Pore helix-S6 interactions are critical in governing current amplitudes of KCNQ3 K+ channels

Two mechanisms have been postulated to underlie KCNQ3 homomeric current amplitudes, which are small compared with those of KCNQ4 homomers and KCNQ2/Q3 heteromers. The first involves differential channel expression governed by the D-helix within the C-terminus. The second suggests similar channel surface expression but an intrinsically unstable KCNQ3 pore. Here, we find H2O2-enhanced oligomerization of KCNQ4 subunits, as reported by nondenaturing polyacrylamide gel electrophoresis, at C643 at the end of the D-helix, where KCNQ3 possesses a histidine. However, H2O2-mediated enhancement of KCNQ4 currents was identical in the C643A mutant, and KCNQ3 H646C produced homomeric or heteromeric (with KCNQ2) currents similar to those of wild-type KCNQ3, ruling out this divergent residue as underlying the small KCNQ3 amplitudes. In KcsA, F103 in S6 is critical for pore-mediated destabilization of the conductive pathway. We found that mutations at the analogous F344 in KCNQ3 dramatically decreased the KCNQ3 currents. Total internal reflection fluorescence imaging revealed only minor differential surface expression among the wild-type and mutant channels. Homology modeling suggests that the effects of the F344 mutants arise from the disruption of the interaction between F344 and A315 in the pore helix. These data support a secondary role of the C-terminus, compared with pore helix-S6 interactions, in governing KCNQ3 current amplitudes.     

Pore determinants of KCNQ3 K+ current expression

KCNQ3 homomeric channels yield very small macroscopic currents compared with other KCNQ channels or KCNQ2/3 heteromers. Two disparate regions of the channels--the C-terminus and the pore region--have been implicated in governing KCNQ current amplitudes. We previously showed that the C-terminus plays a secondary role compared with the pore region. Here, we confirm the critical role of the pore region in determining KCNQ3 currents. We find that mutations at the 312 position in the pore helix of KCNQ3 (I312E, I312K, and I312R) dramatically decreased KCNQ3 homomeric currents as well as heteromeric KCNQ2/3 currents. Evidence that these mutants were expressed in the heteromers includes shifted TEA sensitivity compared with KCNQ2 homomers. To test for differential membrane protein expression, we performed total internal reflection fluorescence imaging, which revealed only small differences that do not underlie the differences in macroscopic currents. To determine whether this mechanism generalizes to other KCNQ channels, we tested the effects of analogous mutations at the conserved I273 position in KCNQ2, with similar results. Finally, we performed homology modeling of the pore region of wild-type and mutant KCNQ3 channels to investigate the putative structural mechanism mediating these results. The modeling suggests that the lack of current in I312E, I312K, and I312R KCNQ3 channels is due to pore helix-selectivity filter interactions that lock the selectivity filter in a nonconductive conformation.     

Molecular cloning and functional expression of KCNQ5, a potassium channel subunit that may contribute to neuronal M-current diversity

We have isolated KCNQ5, a novel human member of the KCNQ potassium channel gene family that is differentially expressed in subregions of the brain and in skeletal muscle. When expressed in Xenopus oocytes, KCNQ5 generated voltage-dependent, slowly activating K(+)-selective currents that displayed a marked inward rectification at positive membrane voltages. KCNQ5 currents were insensitive to the K(+) channel blocker tetraethylammonium but were strongly inhibited by the selective M-current blocker linopirdine. Upon coexpression with the structurally related KCNQ3 channel subunit, current amplitudes increased 4-5-fold. Compared with homomeric KCNQ5 currents, KCNQ3/KCNQ5 currents also displayed slower activation kinetics and less inward rectification, indicating that KCNQ5 combined with KCNQ3 to form functional heteromeric channel proteins. This functional interaction between KCNQ5 and KCNQ3, a component of the M-channel, suggests that KCNQ5 may contribute to a diversity of heteromeric channels underlying native neuronal M-currents.     

