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Background Bi-allelic CYP24A1 mutations can cause idiopathic infantile hypercalcemia (IIH), adult-onset nephrocalcinosis, and possibly bone metabolism disturbances. It is currently unclear if heterozygous carriers experience clinical problems or biochemical abnormalities. Our objective is to gain insight in the biochemical profile and health problems in CYP24A1 heterozygotes. Study design Cross-sectional evaluation of participants. Data of previously reported carriers are reviewed. Setting and participants Outpatient clinic of a tertiary care hospital. Participants were eight family members of an infant with a well-characterized homozygous CYP24A1 mutation c.1186C > T p.(Arg396Trp). Outcomes Serum vitamin D metabolites. Symptoms or biochemical signs of hypercalcemia, hypercalciuria or nephrocalcinosis. Bone health in heterozygous as compared to wild type (WT) subjects. Measurements Genotyping by Sanger sequencing; vitamin D metabolites by liquid chromatography tandem mass spectrometry; renal, calcium and bone markers by biochemical analyses; presence of nephrocalcinosis by renal ultrasound; bone health by dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. Results Six participants were heterozygous carriers of the mutation. None of the heterozygous subjects had experienced IIH. One had a documented history of nephrolithiasis, two others had complaints compatible with this diagnosis. No major differences between WT and heterozygous subjects were found regarding bone health, serum or urinary calcium or 25OHD/24,25(OH)2D ratio. Literature reports on three out of 33 heterozygous cases suffering from IIH. In all three, the 25OHD/24,25(OH)2D ratio was highly elevated. Nephrocalcinosis was frequently reported in family members of IIH cases. Limitations Small sample size, lack of a large control group. Conclusions Our and literature data suggest that most heterozygous CYP24A1 mutation carriers have a normal 25OHD/24,25(OH)2D ratio, are usually asymptomatic and have a normal skeletal status but may possibly be at increased risk of nephrocalcinosis. A review of the available literature suggests that an elevated 25OHD/24,25(OH)2D ratio may be associated with symptoms of IHH, irrespective of carrier status.

Voltage-dependent K(+) channels transfer the voltage sensor movement into gate opening or closure through an electromechanical coupling. To test functionally whether an interaction between the S4-S5 linker (L45) and the cytoplasmic end of S6 (S6(T)) constitutes this coupling, the L45 in hKv1.5 was replaced by corresponding hKv2.1 sequence. This exchange was not tolerated but could be rescued by also swapping S6(T). Exchanging both L45 and S6(T) transferred hKv2.1 kinetics to an hKv1.5 background while preserving the voltage dependence. A one-by-one residue substitution scan of L45 and S6(T) in hKv1.5 further shows that S6(T) needs to be alpha-helical and forms a "crevice" in which residues I422 and T426 of L45 reside. These residues transfer the mechanical energy onto the S6(T) crevice, whereas other residues in S6(T) and L45 that are not involved in the interaction maintain the correct structure of the coupling.

The Kv1–4 families of K+ channels contain a tandem proline motif (P XP) in the S6 helix that is crucial for channel gating. In human Kv1.5, replacing the first proline by an alanine resulted in a nonfunctional channel. This mutant was rescued by introducing another proline at a nearby position, changing the sequence into AVPP. This resulted in a channel that activated quickly (ms range) upon the first depolarization. However, thereafter, the channel became trapped in another gating mode that was characterized by slow activation kinetics (s range) with a shallow voltage dependence. The switch in gating mode was observed even with very short depolarization steps, but recovery to the initial “fast” mode was extremely slow. Computational modeling suggested that switching occurred during channel deactivation. To test the effect of the altered P XP sequence on the mobility of the S6 helix, we used molecular dynamics simulations of the isolated S6 domain of wild type (WT) and mutants starting from either a closed or open conformation. The WT S6 helix displayed movements around the P XP region with simulations starting from either state. However, the S6 with a AVPP sequence displayed flexibility only when started from the closed conformation and was rigid when started from the open state. These results indicate that the region around the P XP motif may serve as a “hinge” and that changing the sequence to AVPP results in channels that deactivate to a state with an alternate configuration that renders them “reluctant” to open subsequently.

