Macroscopic Current Amplitude Research Articles

The mechanism of tuning filler orientation degree in composites based on AC electric field assist: from microscopic dynamical model to macroscopic electrical properties

Using the AC electric field to induce the orientation of nonlinear conductive fillers in composites is an effective solution for alleviating electric field distortion in power modules. However, the mechanism by which the electric field affects the filler dynamic characteristics and the composites’ electrical properties remains unclear. In this paper, the correlation between the microscopic dynamic processes of fillers and the macroscopic current amplitude was analyzed. The results show that the current increases rapidly (0 ∼ 173 s) and then slowly (173 ∼ 869 s) at 600 V mm−1, influenced by the rotation and attraction processes of the fillers. This demonstrates that the orientation stops at about 869 s and the filler orientation state is a key factor in determining the dielectric properties. Secondly, the global orientation evaluation index D for the filler network was proposed, which can also derive the minimum time and energy loss required for preparation. Finally, the impact of different filler orientations on the composites’ conductivity was investigated. In the low electric field stress region, with the average carrier jump distance decreasing from 150.23 to 109.71 nm as the D increases from −0.93 to −0.05. On this basis, materials with nonlinear conductivity gradient distribution can be easily prepared. Before optimization, the electric field stress of the power module at the triple point was 35.79 kV. This composite can reduce the value to 15.42 kV, a decrease of 56.9%, while maintaining good electric field uniformity.

The second PI(3,5)P2 binding site in the S0 helix of KCNQ1 stabilizes PIP2-at the primary PI1 site with potential consequences on intermediate-to-open state transition.

The Phosphatidylinositol 3-phosphate 5-kinase Type III PIKfyve is the main source for selectively generated phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a known regulator of membrane protein trafficking. PI(3,5)P2 facilitates the cardiac KCNQ1/KCNE1 channel plasma membrane abundance and therewith increases the macroscopic current amplitude. Functional-physical interaction of PI(3,5)P2 with membrane proteins and its structural impact is not sufficiently understood. This study aimed to identify molecular interaction sites and stimulatory mechanisms of the KCNQ1/KCNE1 channel via the PIKfyve-PI(3,5)P2 axis. Mutational scanning at the intracellular membrane leaflet and nuclear magnetic resonance (NMR) spectroscopy identified two PI(3,5)P2 binding sites, the known PIP2 site PS1 and the newly identified N-terminal α-helix S0 as relevant for functional PIKfyve effects. Cd2+ coordination to engineered cysteines and molecular modeling suggest that repositioning of S0 stabilizes the channel s open state, an effect strictly dependent on parallel binding of PI(3,5)P2 to both sites.

An amino-terminal point mutation increases EAAT2 anion currents without affecting glutamate transport rates

Excitatory amino acid transporters (EAATs) are prototypical dual function proteins that function as coupled glutamate/Na+/H+/K+ transporters and as anion-selective channels. Both transport functions are intimately intertwined at the structural level: Secondary active glutamate transport is based on elevator-like movements of the mobile transport domain across the membrane, and the lateral movement of this domain results in anion channel opening. This particular anion channel gating mechanism predicts the existence of mutant transporters with changed anion channel properties, but without alteration in glutamate transport. We here report that the L46P mutation in the human EAAT2 transporter fulfills this prediction. L46 is a pore-forming residue of the EAAT2 anion channels at the cytoplasmic entrance into the ion conduction pathway. In whole-cell patch clamp recordings, we observed larger macroscopic anion current amplitudes for L46P than for WT EAAT2. Rapid l-glutamate application under forward transport conditions demonstrated that L46P does not reduce the transport rate of individual transporters. In contrast, changes in selectivity made gluconate permeant in L46P EAAT2, and nonstationary noise analysis revealed slightly increased unitary current amplitudes in mutant EAAT2 anion channels. We used unitary current amplitudes and individual transport rates to quantify absolute open probabilities of EAAT2 anion channels from ratios of anion currents by glutamate uptake currents. This analysis revealed up to 7-fold increased absolute open probability of L46P EAAT2 anion channels. Our results reveal an important determinant of the diameter of EAAT2 anion pore and demonstrate the existence of anion channel gating processes outside the EAAT uptake cycle.

