing domains (VSDs) confer voltage dependence on effector domains of membrane proteins. Ion channels use four VSDs to control a gate in the pore domain, but in the recently discovered phosphatase Ci-VSP, the number of subunits has been unknown. Using single-molecule microscopy to count subunits and voltage clamp fluorometry to detect structural dynamics, we found Ci-VSP to be a monomer, which operates independently, but nevertheless undergoes multiple voltage-dependent conformational transitions.

SUMMARYIn voltage-gated sodium, potassium, and calcium channels the functions of ion conduction and voltage sensing are performed by two distinct structural units: the pore domain and the voltage-sensing domain (VSD). In the Hv1 voltage-gated proton channel, the VSD has the remarkable property of performing both functions. Hv1 was recently found to dimerize and to form channels made of two pores. However, the channels were also found to function when dimerization was prevented, raising a question about the functional role of dimerization. Here we show that the two subunits of the Hv1 dimer influence one another during gating, with positive cooperativity shaping the response to voltage of the two pores. We also find that the two voltage sensors undergo conformational changes that precede pore opening and that these conformational changes are allosterically coupled between the two subunits. Our results point to a major role of dimerization in the modulation of Hv1 activity.

Mutations in PKD1 and TRPP2 account for nearly all cases of autosomal dominant polycystic kidney disease (ADPKD). These 2 proteins form a receptor/ion channel complex on the cell surface. Using a combination of biochemistry, crystallography, and a single-molecule method to determine the subunit composition of proteins in the plasma membrane of live cells, we find that this complex contains 3 TRPP2 and 1 PKD1. A newly identified coiled-coil domain in the C terminus of TRPP2 is critical for the formation of this complex. This coiled-coil domain forms a homotrimer, in both solution and crystal structure, and binds to a single coiled-coil domain in the C terminus of PKD1. Mutations that disrupt the TRPP2 coiled-coil domain trimer abolish the assembly of both the full-length TRPP2 trimer and the TRPP2/PKD1 complex and diminish the surface expression of both proteins. These results have significant implications for the assembly, regulation, and function of the TRPP2/PKD1 complex and the pathogenic mechanism of some ADPKD-producing mutations.autosomal dominant polycystic kidney disease ͉ single-molecule imaging ͉ stoichiometry ͉ transient receptor potential channel ͉ X-ray crystallography

Canonical NMDA receptors assemble from two glycine-binding NR1 subunits with two glutamate-binding NR2 subunits to form glutamate-gated excitatory receptors that mediate synaptic transmission and plasticity. The role of glycine-binding NR3 subunits is less clear. Whereas in Xenopus laevis oocytes, two NR3 subunits coassemble with two NR1 subunits to form a glycine-gated receptor, such a receptor has yet to be found in mammalian cells. Meanwhile, NR1, NR2, and NR3 appear to coassemble into triheteromeric receptors in neurons, but it is not clear whether this occurs in oocytes. To test the rules that govern subunit assembly in NMDA receptors, we developed a single-molecule fluorescence colocalization method. The method focuses selectively on the plasma membrane and simultaneously determines the subunit composition of hundreds of individual protein complexes within an optical patch on a live cell. We find that NR1, NR2, and NR3 follow an exclusion rule that yields separate populations of NR1/NR2 and NR1/NR3 receptors on the surface of oocytes. In contrast, coexpression of NR1, NR3A, and NR3B yields triheteromeric receptors with a fixed stoichiometry of two NR1 subunits with one NR3A and one NR3B. At least part of this regulation of subunit stoichiometry appears to be caused by internal retention. Thus, depending on the mixture of subunits, functional receptors on the cell surface may follow either an exclusion rule or a stoichiometric combination rule, providing an important constraint on functional diversity. Cell-to-cell differences in the rules may help sculpt distinct physiological properties.NR3 ͉ total internal reflection ͉ single-molecule fluorescence ͉ subunit stoichiometry ͉ triheteromeric I on channels, receptors, and other proteins involved in transmembrane signaling are often composed of several different kinds of subunits. The composition can vary depending on the state of the cell and the availability of subunit types, which depends, in turn, on protein production and subcellular localization. Based on subunit availability and interaction affinity, complexes may form in one or more fixed stoichiometries or form with heterogeneous stoichiometries. Bulk assays may not be able to distinguish between these possibilities and could have difficulty in determining the pattern of assembly in specific subcellular compartments, in particular, separating plasma membrane fractions and intracellular membrane compartments. We used an optical approach to determine the subunit composition of integral membrane proteins complexes at the singlemolecule level. Measurements were made simultaneously for many complexes, yielding distributions from which it was possible to deduce the rule that governs complex assembly. The single-molecule approach is compatible with normal (low) levels of protein expression, where the formation of nonnative highorder complexes and aggregation are avoided.Our technique is based on the colocalization of single subunits of a macromolecular complex that are fused to fluorescent protein (FP) tags ...