Deactivation Time Research Articles

Block of Kainate Receptor Desensitization Uncovers a Key Trafficking Checkpoint

A prominent feature of ionotropic glutamate receptors from the AMPA and kainate subtypes is their profound desensitization in response to glutamate-a process thought to protect the neuron from overexcitation. In AMPA receptors, it is well established that desensitization results from rearrangements of the interface formed between agonist-binding domains of adjacent subunits; however, it is unclear how this mechanism applies to kainate receptors. Here we show that stabilization of the binding domain dimer by the generation of intermolecular disulfide bonds apparently blocked desensitization of the kainate receptor GluR6. This result establishes a common desensitization mechanism in both AMPA and kainate receptors. Surprisingly, however, surface expression of these nondesensitizing mutants was drastically reduced and did not depend on channel activity. Therefore, in addition to its role at the synapse, we now propose an intracellular role for desensitization in controlling maturation and trafficking of glutamate receptors.

Mallotoxin Is a Novel HumanEther-a-go-go-Related Gene (hERG) Potassium Channel Activator

Human ether-a-go-go-related gene (hERG) encodes a rapidly activating delayed rectifier potassium channel that plays important roles in cardiac action potential repolarization. Although many drugs and compounds block hERG channels, activators of the channel have only recently been described. Three structurally diverse synthetic compounds have been reported to activate hERG channels by altering deactivation or inactivation or by unidentified mechanisms. Here, we describe a novel, naturally occurring hERG channel activator, mallotoxin (MTX). The effects of MTX on hERG channels were investigated using the patch-clamp technique. MTX increased both step and tail hERG currents with EC(50) values of 0.34 and 0.52 microM, respectively. MTX leftward shifted the voltage dependence of hERG channel activation to less depolarized voltages ( approximately 24 mV at 2.5 microM). In addition, MTX increased hERG deactivation time constants. MTX did not change the half-maximal inactivation voltage of the hERG channel, but it reduced the slope of the voltage-dependent inactivation curve. All of these factors contribute to the enhanced activity of hERG channels. During a voltage-clamp protocol using prerecorded cardiac action potentials, 2.5 microM MTX increased the total potassium ions passed through hERG channels by approximately 5-fold. In conclusion, MTX activates hERG channels through distinct mechanisms and with significantly higher potency than previously reported hERG channel activators.

Differential Regulation of Two Distinct Voltage-Dependent Sodium Currents by Group III Metabotropic Glutamate Receptor Activation in Insect Pacemaker Neurons

Using whole cell patch-clamp technique and immunocytochemistry on adult dorsal unpaired median (DUM) neurons isolated from the cockroach Periplaneta americana CNS, we reported the characterization of a native mGluR, sharing pharmacological properties with vertebrate metabotropic glutamate receptor III (mGluRIII) that regulated voltage-dependent sodium current (I(Na)). The global I(Na) was dissociated by means of l-glutamate sensitivity, deactivation time constant, voltage dependence of activation and inactivation, recovery from inactivation, and intracellular regulation process. These two currents were respectively designated I(Na1) and I(Na2) for l-glutamate-sensitive and -insensitive sodium currents. l-glutamate selectively reduced I(Na1) by an increase of intracellular cAMP level. Using different activators and/or inhibitors of G proteins and cAMP/PKA cascade, together with St-Ht31 (an inhibitor of PKA binding to AKAP) and AKAP-79 antibodies, we established that mGluRIII was linked to I(Na1) by a Gi/o and a suspected Gs protein. According to the activated signaling pathway, l-glutamate elevated the cAMP level, which thereby activated cytosolic PKA and released PKA bound to AKAP. As expected from both biophysical and pharmacological studies, we showed that, through an inhibition of I(Na1), l-glutamate increased DUM neuron spontaneous electrical activity. These results indicated that such mGluRIII-activated dual processes provided a new physiological control of pacemaker neuronal firing.