The Selectivity Filter Is Involved in the U-Type Inactivation Process of Kv2.1 and Kv3.1 Channels

Voltage-gated potassium (Kv) channels display several types of inactivation processes, including N-, C-, and U-types. C-type inactivation is attributed to a nonconductive conformation of the selectivity filter (SF). It has been proposed that the activation gate and the channel’s SF are allosterically coupled because the conformational changes of the former affect the structure of the latter and vice versa. The second threonine of the SF signature sequence (e.g., TTVGYG) has been proven to be essential for this allosteric coupling. To further study the role of the SF in U-type inactivation, we substituted the second threonine of the TTVGYG sequence by an alanine in the hKv2.1 and hKv3.1 channels, which are known to display U-type inactivation. Both hKv2.1-T377A and hKv3.1-T400A yielded channels that were resistant to inactivation, and as a result, they displayed noninactivating currents upon channel opening; i.e., hKv2.1-T377A and hKv3.1-T400A remained fully conductive upon prolonged moderate depolarizations, whereas in wild-type hKv2.1 and hKv3.1, the current amplitude typically reduces because of U-type inactivation. Interestingly, increasing the extracellular K+ concentration increased the macroscopic current amplitude of both hKv2.1-T377A and hKv3.1-T400A, which is similar to the response of the homologous T to A mutation in Shaker and hKv1.5 channels that display C-type inactivation. Our data support an important role for the second threonine of the SF signature sequence in the U-type inactivation gating of hKv2.1 and hKv3.1.

Identification of a proton sensor that regulates conductance and open time of single hERG channels

The hERG potassium channel influences ventricular action potential duration. Extracellular acidosis occurs in pathological states including cardiac ischaemia. It reduces the amplitude of hERG current and speeds up deactivation, which can alter cardiac excitability. This study aimed to identify the site of action by which extracellular protons regulate the amplitude of macroscopic hERG current. Recordings of macroscopic and single hERG1a and 1b channel activity, mutagenesis, and the recent cryoEM structure for hERG were employed. Single hERG1a and 1b channels displayed open times that decreased with membrane depolarization, suggestive of a blocking mechanism that senses approximately 20% of the membrane electric field. This mechanism was sensitive to pH; extracellular acidosis reduced both hERG1a and1b channel open time and conductance. The effects of acidosis on macroscopic current amplitude and deactivation displayed different sensitivities to protons. Point mutation of a pair of residues (E575/H578) in the pore turret abolished the acidosis-induced decrease of current amplitude, without affecting the change in current deactivation. In single hERG1a channel recordings, the conductance of the double-mutant channel was unaffected by extracellular acidosis. These findings identify residues in the outer turret of the hERG channel that act as a proton sensor to regulate open time and channel conductance.

Stargazin and \u03b34 slow the channel opening and closing rates of GluA4 AMPA receptors

As auxiliary subunits, transmembrane AMPA receptor regulatory proteins (TARPs) are known to enhance macroscopic current amplitude and alter kinetic properties of AMPA receptors on slow time scale, such as desensitization rate. Whether TARPs affect the rate of AMPA channel opening and closing, however, remains elusive. Using a laser-pulse photolysis technique, we investigated the effect of γ-2 (stargazin, a type 1a TARP) and γ-4 (a type 1b TARP) on the channel-opening and channel-closing rate constants (i.e., kop and kcl) of GluA4 homomeric channels. We found both TARPs slow the kop and kcl by 4-fold and 3-fold, respectively, without appreciable change of channel-opening probability, as compared with GluA4 channel alone. On the other hand, γ-4 has a stronger effect on slowing the channel desensitization rate than γ-2; yet, γ-2 causes a much more pronounced left shift of the dose-response relationship by increasing its affinity towards glutamate than γ-4. Our study shows that on the faster time scale, the major impact of TARP association with GluA4 is to lengthen the lifetime of the open channel, which is slow to form, to allow a larger charge transfer through the open channel that closes more slowly, without appreciable change of channel opening probability.