Structure and assembly mechanism for heteromeric kainate receptors

Native glutamate receptor ion channels are tetrameric assemblies containing two or more different subunits. NMDA receptors are obligate heteromers formed by coassembly of two or three divergent gene families. While some AMPA and kainate receptors can form functional homomeric ion channels, the KA1 and KA2 subunits are obligate heteromers which?function only in combination with GluR5-7. The mechanisms controlling glutamate receptor assembly involve an initial step in which the amino terminal domains (ATD) assemble as dimers. Here, we establish by sedimentation velocity that the ATDs of GluR6 and KA2 coassemble as a heterodimer of K(d) 11?nM, 32,000-fold lower than the K(d) for homodimer formation by KA2; we solve crystal structures for the GluR6/KA2 ATD heterodimer and heterotetramer assemblies. Using these structures as a guide, we perform a mutant cycle analysis to probe the energetics of assembly and show that high-affinity ATD interactions are required for biosynthesis of functional heteromeric receptors.     

Stoichiometry and assembly of olfactory cyclic nucleotide-gated channels

Native ion channels are precisely tuned to their physiological role in neuronal signaling. This tuning frequently involves the controlled assembly of heteromeric channels comprising multiple types of subunits. Cyclic nucleotide-gated (CNG) channels of olfactory neurons are tetramers and require three types of subunits, CNGA2, CNGA4, and CNGB1b, to exhibit properties necessary for olfactory transduction. Using fluorescently tagged subunits and fluorescence resonance energy transfer (FRET), we find the subunit composition of heteromeric olfactory channels in the surface membrane is fixed, with 2:1:1 CNGA2:CNGA4:CNGB1b. Furthermore, when expressed individually with CNGA2, CNGA4 and CNGB1b subunits were still present in only a single copy and, when expressed alone, did not self-assemble. These results suggest that the precise assembly of heteromeric olfactory channels results from a mechanism where CNGA4 and CNGB1b subunits have a high affinity for CNGA2 but not for self-assembly, precluding more than one CNGA4 or CNGB1b subunit in the channel complex.     

The SK3 subunit of small conductance Ca2+-activated K+ channels interacts with both SK1 and SK2 subunits in a heterologous expression system

The aim of this study was to determine whether functional heteromeric channels can be formed by co-assembly of rat SK3 (rSK3) potassium channel subunits with either SK1 or SK2 subunits. First, to determine whether rSK3 could co-assemble with rSK2 we created rSK3VK (an SK3 mutant insensitive to block by UCL 1848). When rSK3VK was co-expressed with rSK2 the resulting currents had an intermediate sensitivity to UCL 1848 (IC50 of approximately 5 nM compared with 120 pM for rSK2 and >300 nM for rSK3VK), suggesting that rSK3 and rSK2 can form functional heteromeric channels. To detect co-assembly of SK3 with SK1, we initially used a dominant negative construct of the human SK1 subunit (hSK1YP). hSK1YP dramatically reduced the SK3 current, supporting the idea that SK3 and SK1 subunits also interact. To determine whether these assemblies were functional we created rSK3VF, an rSK3 mutant with an enhanced affinity for tetraethylammonium chloride (TEA) (IC50 of 0.3 mM). Co-transfection of rSK3VF and hSK1 produced currents with a sensitivity to TEA not different from that of hSK1 alone (IC50 approximately 15 mM). These results suggest that hSK1 does not produce functional cell-surface assemblies with SK3. Antibody-staining experiments suggested that hSK1 may reduce the number of functional SK3 subunits reaching the cell surface. Additional experiments showed that co-expression of the rat SK1 gene with SK3 also dramatically suppressed SK current. The pharmacology of the residual current was consistent with that of homomeric SK3 assemblies. These results demonstrate interactions that cause changes in protein trafficking, cell surface expression, and channel pharmacology and strongly suggest heteromeric assembly of SK3 with the other SK channel subunits.     