KCNQ/M‐currents contribute to the resting membrane potential in rat visceral sensory neurons

The M-current is a slowly activating, non-inactivating potassium current that has been shown to be present in numerous cell types. In this study, KCNQ2, Q3 and Q5, the molecular correlates of M-current in neurons, were identified in the visceral sensory neurons of the nodose ganglia from rats through immunocytochemical studies. All neurons showed expression of each of the three proteins. In voltage clamp studies, the cognition-enhancing drug linopirdine (1-50 microM) and its analogue, XE991 (10 microM), quickly and irreversibly blocked a small, slowly activating current that had kinetic properties similar to KCNQ/M-currents. This current activated between -60 and -55 mV, had a voltage-dependent activation time constant of 208 +/- 12 ms at -20 mV, a deactivation time constant of 165 +/- 24 ms at -50 mV and V1/2 of -24 +/- 2 mV, values which are consistent with previous reports for endogenous M-currents. In current clamp studies, these drugs also led to a depolarization of the resting membrane potential at values as negative as -60 mV. Flupirtine (10-20 microM), an M-current activator, caused a 3-14 mV leftward shift in the current-voltage relationship and also led to a hyperpolarization of resting membrane potential. These data indicate that the M-current is present in nodose neurons, is activated at resting membrane potential and that it is physiologically important in regulating excitability by maintaining cells at negative voltages.

An Interaction Involving an Arginine Residue in the Cytoplasmic Domain of the 5-HT3A Receptor Contributes to Receptor Desensitization Mechanism

A large cytoplasmic domain accounts for approximately one-third of the entire protein of one superfamily of ligand-gated membrane ion channels, which includes nicotinic acetylcholine (nACh), gamma-aminobutyric acid type A (GABA(A)), serotonin type 3 (5-HT3), and glycine receptors. Desensitization is one functional feature shared by these receptors. Because most molecular studies of receptor desensitization have focused on the agonist binding and channel pore domains, relatively little is known about the role of the large cytoplasmic domain (LCD) in this process. To address this issue, we sequentially deleted segments of the LCD of the 5-HT3A receptor and examined the function of the mutant receptors. Deletion of a small segment that contains three amino acid residues (425-427) significantly slowed the desensitization kinetics of the 5-HT3A receptor. Both deletion and point mutation of arginine 427 altered desensitization kinetics in a manner similar to that of the (425-427) deletion without significantly changing the apparent agonist affinity. The extent of receptor desensitization was positively correlated with the polarity of the amino acid residue at 427: the desensitization accelerates with increasing polarity. Whereas the R427L mutation produced the slowest desensitization, it did not significantly alter single channel conductance of 5-HT3A receptor. Thus, the arginine 427 residue in the LCD contributes to 5-HT3A receptor desensitization, possibly through forming an electrostatic interaction with its neighboring residues. Because the polarity of the amino acid residue at 427 is highly conserved, such a desensitization mechanism may occur in other members of the Cys-loop family of ligand-gated ion channels.

Interaction between cyclodextrin and neuronal membrane results in modulation of GABAA receptor conformational transitions

Cyclodextrins (CDs) are nanostructures widely applied in biotechnology and chemistry. Owing to partially hydrophobic character, CDs interact with biological membranes. While the mechanisms of CDs interactions with lipids were widely studied, their effects on proteins are less understood. In the present study we investigated the effects of beta cyclodextrin (betaCD) on GABA(A) receptor (GABA(A)R) gating. To reliably resolve the kinetics of conformational transitions, currents were elicited by ultrafast gamma-aminobutyric acid (GABA) applications to outside-out patches from rat cultured hippocampal neurons. betaCD increased the amplitude of responses to saturating GABA concentration ([GABA]) in a dose-dependent manner and this effect was accompanied by profound alterations in the current kinetics. Current deactivation was slowed down by betaCD but this effect was biphasic with a maximum at around 0.5 mM betaCD. While the fast deactivation time constant was monotonically slowed down within considered betaCD concentration range, the slow component first increased and then, at millimolar betaCD concentration, decreased. The rate and extent of desensitization was decreased by betaCD in a dose-dependent manner. The analysis of current responses to nonsaturating [GABA] indicated that betaCD affected the GABA(A)R agonist binding site by slowing down the unbinding rate. Modulation of GABA(A)R desensitization and binding showed different concentration-dependence suggesting different modualtory sites with higher affinity of the latter one. All the betaCD effects were fully reversible indicating that cholesterol uptake into betaCD was not the primary mechanism. We conclude that betaCD is a strong modulator of GABA(A)R conformational transitions.