Singlet oxygen modification abolishes voltage-dependent inactivation of the sea urchin spHCN channel.

Photochemically or metabolically generated singlet oxygen (1O2) reacts broadly with macromolecules in the cell. Because of its short lifetime and working distance, 1O2 holds potential as an effective and precise nanoscale tool for basic research and clinical practice. Here we investigate the modification of the spHCN channel that results from photochemically and chemically generated 1O2 The spHCN channel shows strong voltage-dependent inactivation in the absence of cAMP. In the presence of photosensitizers, short laser pulses transform the gating properties of spHCN by abolishing inactivation and increasing the macroscopic current amplitude. Alanine replacement of a histidine residue near the activation gate within the channel's pore abolishes key modification effects. Application of a variety of chemicals including 1O2 scavengers and 1O2 generators supports the involvement of 1O2 and excludes other reactive oxygen species. This study provides new understanding about the photodynamic modification of ion channels by 1O2 at the molecular level.

The Effects of Extracellular Protons on the hERG Potassium Channel

The α-subunit of channels mediating the cardiac rapid delayed rectifier current (IKr) is encoded by the human Ether-à-go-go-Related Gene (hERG). Macroscopic hERG current (IhERG) amplitude is reduced and deactivation kinetics are accelerated with extracellular acidosis. We have investigated the single channel basis for the effects of acidic external pH (pHe) on the isoforms of IhERG expressed in myocytes (hERG1a and 1b). Patch clamp recordings were made at room temperature with the extracellular superfusate (whole-cell) or pipette solution (cell attached) acidified to pH 6.3 compared with control (pH 7.4). A decrease in pHe to 6.3 caused acceleration in deactivation and a reduction in maximal whole-cell conductance of ∼34% for IhERG1a (n=8 cells) and of ∼36% for IhERG1b (n=5 cells). Single channel recordings were made with isotonic potassium (140 mM) bathing the cells and in the electrode. Channel amplitude and open state kinetics were measured at a series of repolarisation voltages following a depolarising command to +40mV. Slope conductance values derived from amplitude current-voltage relationships between −120 and −40mV were 12.3±0.2pS for pH 7.4 (n=10 cells) and 9.3±0.1pS for pH 6.3 (n=9 cells) (P<0.01, two-tailed t-test) for hERG1a. The corresponding values for hERG1b were 11.4±0.2pS for pH 7.4 (n=6 cells) and 7.6±0.4pS for pH 6.3 (n=5 cells) (P<0.0001; two-tailed t-test). Open-time kinetics at −120mV for hERG1a were reduced from 8.49±1.0ms in control (n=8 cells) to 4.2±0.4ms in pHe 6.3 (n=7 cells) (P<0.05; two-tailed t-test). The hERG1b open state kinetics were 5.7±0.6ms in pHe 7.4 (n=6 cells) and reduced to 3.1±0.7ms in pHe 6.3 (n=5 cells) (P<0.05; two-tailed t-test). Thus, it can be concluded that a reduction in the single channel conductance and acceleration of open-times contribute to the attenuation of macroscopic IhERG when exposed to acidic pHe.