Decreased subunit stability as a novel mechanism for potassium current impairment by a KCNQ2 C terminus mutation causing benign familial neonatal convulsions

KCNQ2 and KCNQ3 K+ channel subunits underlie the muscarinic-regulated K+ current (I(KM)), a widespread regulator of neuronal excitability. Mutations in KCNQ2- or KCNQ3-encoding genes cause benign familiar neonatal convulsions (BFNCs), a rare autosomal-dominant idiopathic epilepsy of the newborn. In the present study, we have investigated, by means of electrophysiological, biochemical, and immunocytochemical techniques in transiently transfected cells, the consequences prompted by a BFNC-causing 1-bp deletion (2043deltaT) in the KCNQ2 gene; this frameshift mutation caused the substitution of the last 163 amino acids of the KCNQ2 C terminus and the extension of the subunit by additional 56 residues. The 2043deltaT mutation abolished voltage-gated K+ currents produced upon homomeric expression of KCNQ2 subunits, dramatically reduced the steady-state cellular levels of KCNQ2 subunits, and prevented their delivery to the plasma membrane. Metabolic labeling experiments revealed that mutant KCNQ2 subunits underwent faster degradation; 10-h treatment with the proteasomal inhibitor MG132 (20 microm) at least partially reversed such enhanced degradation. Co-expression with KCNQ3 subunits reduced the degradation rate of mutant KCNQ2 subunits and led to their expression on the plasma membrane. Finally, co-expression of KCNQ2 2043deltaT together with KCNQ3 subunits generated functional voltage-gated K+ currents having pharmacological and biophysical properties of heteromeric channels. Collectively, the present results suggest that mutation-induced reduced stability of KCNQ2 subunits may cause epilepsy in neonates.     

Epithelial Na+ channel subunit stoichiometry

Ion channels, including the epithelial Na(+) channel (ENaC), are intrinsic membrane proteins comprised of component subunits. Proper subunit assembly and stoichiometry are essential for normal physiological function of the channel protein. ENaC comprises three subunits, alpha, beta, and gamma, that have common tertiary structures and much amino acid sequence identity. For maximal ENaC activity, each subunit is required. The subunit stoichiometry of functional ENaC within the membrane remains uncertain. We combined a biophysical approach, fluorescence intensity ratio analysis, used to assess relative subunit stoichiometry with total internal reflection fluorescence microscopy, which enables isolation of plasma membrane fluorescence signals, to determine the limiting subunit stoichiometry of ENaC within the plasma membrane. Our results demonstrate that membrane ENaC contains equal numbers of each type of subunit and that at steady state, subunit stoichiometry is fixed. Moreover, we find that when all three ENaC subunits are coexpressed, heteromeric channel formation is favored over homomeric channels. Electrophysiological results testing effects of ENaC subunit dose on channel activity were consistent with total internal reflection fluorescence/fluorescence intensity ratio findings and confirmed preferential formation of heteromeric channels containing equal numbers of each subunit.     

Assembly stoichiometry of the GluK2/GluK5 kainate receptor complex

Ionotropic glutamate receptors assemble as homo- or heterotetramers. One well-studied heteromeric complex is formed by the kainate receptor subunits GluK2 and GluK5. Retention motifs prevent trafficking of GluK5 homomers to the plasma membrane, but coassembly with GluK2 yields functional heteromeric receptors. Additional control over GluK2/GluK5 assembly seems to be exerted by the aminoterminal domains, which preferentially assemble into heterodimers as isolated domains. However,the stoichiometry of the full-length GluK2/GluK5 receptor complex has yet to be determined, as is the case for all non-NMDA glutamate receptors. Here, we address this question, using a single-molecule imaging technique that enables direct counting of the number of each GluK subunit type in homomeric and heteromeric receptors in the plasma membranes of live cells. We show that GluK2 and GluK5 assemble with 2:2 stoichiometry. This is an important step toward understanding the assembly mechanism, architecture, and functional consequences of heteromer formation in ionotropic glutamate receptors.     