Mechanisms for Picrotoxin Block of α2 Homomeric Glycine Receptors

It is well known that the convulsant alkaloid picrotoxin (PTX) can inhibit neuronal gamma-aminobutyric acid (GABA) and homomeric glycine receptors (GlyR). However, the mechanism for PTX block of alpha(2) homomeric GlyR is still unclear compared with that of alpha(1) homomeric GlyR, GABA(A), and GABA(C) receptors. Furthermore, PTX effects on GlyR kinetics have been poorly explored at the single-channel level. Hence, we used the patch-clamp technique in the outside-out configuration to investigate the mechanism of PTX suppression of currents carried by alpha(2) homomeric GlyRs stably transfected into Chinese hamster ovary cells. PTX inhibited the alpha(2) homomeric GlyR current elicited by glycine in a concentration-dependent and voltage-independent manner. Both competitive and noncompetitive mechanisms were observed. PTX decreased the mean open time of the GlyR channel in a concentration-dependent manner, suggesting that PTX can block channel openings and bind to the receptor in the open channel conformation. When PTX and glycine were co-applied, a small rebound current was observed during drug washout. Application of PTX during the deactivation phase of glycine-induced currents eliminated the rebound current and accelerated the deactivation time course in a concentration-dependent manner. PTX could not bind to the unbound conformation of GlyR, but could be trapped at its binding site when the channel closed during glycine dissociation. Based on these observations, we propose a kinetic Markov model in which PTX binds to the alpha(2) homomeric GlyR in both the open channel state and the fully liganded closed state. Our data suggest a new allosteric mechanism for PTX inhibition of wild-type homomeric alpha(2) GlyR.

Autonomous artificial nanomotor powered by sunlight

Light excitation powers the reversible shuttling movement of the ring component of a rotaxane between two stations located at a 1.3-nm distance on its dumbbell-shaped component. The photoinduced shuttling movement, which occurs in solution, is based on a "four-stroke" synchronized sequence of electronic and nuclear processes. At room temperature the deactivation time of the high-energy charge-transfer state obtained by light excitation is approximately 10 micros, and the time period required for the ring-displacement process is on the order of 100 micros. The rotaxane behaves as an autonomous linear motor and operates with a quantum efficiency up to approximately 12%. The investigated system is a unique example of an artificial linear nanomotor because it gathers together the following features: (i) it is powered by visible light (e.g., sunlight); (ii) it exhibits autonomous behavior, like motor proteins; (iii) it does not generate waste products; (iv) its operation can rely only on intramolecular processes, allowing in principle operation at the single-molecule level; (v) it can be driven at a frequency of 1 kHz; (vi) it works in mild environmental conditions (i.e., fluid solution at ambient temperature); and (vii) it is stable for at least 10(3) cycles.

Characterization of M-Current in Ventral Tegmental Area Dopamine Neurons

M-current (I(M)) is a voltage-gated potassium current (KCNQ type) that affects neuronal excitability and is modulated by some drugs of abuse. Ventral tegmental area (VTA) dopamine (DA) neurons are important for the reinforcing effects of drugs of abuse. Therefore we studied I(M) in acutely dissociated rat DA VTA neurons with nystatin-perforated patch recording. The standard deactivation protocol was used to measure I(M) during voltage-clamp recording with hyperpolarizing voltage steps to -65 mV (in 10-mV increments) from a holding potential of -25 mV. I(M) amplitude was voltage dependent and maximal current amplitude was detected at -45 mV. The deactivation time constant of I(M) was voltage dependent and became shorter at more negative voltages. The I(M)/KCNQ antagonist XE991 (0.3-30 microM) caused a concentration-dependent reduction in I(M) amplitude with an IC(50) of 0.71 microM. Tetraethylammonium (TEA, 0.3-10 mM) caused a concentration-dependent inhibition of I(M) with an IC(50) of 1.56 mM. In current-clamp recordings, all DA VTA neurons were spontaneously active. Analysis of evoked action potential shape indicated that XE991 (1-10 microM) reduced the fast and slow components of the spike afterhyperpolarization (AHP) without affecting the middle component of the AHP. Action potential amplitude, duration, and threshold were not affected by XE991. In addition, 10 microM XE991 significantly shortened the interspike intervals in evoked spike trains. In conclusion, I(M) is active near threshold in DA VTA neurons, is blocked by XE991 (10 microM) and TEA (10 mM), may contribute to the shape of the AHP, and may decrease excitability of these neurons.