Patch-Clamp Fluorometry based Determination of Relative Ion Permeability for HCN Channels

Determining the relative permeability of different ions is a classical topic for channel biophysics. Two traditional methods have been used are: 1) single channel recording method, and 2) the reversal potential measurement based on the Goldman-Hodgkin-Katz (GHK) equation. Both methods have been used extensively, but certain limitations such as the extremely small single channel conductance prevent the broad application of either method to every type of channels. For the GHK/reversal potential method, when the differential ion concentrations across the cell membrane lead to changes in channel gating property or local biophysical parameters such as pH, the interpretation of the results could be complicated. For the determination of the relative permeability of ammonium (NH4+) compared to another ion, such as potassium (K), the GHK/reversal potential method may not produce accurate results because NH4+ in mill molar range produces a change in local pH on other side of the membrane. The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are known to be sensitive to different levels of pH. Here we have proposed a novel method for determining the relative permeability of different ions based on the technique of patch-clamp fluorometry (PCF), which enables simultaneous measurements of macroscopic current amplitude and fluorescence intensity. The slope of the current amplitude vs. fluorescence intensity is directly correlated to the relative ionic permeability. We applied this PCF-based method to the EGFP-tagged HCN channels and determined the ionic selectivity of K, Na+, and NH4+. The PK/PNa was found to be 2.92, the PK/PNa in the absence of any potassium ions was found to be 39, and the PK/PNH4 was found to be 15. Our results were consistent with previous reports. The PCF-based approach holds the potential as an alternative approach for determining the relative ion permeability.

Impaired surface membrane insertion of homo- and heterodimeric human muscle chloride channels carrying amino-terminal myotonia-causing mutations.

Mutations in the muscle chloride channel gene (CLCN1) cause myotonia congenita, an inherited condition characterized by muscle stiffness upon sudden forceful movement. We here studied the functional consequences of four disease-causing mutations that predict amino acid substitutions Q43R, S70L, Y137D and Q160H. Wild-type (WT) and mutant hClC-1 channels were heterologously expressed as YFP or CFP fusion protein in HEK293T cells and analyzed by whole-cell patch clamp and fluorescence recordings on individual cells. Q43R, Y137D and Q160H, but not S70L reduced macroscopic current amplitudes, but left channel gating and unitary current amplitudes unaffected. We developed a novel assay combining electrophysiological and fluorescence measurements at the single-cell level in order to measure the probability of ion channel surface membrane insertion. With the exception of S70L, all tested mutations significantly reduced the relative number of homodimeric hClC-1 channels in the surface membrane. The strongest effect was seen for Q43R that reduced the surface insertion probability by more than 99% in Q43R homodimeric channels and by 92 ± 3% in heterodimeric WT/Q43R channels compared to homodimeric WT channels. The new method offers a sensitive approach to investigate mutations that were reported to cause channelopathies, but display only minor changes in ion channel function.

Assessment of the functionality and stability of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology

In our previous study we examined the functionality and stability of nicotinic acetylcholine receptor (nAChR)-detergent complexes (nAChR-DCs) from affinity-purified Torpedo californica (Tc) using fluorescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) and planar lipid bilayer (PLB) recordings for phospholipid and cholesterol like detergents. In the present study we enhanced the functional characterization of nAChR-DCs by recording macroscopic ion channel currents in Xenopus oocytes using the two electrode voltage clamp (TEVC). The use of TEVC allows for the recording of macroscopic currents elicited by agonist activation of nAChR-DCs that assemble in the oocyte plasma membrane. Furthermore, we examined the stability of nAChR-DCs, which is obligatory for the nAChR crystallization, using a 30day FRAP assay in LCP for each detergent. The present results indicate a marked difference in the fractional fluorescence recovery (ΔFFR) within the same detergent family during the 30day period assayed. Within the cholesterol analog family, sodium cholate and CHAPSO displayed a minimum ΔFFR and a mobile fraction (MF) over 80%. In contrast, CHAPS and BigCHAP showed a marked decay in both the mobile fraction and diffusion coefficient. nAChR-DCs containing phospholipid analog detergents with an alkylphosphocholine (FC) and lysofoscholine (LFC) of 16 carbon chains (FC-16, LFC-16) were more effective in maintaining a mobile fraction of over 80% compared to their counterparts with shorter acyl chain (C12, C14). The significant differences in macroscopic current amplitudes, activation and desensitization rates among the different nAChR-DCs evaluated in the present study allow to dissect which detergent preserves both, agonist activation and ion channel function. Functionality assays using TEVC demonstrated that LFC16, LFC14, and cholate were the most effective detergents in preserving macroscopic ion channel function, however, the nAChR-cholate complex display a significant delay in the ACh-induce channel activation. In summary, these results suggest that the physical properties of the lipid analog detergents (headgroup and acyl chain length) are the most effective in maintaining both the stability and functionality of the nAChR in the detergent solubilized complex.