Dual-color quantum dot detection of a heterotetrameric potassium channel (hKCa3.1)

Potassium channels play a key role in establishing the cell membrane potential and are expressed ubiquitously. Today, more than 70 mammalian K(+) channel genes are known. The diversity of K(+) channels is further increased by the fact that different K(+) channel family members may assemble to form heterotetramers. We present a method based on fluorescence microscopy to determine the subunit composition of a tetrameric K(+) channel. We generated artificial "heteromers" of the K(+) channel hK(Ca)3.1 by coexpressing two differently tagged hK(Ca)3.1 constructs containing either an extracellular hemagglutinin (HA) or an intracellular V5 epitope. hK(Ca)3.1 channel subunits were detected in the plasma membrane of MDCK-F cells or HEK293 cells by labeling the extra- and intracellular epitopes with differently colored quantum dots (QDs). As previously shown for the extracellular part of hK(Ca)3.1 channels, its intracellular domain can also bind only one QD label at a time. When both channel subunits were coexpressed, 27.5 ± 1.8% and 24.9 ± 2.1% were homotetramers consisting of HA- and V5-tagged subunits, respectively. 47.6 ± 3.2% of the channels were heteromeric and composed of both subunits. The frequency distribution of HA- and V5-tagged homo- and heteromeric hK(Ca)3.1 channels is reminiscent of the binomial distribution (a + b)(2) = a(2) + 2ab + b(2). Along these lines, our findings are consistent with the notion that hK(Ca)3.1 channels are assembled from two homomeric dimers and not randomly from four independent subunits. We anticipate that our technique will be applicable to other heteromeric membrane proteins, too.     

Thermosensitive TRPV channel subunits coassemble into heteromeric channels with intermediate conductance and gating properties

Heat-sensitive transient receptor potential (TRP) channels (TRPV1-4) form the major cellular sensors for detecting temperature increases. Homomeric channels formed by thermosensitive TRPV subunits exhibit distinct temperature thresholds. While these subunits do share significant sequence similarity, whether they can coassemble into heteromeric channels has been controversial. In the present study we investigated the coassembly of TRPV subunits using both spectroscopy-based fluorescence resonance energy transfer (FRET) and single-channel recordings. Fluorescent protein-tagged TRPV subunits were coexpressed in HEK 293 cells; FRET between different subunits was measured as an indication of the formation of heteromeric channels. We observed strong FRET when fluorescence signals were collected selectively from the plasma membrane using a "spectra FRET" approach but much weaker or no FRET from intracellular fluorescence. In addition, no FRET was detected when TRPV subunits were coexpressed with members of the TRPM subfamily or CLC-0 chloride channel subunits. These results indicate that a substantial fraction of TRP channels in the plasma membrane of cotransfected cells were heteromeric. Single-channel recordings confirmed the existence of multiple heteromeric channel forms. Interestingly, heteromeric TRPV channels exhibit intermediate conductance levels and gating kinetic properties. As these subunits coexpress both in sensory neurons and in other tissues, including heart and brain, coassembly between TRPV subunits may contribute to greater functional diversity.     

Regulation of the voltage-gated K(+) channels KCNQ2/3 and KCNQ3/5 by ubiquitination. Novel role for Nedd4-2

The muscarine-sensitive K(+) current (M-current) stabilizes the resting membrane potential in neurons, thus limiting neuronal excitability. The M-current is mediated by heteromeric channels consisting of KCNQ3 subunits in association with either KCNQ2 or KCNQ5 subunits. The role of KCNQ2/3/5 in the regulation of neuronal excitability is well established; however, little is known about the mechanisms that regulate the cell surface expression of these channels. Ubiquitination by the Nedd4/Nedd4-2 ubiquitin ligases is known to regulate a number of membrane ion channels and transporters. In this study, we investigated whether Nedd4/Nedd4-2 could regulate KCNQ2/3/5 channels. We found that the amplitude of the K(+) currents mediated by KCNQ2/3 and KCNQ3/5 were reduced by Nedd4-2 (but not Nedd4) in a Xenopus oocyte expression system. Deletion experiments showed that the C-terminal region of the KCNQ3 subunit is required for the Nedd4-2-mediated regulation of the heteromeric channels. Glutathione S-transferase fusion pulldowns and co-immunoprecipitations demonstrated a direct interaction between KCNQ2/3 and Nedd4-2. Furthermore, Nedd4-2 could ubiquitinate KCNQ2/3 in transfected cells. Taken together, these data suggest that Nedd4-2 is potentially an important regulator of M-current activity in the nervous system.     