Temporal cDNA microarray analysis of gene expression in human hepatocellular carcinoma upon radiation exposure

Radiotherapy is not commonly used for the treatment of human hepatocellular carcinoma, due to its poor response rate and poor tolerance of normal liver to ionizing radiation. Recently developed microarray technology makes it possible to verify genes responsive to anticancer therapy of human cancers by simultaneous analysis of gene expression profiles. In the present study, the expression profile of radiation-responsive genes in human hepatocellular carcinoma was evaluated through time-dependent cDNA microarray analysis of expressional variation, following exposure to ionizing radiation. Upon exposure to radiation, more than 13% of genes in both radiation-resistant and -sensitive cells responded to radiation. Time-dependent analysis of radiation-responsive genes revealed that, irrespective of radiation sensitivity, greatly different subsets of genes sequentially participated in cellular response to radiation at their specific activation or deactivation time points. The majority of radiation-responsive genes were differentially but not commonly expressed between radiation-resistant and -sensitive cells. When these differentially regulated genes were classified according to their physiological and functional characteristics and radiation sensitivity, it was prominently obvious that DNA repair-promoting genes were up-regulated in radio-resistant cells and down-regulated or unchanged in radiation-sensitive cells. The present findings indicate that different subsets of genes are sequentially working and DNA repair capacity may control the radiation sensitivity of human hepatocellular carcinoma cells more than any other physiological factor.

Phosphorylation of the A-kinase-anchoring Protein Yotiao Contributes to Protein Kinase A Regulation of a Heart Potassium Channel

Regulation of the heart by the sympathetic nervous system, fundamental to the physiological response to stress and exercise, requires coordinated phosphorylation of multiple downstream molecular targets, including the I(Ks) (slowly activating potassium current) channel. Sympathetic nervous system stimulation increases intracellular cAMP for which targeted regulation is directed in large part by distinct scaffold or anchoring proteins. Yotiao is an A-kinase-anchoring protein (AKAP) that recruits the cyclic AMP-dependent protein kinase (protein kinase A (PKA)) and protein phosphatase 1 to the carboxyl terminus of the I(Ks) channel to form a molecular complex and control its phosphorylation state, crucial to the cardiac cellular response to sympathetic nervous system stimulation. Here we report that Yotiao itself is a substrate for PKA phosphorylation, and we identify a Yotiao amino-terminal (N-T) residue (Ser-43) that is PKA-phosphorylated in response to beta-adrenergic receptor stimulation. The replacement of Ser-43 by Ala ablates the PKA phosphorylation of N-T Yotiao and markedly diminishes the functional response of the wild type and pseudo-phosphorylated I(Ks) channel to cAMP but neither prevents the PKA phosphorylation of KCNQ1 nor its binding to Yotiao. These results suggest, for the first time, a critical role for the PKA phosphorylation of an AKAP in the functional regulation of an ion channel protein and postphosphorylation allosteric modulation of the I(Ks) channel by Yotiao.

Electroluminescent properties of organic light-emitting diodes using BAlq and Alq 3 co-evaporation layer

We applied co-evaporation films (BAlq:Alq 3) as an emission layer of OLED and studied their energy carrier transfer process. Energy was partially transferred from excited BAlq to Alq 3 by PL spectra. We also investigated its light-emission mechanism using a co-evaporation layer in OLED. Energy transfer occurs from NPD to Alq 3 or suppression of electron injection into NPD takes place. Furthermore, the hole is injected into the BAlq within 20-nm distance from the NPD/BAlq interface injected holes can move to as much as 10 nm from the interface. Doping Alq 3 in BAlq changes the number of electrons that are injected into BAlq because of its electron trapping against BAlq. We also suggest that the rate constant of energy transfer from NPD to Alq 3 is shorter than the radiative deactivation time of NPD. The number of NPD excitons generated by recombination is less than that of deactivating Alq 3.