Human CLC-K Channels Require Palmitoylation of Their Accessory Subunit Barttin to Be Functional

CLC-K/barttin chloride channels are essential for NaCl re-absorption in Henle's loop and for potassium secretion by the stria vascularis in the inner ear. Here, we studied the posttranslational modification of such channels by palmitoylation of their accessory subunit barttin. We found that barttin is palmitoylated in vivo and in vitro and identified two conserved cysteine residues at positions 54 and 56 as palmitoylation sites. Point mutations at these two residues reduce the macroscopic current amplitudes in cells expressing CLC-K/barttin channels proportionally to the relative reduction in palmitoylated barttin. CLC-K/barttin expression, plasma membrane insertion, and single channel properties remain unaffected, indicating that these mutations decrease the number of active channels. R8W and G47R, two naturally occurring barttin mutations identified in patients with Bartter syndrome type IV, reduce barttin palmitoylation and CLC-K/barttin channel activity. Palmitoylation of the accessory subunit barttin might thus play a role in chloride channel dysfunction in certain variants of Bartter syndrome. We did not observe pronounced alteration of barttin palmitoylation upon increased salt and water intake or water deprivation, indicating that this posttranslational modification does not contribute to long term adaptation to variable water intake. Our results identify barttin palmitoylation as a novel posttranslational modification of CLC-K/barttin chloride channels.

Novel Kv7.1-Phosphatidylinositol 4,5-Bisphosphate Interaction Sites Uncovered by Charge Neutralization Scanning

Kv7.1 to Kv7.5 α-subunits belong to the family of voltage-gated potassium channels (Kv). Assembled with the β-subunit KCNE1, Kv7.1 conducts the slowly activating potassium current IKs, which is one of the major currents underlying repolarization of the cardiac action potential. A known regulator of Kv7 channels is the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 increases the macroscopic current amplitude by stabilizing the open conformation of 7.1/KCNE1 channels. However, knowledge about the exact nature of the interaction is incomplete. The aim of this study was the identification of the amino acids responsible for the interaction between Kv7.1 and PIP2. We generated 13 charge neutralizing point mutations at the intracellular membrane border and characterized them electrophysiologically in complex with KCNE1 under the influence of diC8-PIP2. Electrophysiological analysis of corresponding long QT syndrome mutants suggested impaired PIP2 regulation as the cause for channel dysfunction. To clarify the underlying structural mechanism of PIP2 binding, molecular dynamics simulations of Kv7.1/KCNE1 complexes containing two PIP2 molecules in each subunit at specific sites were performed. Here, we identified a subset of nine residues participating in the interaction of PIP2 and Kv7.1/KCNE1. These residues may form at least two binding pockets per subunit, leading to the stabilization of channel conformations upon PIP2 binding.

Alternating Access to the Transmembrane Domain of the ATP-binding Cassette Protein Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7)

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.