The Role of the Carboxyl Terminus Helix C-D Linker in Regulating KCNQ3 K+ Current Amplitudes by Controlling Channel Trafficking

In the central and peripheral nervous system, the assembly of KCNQ3 with KCNQ2 as mostly heteromers, but also homomers, underlies “M-type” currents, a slowly-activating voltage-gated K⁺ current that plays a dominant role in neuronal excitability. KCNQ3 homomers yield much smaller currents compared to KCNQ2 or KCNQ4 homomers and KCNQ2/3 heteromers. This smaller current has been suggested to result either from divergent channel surface expression or from a pore that is more unstable in KCNQ3. Channel surface expression has been shown to be governed by the distal part of the C-terminus in which helices C and D are critical for channel trafficking and assembly. A sequence alignment of this region in KCNQ channels shows that KCNQ3 possesses a longer linker between helix C and D compared to the other KCNQ subunits. Here, we investigate the role of the extra residues of this linker on KCNQ channel expression. Deletion of these residues increased KCNQ3 current amplitudes. Total internal reflection fluorescence imaging and plasma membrane protein assays suggest that the increase in current is due to a higher surface expression of the channels. Conversely, introduction of the extra residues into the linker between helices C and D of KCNQ4 reduced current amplitudes by decreasing the number of KCNQ4 channels at the plasma membrane. Confocal imaging suggests a higher fraction of channels, which possess the extra residues of helix C-D linker, were retained within the endoplasmic reticulum. Such retention does not appear to lead to protein accumulation and activation of the unfolded protein response that regulates protein folding and maintains endoplasmic reticulum homeostasis. Taken together, we conclude that extra helix C-D linker residues play a role in KCNQ3 current amplitudes by controlling the exit of the channel from the endoplasmic reticulum.     

Identification of a domain involved in ATP-gated ionotropic receptor subunit assembly.

P2X receptors are ATP-gated ion channels found in a variety of tissues and cell types. Seven different subunits (P2X(1)-P2X(7)) have been molecularly cloned and are known to form homomeric, and in some cases heteromeric, channel complexes. However, the molecular determinants leading to the assembly of subunits into P2X receptors are unknown. To address this question we utilized a co-immunoprecipitation assay in which epitope-tagged deletion mutants and chimeric constructs were examined for their ability to co-associate with full-length P2X subunits. Deletion mutants of the P2X(2) receptor subunit were expressed individually and together with P2X(2) or P2X(3) receptor subunits in HEK 293 cells. Deletion of the amino terminus up to the first transmembrane domain (amino acid 28) and beyond (to amino acid 51) did not prevent subunit assembly. Analysis of the carboxyl terminus demonstrated that mutants missing the portion of the protein downstream of the second transmembrane domain could also still co-assemble. However, a mutant terminating 25 amino acids before the second transmembrane domain could not assemble with other subunits or itself, implicating the missing region of the protein in assembly. This finding was supported and extended by data utilizing a chimera strategy that indicated TMD2 is a critical determinant of P2X subunit assembly.     