Neuronal Kir3.1/Kir3.2a channels coupled to serotonin 1A and muscarinic m2 receptors are differentially modulated by the ‘short’ RGS3 isoform

‘Regulators of G protein signaling’ (RGS proteins) have profound effects on ion channels regulated by G protein-coupled receptor (GPCR) signaling, including the G protein-gated inwardly rectifying K + (GIRK) channels that inhibit excitability of neuronal, endocrine, and cardiac cells. Here we describe the effects of an alternatively spliced ‘short’ RGS3 isoform (RGS3s) in comparison to RGS4, on the temporal and steady-state gating properties of neuronal GIRK channels (Kir3.1/Kir3.2a) activated by either serotonin 1A (5-HT 1A) receptors or muscarinic m2 receptors expressed in Chinese hamster ovary (CHO-K1) cells. RGS3s is abundantly expressed in brain and contains a unique short N-terminus via alternative splicing that distinguishes it from other RGS3 isoforms as well as other members of the B/R4 RGS gene subfamily. Our results indicate that RGS3s and RGS4 similarly affect the temporal and steady-state gating properties of 5-HT 1A receptor-coupled Kir3.1/Kir3.2a channels, but differentially modulate muscarinic m2 receptor-coupled channels. RGS3s caused a significant ∼45% reduction in the maximal acetylcholine (ACh)-evoked GIRK current amplitude and a marked shift in the steady-state ACh dose–response relation indicative of a reduction in functionally coupled m2 receptor-GIRK channel complexes. Yet RGS3s still accelerated the m2 receptor-dependent GIRK activation, deactivation, and acute desensitization time course consistent with the RGS-enhanced GAP activity that was also observed with RGS4. Several mechanisms that may contribute to the receptor-dependent effects of RGS3s are discussed with particular attention to the role of the distinct N-terminal domain. Our findings highlight the potential impact of selective RGS-GPCR interactions on neuronal GIRK channel function that may affect the properties of inhibitory postsynaptic potentials activated by different GPCR-GIRK channel complexes.

Modulating effect of ginseng saponins on heterologously expressed HERG currents in Xenopus oocytes

To examine the effects of ginseng saponins on the heterologously expressed human ether-a-go-go related gene (HERG) that encodes the rapid component of the delayed rectifier K+ channel. A two-electrode voltage clamp technique was used. HERG currents were recorded in Xenopus oocytes injected with HERG cRNA. Crude saponins of Korean red ginseng (GS) induced a minimal increase of the maximal HERG conductance without changes in the voltage-dependent HERG current activation and inactivation curves. GS, however, decelerated HERG current deactivation in a concentration-dependent manner, which was more noticeable with panaxitriol (PT) than panaxidiol (PD). Consistently, ginseng saponins increased the HERG deactivation time constants with the order of potency of Rg1 (a major component of PT)>Rf1>Rb1 (a major component of PD). Re had little effect on HERG deactivation. During a cardiac action potential, GS increased the outward HERG current. Ginseng saponins enhance HERG currents, which could be in part a possible mechanism of the shortening cardiac action potential of ginseng saponins.