The GABRA6 mutation, R46W, associated with childhood absence epilepsy, alters α6β2γ2 and α6β2δ GABAA receptor channel gating and expression

A GABA(A) receptor α6 subunit mutation, R46W, was identified as a susceptibility gene that may contribute to the pathogenesis of childhood absence epilepsy (CAE), but the molecular basis for alteration of GABA(A) receptor function is unclear. The R46W mutation is located in a region homologous to a GABA(A) receptor γ2 subunit missense mutation, R82Q, that is associated with CAE and febrile seizures in humans. To determine how this mutation reduces GABAergic inhibition, we expressed wild-type (α6β2γ2L and α6β2δ) and mutant (α6(R46W)β2γ2L and α6(R46W)β2δ) receptors in HEK 293T cells and characterize their whole-cell and single-channel currents, and surface and total levels. We demonstrated that gating and assembly of both α6(R46W)β2γ2L and α6(R46W)β2δ receptors were impaired. Compared to wild-type currents, α6(R46W)β2γ2L and α6(R46W)β2δ receptors had a reduced current density, α6(R46W)β2γ2L currents desensitized to a greater extent and deactivated at a slower rate, α6(R46W)β2δ receptors did not desensitize but deactivated faster and both α6(R46W)β2γ2L and α6(R46W)β2δ single-channel current mean open times and burst durations were reduced. Surface levels of coexpressed α6(R46W), β2 and δ, but not γ2L, subunits were decreased. 'Heterozygous' coexpression of α6(R46W) and α6 subunits with β2 and γ2L subunits produced intermediate macroscopic current amplitudes by increasing incorporation of wild-type and decreasing incorporation of mutant subunits into receptors trafficked to the surface. Finally, these findings suggest that similar to the γ2(R82Q) mutation, the CAE-associated α6(R46W) mutation could cause neuronal disinhibition and thus increase susceptibility to generalized seizures through a reduction of αβγ and αβδ receptor function and expression.

A shortcut to a skeletal muscle DHPR knock‐in?

A shortcut to a skeletal muscle DHPR knock‐in?

Alternative splicing determines mRNA translation initiation and function of human K2P10.1 K+ channels

Potassium-selective ion channels regulate cardiac and neuronal excitability by stabilizing the resting membrane potential and by modulating shape and frequency of action potentials. The delicate control of membrane voltage requires structural and functional diversity of K+ channel subunits expressed in a given cell. Here we reveal a previously unrecognized biological mechanism. Tissue-specific mRNA splicing regulates alternative translation initiation (ATI) of human K(2P)10.1 K+ background channels via recombination of 5 nucleotide motifs. ATI-dependent expression of full-length protein or truncated subunits initiated from two downstream start codons determines macroscopic current amplitudes and biophysical properties of hK(2P)10.1 channels. The interaction between hK(2P)10.1 mRNA splicing, translation and function increases K+ channel complexity and is expected to contribute to electrophysiological plasticity of excitable cells.

Optimization of the Degenerated Interfacial ATP Binding Site Improves the Function of Disease-related Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, an ATP binding cassette (ABC) protein whose defects cause the deadly genetic disease cystic fibrosis (CF), encompasses two nucleotide binding domains (NBD1 and NBD2). Recent studies indicate that in the presence of ATP, the two NBDs coalesce into a dimer, trapping an ATP molecule in each of the two interfacial composite ATP binding sites (site 1 and site 2). Experimental evidence also suggests that CFTR gating is mainly controlled by ATP binding and hydrolysis in site 2, whereas site 1, which harbors several non-canonical substitutions in ATP-interacting motifs, is considered degenerated. The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. We further showed that the same maneuver also improved the function of WT-CFTR and the most common CF-associated ΔF508 channels, both of which rely on site 2 for gating control. Thus, our results demonstrated that the degenerated site 1 can be rebuilt to complement or support site 2 for CFTR function. Possible approaches for developing CFTR potentiators targeting site 1 will be discussed.

P45 Neonatal outcome in late-preterm hypertensive pregnancies – is there an optimal timing for delivery?