Interleukin-5 receptor subunit oligomerization and rearrangement revealed by fluorescence resonance energy transfer imaging

Interleukin (IL)-5 exerts hematopoietic functions through binding to the IL-5 receptor subunits, alpha and betac. Specific assembly steps of full-length subunits as they occur in cell membranes, ultimately leading to receptor activation, are not well understood. We tracked the oligomerization of IL-5 receptor subunits using fluorescence resonance energy transfer (FRET) imaging. Full-length IL-5Ralpha and betac were expressed in Phoenix cells as chimeric proteins fused to enhanced cyan or yellow fluorescent protein (CFP or YFP, respectively). A time- and dose-dependent increase in FRET signal between IL-5Ralpha-CFP and betac-YFP was observed in response to IL-5, indicative of heteromeric receptor alpha-betac subunit interaction. This response was inhibited by AF17121, a peptide antagonist of IL-5Ralpha. Substantial FRET signals with betac-CFP and betac-YFP co-expressed in the absence of IL-5Ralpha demonstrated that betac subunits exist as preformed homo-oligomers. IL-5 had no effect on this betac-alone FRET signal. Interestingly, the addition of IL-5 to cells co-expressing betac-CFP, betac-YFP, and nontagged IL-5Ralpha led to further increase in FRET efficiency. Observation of preformed betac oligomers fits with the view that this form can lead to rapid cellular responses upon IL-5 stimulation. The IL-5-induced effects on betac assembly in the presence of nontagged IL-5Ralpha provide direct evidence that IL-5 can cause higher order rearrangements of betac homo-oligomers. These results suggest that IL-5 and perhaps other betac cytokines (IL-3 and granulocyte/macrophage colony-stimulating factor) trigger cellular responses by the sequential binding of cytokine ligand to the specificity receptor (subunit alpha), followed by binding of the ligand-subunit alpha complex to, and consequent rearrangement of, a ground state form of betac oligomers.     

Fluorescence resonance energy transfer analysis of subunit assembly of the ASIC channel

Acid-sensing ion channels (ASICs) are believed to be homo- or heteromeric complexes, which have been verified by classical methods such as co-immunoprecipitation or electrophysiological assays. However, the exact subunit combinations of ASICs in living cells have not been established yet. Here, we apply assays based on fluorescence resonance energy transfer (FRET) between GFP color mutants CFP and YFP to investigate ASIC assembly directly in living cells. Homomerization as well as heteromerization of different combinations of ASIC subunits were found. In addition, our results suggest the formation of heteromeric 1a/2a channels of stoichiometry consisting of at least two 1a subunits and two 2a subunits. Similar stoichiometry was observed from heteromeric 1a/2b and 2a/2b channels. Our results imply that these heteromeric ASIC channels contain at least four subunits.     

Achromatopsia-associated mutation in the human cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit alters the ligand sensitivity and pore properties of heteromeric channels

Cone photoreceptor cyclic nucleotide-gated (CNG) channels are thought to form by assembly of two different subunit types, CNGA3 and CNGB3. Recently, mutations in the gene encoding the CNGB3 subunit have been linked to achromatopsia in humans. Here we describe the functional consequences of two achromatopsia-associated mutations in human CNGB3 (hCNGB3). Co-expression in Xenopus oocytes of human CNGA3 (hCNGA3) subunits with hCNGB3 subunits containing an achromatopsia-associated mutation in the S6 transmembrane domain (S435F) generated functional heteromeric channels that exhibited an increase in apparent affinity for both cAMP and cGMP compared with wild type heteromeric channels. In contrast, co-expression of a presumptive null mutation of hCNGB3 (T383f.s.Delta C) with hCNGA3 produced channels with properties indistinguishable from homomeric hCNGA3 channels. The effect of hCNGB3 S435F subunits on cell-surface expression of green fluorescent protein-tagged hCNGA3 subunits and of non-tagged hCNGA3 subunits on surface expression of green fluorescent protein-hCNGB3 S435F subunits were similar to those observed for wild type hCNGB3 subunits, suggesting that the mutation does not grossly disturb subunit assembly or plasma membrane targeting. The S435F mutation was also found to produce changes in the pore properties of the channel, including decreased single channel conductance and decreased sensitivity to block by l-cis-diltiazem. Overall, these results suggest that the functional properties of cone CNG channels may be altered in patients with the S435F mutation, providing evidence supporting the pathogenicity of this mutation in humans. Thus, achromatopsia may arise from a disturbance of cone CNG channel gating and permeation or from the absence of functional CNGB3 subunits.     