Differential Effects of β1 and β2 Subunits on BK Channel Activity

High conductance, calcium- and voltage-activated potassium (BK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (β) subunits. β1 and β2 subunits increase apparent channel calcium sensitivity. The β1 subunit also decreases the voltage sensitivity of the channel and the β2 subunit produces an N-type inactivation of BK currents. We further characterized the effects of the β1 and β2 subunits on the calcium and voltage sensitivity of the channel, analyzing the data in the context of an allosteric model for BK channel activation by calcium and voltage (Horrigan and Aldrich, 2002). In this study, we used a β2 subunit without its N-type inactivation domain (β2IR). The results indicate that the β2IR subunit, like the β1 subunit, has a small effect on the calcium binding affinity of the channel. Unlike the β1 subunit, the β2IR subunit also has no effect on the voltage sensitivity of the channel. The limiting voltage dependence for steady-state channel activation, unrelated to voltage sensor movements, is unaffected by any of the studied β subunits. The same is observed for the limiting voltage dependence of the deactivation time constant. Thus, the β1 subunit must affect the voltage sensitivity by altering the function of the voltage sensors of the channel. Both β subunits reduce the intrinsic equilibrium constant for channel opening (L 0). In the allosteric activation model, the reduction of the voltage dependence for the activation of the voltage sensors accounts for most of the macroscopic steady-state effects of the β1 subunit, including the increase of the apparent calcium sensitivity of the BK channel. All allosteric coupling factors need to be increased in order to explain the observed effects when the α subunit is coexpressed with the β2IR subunit.

Reduced voltage dependence of inactivation in the SCN5A sodium channel mutation delF1617

Deletion of a phenylalanine at position 1617 (delF1617) in the extracellular linker between segments S3 and S4 in domain IV of the human heart Na(+) channel (hH1a) has been tentatively associated with long QT syndrome type 3 (LQT3). In a mammalian cell expression system, we compared whole cell, gating, and single-channel currents of delF1617 with those of wild-type hH1a. The half points of the peak activation-voltage curve for the two channels were similar, as were the deactivation time constants at hyperpolarized test potentials. However, delF1617 demonstrated a significant negative shift of -7 mV in the half point of the voltage-dependent Na(+) channel availability curve compared with wild type. In addition, both the time course of decay of Na(+) current (I(Na)) and two-pulse development of inactivation of delF1617 were faster at negative test potentials, whereas they tended to be slower at positive potentials compared with wild type. Mean channel open times for delF1617 were shorter at potentials <0 mV, whereas they were longer at potentials >0 mV compared with wild type. Using anthopleurin-A, a site-3 toxin that inhibits movement of segment S4 in domain IV (S4-DIV), we found that gating charge contributed by the S4-DIV in delF1617 was reduced 37% compared with wild type. We conclude that deletion of a single amino acid in the S3-S4 linker of domain IV alters the voltage dependence of fast inactivation via a reduction in the gating charge contributed by S4-DIV and can cause either a gain or loss of I(Na), depending on membrane potential.

Molecular Determinants of Glycine-Independent Desensitization of NR1/NR2A Receptors

Glycine-independent desensitization is thought to be an important regulatory mechanism for the function of N-methyl-D-aspartate (NMDA) receptors. Previous studies have suggested that the molecular determinants for glycine-independent desensitization are located at two distinct domains of NR2A, i.e., the amino-terminal domain (ATD) and the pre-M1 domain. Since the glycine-independent desensitization described in these earlier studies was a mixture of glycine-independent desensitization and zinc-dependent apparent desensitization, the exact role of these two domains in glycine-independent desensitization remains in question. In the present study, we show that deletion of the ATD of NR2A or mutating the pre-M1 region of NR2A causes no detectable changes in the degree or the time constant of glycine-independent desensitization. Therefore, the ATD and the pre-M1 domain of NR2A play no significant role in glycine-independent desensitization of NR1/NR2A receptors. On the other hand, several residues in the lurcher motif of either NR1 or NR2A are critical for the glycine-independent desensitization of NR1/NR2A receptors. In addition to NR1a(A653T) and NR2A(A651T), NR1a(T648C), NR1a(A649C), NR2A(T646C), and NR2A(A647C) show significantly reduced glycine-independent desensitization. Since all these mutations also alter the proton sensitivity or deactivation time constants of NR1/NR2A receptors, our data suggest that the channel gating and desensitization of NMDA receptors share common molecular determinants and that the lurcher motif of NR1 and NR2A is critical for both processes.