KCNQ1–5 (Kv7.1–7.5) subunits assemble to form a variety of functional K+ channels in the nervous system, heart, and epithelia. KCNQ1 and KCNQ4 homomers and KCNQ2/3 heteromers yield large currents, whereas KCNQ2 and KCNQ3 homomers yield small currents. Since the unitary conductance of KCNQ3 is five- to 10-fold greater than that of KCNQ4 or KCNQ1, these differences are even more striking. To test for differential membrane protein expression, we performed biotinylation and total internal reflection fluorescence imaging assays; however, both revealed only small differences among the channels, leading us to investigate other mechanisms at work. We probed the molecular determinants governing macroscopic current amplitudes, with focus on the turret and pore-loop domains of KCNQ1 and KCNQ3. Elimination of the putative N289 glycosylation site in KCNQ1 reduced current density by ∼56%. A chimera consisting of KCNQ3 with the turret domain (TD) of KCNQ1 increased current density by about threefold. Replacement of the proximal half of the TD in KCNQ3 with that of KCNQ1 increased current density by fivefold. A triple chimera containing the TD of KCNQ1 and the carboxy terminus of KCNQ4 yielded current density 10- or sixfold larger than wild-type KCNQ3 or KCNQ1, respectively, suggesting that the effects on current amplitudes of the TD and the carboxy-terminus are additive. Critical was the role of the intracellular TEA+-binding site. The KCNQ3 (A315T) swap increased current density by 10-fold, and the converse KCNQ1 (T311A) swap reduced it by 10-fold. KCNQ3 (A315S) also yielded greatly increased current amplitudes, whereas currents from mutant A315V channels were very small. The KCNQ3 (A315T) mutation increased the sensitivity of the channels to external Ba2+ block by eight- to 28-fold, consistent with this mutation altering the structure of the selectivity filter. To investigate a structural hypothesis for the effects of these mutations, we performed homology modeling of the pore region of wild-type and mutant KCNQ3 channels, using KvAP as a template. The modeling suggests a critical stabilizing interaction between the pore helix and the selectivity filter that is absent in wild-type KCNQ3 and the A315V mutant, but present in the A315T and A315S mutants. We conclude that KCNQ3 homomers are well expressed at the plasma membrane, but that most wild-type channels are functionally silent, with rearrangements of the pore-loop architecture induced by the presence of a hydroxyl-containing residue at the 315 position “unlocking” the channels into a conductive conformation.

A Conserved Aspartate Determines Pore Properties of Anion Channels Associated with Excitatory Amino Acid Transporter 4 (EAAT4)

Excitatory amino acid transporter (EAAT) glutamate transporters function not only as secondary active glutamate transporters but also as anion channels. Recently, a conserved aspartic acid (Asp(112)) within the intracellular loop near to the end of transmembrane domain 2 was proposed as a major determinant of substrate-dependent gating of the anion channel associated with the glial glutamate transporter EAAT1. We studied the corresponding mutation (D117A) in another EAAT isoform, EAAT4, using heterologous expression in mammalian cells, whole cell patch clamp, and noise analysis. In EAAT4, D117A modifies unitary conductances, relative anion permeabilities, as well as gating of associated anion channels. EAAT4 anion channel gating is characterized by two voltage-dependent gating processes with inverse voltage dependence. In wild type EAAT4, external l-glutamate modifies the voltage dependence as well as the minimum open probabilities of both gates, resulting in concentration-dependent changes of the number of open channels. Not only transport substrates but also anions affect wild type EAAT4 channel gating. External anions increase the open probability and slow down relaxation constants of one gating process that is activated by depolarization. D117A abolishes the anion and glutamate dependence of EAAT4 anion currents and shifts the voltage dependence of EAAT4 anion channel activation by more than 200 mV to more positive potentials. D117A is the first reported mutation that changes the unitary conductance of an EAAT anion channel. The finding that mutating a pore-forming residue modifies gating illustrates the close linkage between pore conformation and voltage- and substrate-dependent gating in EAAT4 anion channels.