Gating and flickery block differentially affected by rubidium in homomeric KCNQ1 and heteromeric KCNQ1/KCNE1 potassium channels

The voltage-gated potassium channel KCNQ1 associates with the small KCNE1 subunit to form the cardiac IKs delayed rectifier potassium current and mutations in both genes can lead to the long QT syndrome. KCNQ1 can form functional homotetrameric channels, however with drastically different biophysical properties compared to heteromeric KCNQ1/KCNE1 channels. We analyzed gating and conductance of these channels expressed in Xenopus oocytes using the two-electrode voltage-clamp and the patch-clamp technique and high extracellular potassium (K) and rubidium (Rb) solutions. Inward tail currents of homomeric KCNQ1 channels are increased about threefold upon substitution of 100 mM potassium with 100 mM rubidium despite a smaller rubidium permeability, suggesting an effect of rubidium on gating. However, the kinetics of tail currents and the steady-state activation curve are only slightly changed in rubidium. Single-channel amplitude at negative voltages was estimated by nonstationary noise analysis, and it was found that rubidium has only a small effect on homomeric channels (1.2-fold increase) when measured at a 5-kHz bandwidth. The apparent single-channel conductance was decreased after filtering the data at lower cutoff frequencies indicative of a relatively fast "flickery/block" process. The relative conductance in rubidium compared to potassium increased at lower cutoff frequencies (about twofold at 10 Hz), suggesting that the main effect of rubidium is to decrease the probability of channel blockage leading to an increase of inward currents without large changes in gating properties. Macroscopic inward tail currents of heteromeric KCNQ1/KCNE1 channels in rubidium are reduced by about twofold and show a pronounced sigmoidal time course that develops with a delay similar to the inactivation process of homomeric KCNQ1, and is indicative of the presence of several open states. The single channel amplitude of heteromers is about twofold smaller in rubidium than in potassium at a bandwidth of 5 kHz. Filtering at lower cutoff frequencies reduces the apparent single-channel conductance, the ratio of the conductance in rubidium versus potassium is, however, independent of the cutoff frequency. Our results suggest the presence of a relatively rapid process (flicker) that can occur almost independently of the gating state. Occupancy by rubidium at negative voltages favors the flicker-open state and slows the flickering rate in homomeric channels, whereas rubidium does not affect the flickering in heteromeric channels. The effects of KCNE1 on the conduction properties are consistent with an interaction of KCNE1 in the outer vestibule of the channel.     

Agonist binding to the GluK5 subunit is sufficient for functional surface expression of heteromeric GluK2/GluK5 kainate receptors

Trafficking of ionotropic glutamate receptors to the plasma membrane commonly requires occupation of the agonist binding sites. This quality control check does not typically involve receptor activation, as binding by competitive antagonists or to non-functional channels may also permit surface expression. The tetrameric kainate receptors can be assembled from five different subunits (GluK1–GluK5). While the “low-affinity” GluK1-3 subunits are able to produce functional homomeric receptors, the “high-affinity” GluK4 and GluK5 subunits require co-assembly with GluK1, 2, or 3 for surface expression. These two different types of subunits have distinct functional roles in the receptor. Therefore, we examined the relative importance of occupancy of the agonist site of the GluK2 or GluK5 subunit for surface expression of heteromeric receptors. We created subunits with a mutation within the S2 ligand-binding domain which decreased agonist affinity. Mutations at this site reduced functional surface expression of homomeric GluK2 receptors, but surface expression of these receptors could be increased with either a competitive antagonist or co-assembly with wild-type GluK5. In contrast, mutations in the GluK5 subunit reduced the production of functional heteromeric receptors at the membrane, and could not be rescued with either an antagonist or wild-type GluK2. These findings indicate that ligand binding to only the GluK5 subunit is both necessary and sufficient to allow trafficking of recombinant GluK2/K5 heteromers to the cell membrane, but that occupancy of the GluK2 site alone is not. Our results suggest a distinct role for the GluK5 subunit in regulating surface expression of heteromeric kainate receptors.     

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.