Effects of Oxypeucedanin on hKv1.5 and Action Potential Duration

A furocoumarin derivative, oxypeucedanin, was purified from Angelica dahurica, and its effects on the human Kv1.5 (hKv1.5) channel and on the cardiac action potential duration (APD), were examined using the patch-clamp technique and the conventional microelectrode technique. Oxypeucedanin inhibited the hKv1.5 current in a concentration-dependent manner, with an IC(50) value of 76 nM, while it had no effect on human eag-related gene (HERG) current. Oxypeucedanin induced an initial fast decline of hKv1.5 current during depolarizations. The inhibition of hKv1.5 channel by oxypeucedanin was voltage-dependent, especially at depolarizing pulses between -40 and 0 mV which corresponds to the voltage range of the channel's opening. Oxypeucedanin also slowed the deactivation time course, resulting in a tail crossover phenomenon. Additionally, oxypeucedanin prolonged the APD of rat atrial and ventricular muscles in a dose-dependent manner. These results suggest that oxypeucedanin is a kind of open-channel blocker of the hKv1.5 channel and it prolongs the APD; therefore, it is an excellent candidate as an antiarrhythmic drug for atrial fibrillation.

Novel C-terminus frameshift mutation, 1122fs/147, of HERG in LQT2: additional amino acids generated by frameshift cause accelerated inactivation

Objective. – The function of the C-terminus region of the human ether-a-go-go related gene (HERG) has not been well characterized except for its involvement in trafficking. To understand further the role of C-terminus region, we performed a functional analysis of a novel frameshift mutation (1122fs/147) identified in a Japanese long QT syndrome 2 (LQT2) patient who had recurrent episodes of syncope. Methods. – Wild type (WT) and mutant HERG plasmids were transfected into human embryonic kidney (HEK-293) cells, and whole-cell current was recorded by the patch-clamp technique. Confocal microscopy was performed to examine the membrane distribution of channel protein using a green fluorescent protein tagged to the N-terminus of HERG. Results. – The mutant 1122fs/147 alone could express current, but reduced density by 74% of control. No dominant negative effect was noted with co-expression of WT and 1122fs/147. Activation and deactivation time constants were not changed, while inactivation was accelerated in 1122fs/147 compared to WT, and V 1/2 of steady-state inactivation curve shifted by 11 mV in the negative direction. Current density of 1123stop mutant revealed 49% reduction compared to WT and showed no shift in steady-state inactivation. Confocal microscopy revealed reduced protein expression on the cell surface both in 1122fs/147 and 1123stop mutants compared to WT. Conclusion. – Frameshift mutation at the C-terminus region with additional 147 amino acids evoked a loss of function of the HERG channel. A negative shift in steady-state inactivation induced by the additional 147 amino acids and trafficking defect contribute to a reduced current amplitude of 1122fs/147.

Differential Effects on Current Kinetics by Point Mutations in the lurcher Motif of NR1/NR2A Receptors

The extracellular proton site has emerged as the key site in regulating N-methyl-d-aspartate (NMDA) receptor function. However, the molecular determinants for the proton-sensitive gating of NMDA receptors are still not clearly delineated. The highly conserved lurcher motif plays an important role in determining the proton sensitivity of NMDA receptors. Mutations of several residues in the lurcher motif of either NR1 or NR2A significantly reduce proton sensitivity of recombinant NR1/NR2A receptors. It remains uncertain how these residues play a role in proton inhibition of NMDA receptors. Mutations of these residues could directly reduce the proton affinity. Alternatively, they could alter the proton IC(50) indirectly by increasing channel open probability. In the present study, we recorded the macroscopic NMDA currents in HEK 293 cells with a piezo-based rapid solution exchange system. We show that zinc slows the deactivation of NR1a(A653T)/NR2A receptors and NR1a/NR2A(A651T) receptors. However, NR1a(T648C)/NR2A, NR1a/NR2A(T646C), NR1a(A649C)/NR2A, NR1a/NR2A(A647C), and NR1a(A653T)/NR2A exhibit significantly slower rise time and deactivation time constants under nominally zinc-free conditions. Our data suggest that the channel open probability for these mutant receptors may be significantly increased. The reduction in proton sensitivity by these mutations could be accounted for, at least partially, by the increased channel open probability. In contrast, NR1a/NR2A(A651T) exhibits normal macroscopic currents, suggesting that the reduction of proton sensitivity by this mutation cannot be attributed to any significant change of open probability. Further experiments are needed to determine the exact role of this residue in proton-sensitive gating of NMDA receptors.