During two hours of observation, a female Blackbird made approximately 30 attempts to catch fish, 20 of which were successful. The bird watched and followed a minnow shoal in shallow parts of the river, hopping from stone to stone in a way that resembled its normal behaviour when catching earthworms. The bird only caught small individuals (5–7 cm). Larger individuals were present in the river but were not seen within the shoal and were not attacked.

Co-assembly of KCNQ1 α-subunits with KCNE1 β-subunits results in the channel complex underlying the cardiac IKs current in vivo. Like other voltage-gated K+ channels, KCNQ1 has a tetrameric configuration. The S6 segment of each subunit lines the ion channel pore with the lower part forming the activation gate. To determine residues involved in protein–protein interactions in the C-terminal part of S6 (S6T), alanine and tryptophan perturbation scans were performed from residue 348–362 in the KCNQ1 channel. Several residues were identified to be relevant in channel gating, as substitutions affected the activation and/or deactivation process. Some mutations (F351A and V355W) drastically altered the gating characteristics of the resultant KCNQ1 channel, to the point of mimicking the IKs current. Furthermore, mutagenesis of residue L353 to an alanine or a charged residue impaired normal channel closure upon hyperpolarization, generating a constitutively open phenotype. This indicates that the L353 residue is essential for stabilizing the closed conformation of the channel gate. These findings together with the identification of several LQT1 mutations in the S6 C-terminus of KCNQ1 underscore the relevance of this region in KCNQ1 and IKs channel gating.

The KChIP1b splice variant has been shown to induce slow recovery from inactivation for Kv4.2 whereas KChIP1a enhanced the recovery. Both splice variants differ only by the insertion of the exon1b, rich in aromatic residues (5/11). We analysed in detail the modifications of Kv4.2 gating induced by the KChIP1b splice variant and the role for the aromatic cluster in KChIP1b in inducing these changes. By substituting alanine for the aromatic residues individually or in combination, we could convert the KChIP1b recovery behaviour into that of KChIP1a. The replacement of one or two aromatic residues resulted in a partial restitution of the KChIP1a recovery behaviour. When three aromatic residues were replaced in the exon1b, the recovery from inactivation was fast with time constants that were similar to those obtained with KChIP1a. Moreover, similar findings were observed for closed state inactivation and for the voltage dependence of inactivation. Thus, reduction of the side chain bulkiness in exon1b resulted in the conversion of the KChIP1b phenotype into the KChIP1a phenotype. These results indicate that the aromatic cluster in exon1b modulates the transitions towards and from the closed inactivated states and the steady state distribution over the respective states.

Human ether-a-go-go-related gene (HERG) potassium channels contribute to the repolarization of the cardiac action potential and display unique gating properties with slow activation and fast inactivation kinetics. Deletions in the N-terminal ‘proximal’ domain (residues 135–366) have been shown to induce hyperpolarizing shifts in the voltage dependence of activation, suggesting that it modulates activation. However, we did not observe a hyperpolarizing shift with a subtotal deletion designed to preserve the local charge distribution, and other deletions narrowed the region to the KIKER containing sequence 362–372. Replacing the positively charged residues of this sequence by negative ones (EIEEE) resulted in a −45mV shift of the voltage dependence of activation. The shifts were intermediate for individual charge reversals, whereas E365R resulted in a positive shift. Furthermore, the shifts in the voltage dependence were strongly correlated with the net charge of the KIKER region. The apparent speeding of the activation was attributable to the shifted voltage dependence of activation. Additionally, the introduction of negative charges accelerated the intermediate voltage-independent forward rate constant. We propose that the modulatory effects of the proximal domain on HERG gating are largely electrostatic, localized to the charged KIKER sequence.

The subunit Kv6.3 encodes a voltage-gated potassium channel belonging to the group of electrically silent Kv subunits, i.e. subunits that do not form functional homotetrameric channels. The lack of current, caused by retention in the endoplasmic reticulum (ER), was overcome by coexpression with Kv2.1. To investigate whether a specific section of Kv6.3 was responsible for ER retention, we constructed chimeric subunits between Kv6.3 and Kv2.1, and analysed their subcellular localization and functionality. The results demonstrate that the ER retention of Kv6.3 is not caused by the N-terminal A and B box (NAB) domain nor the intracellular N- or C-termini, but rather by the S1–S6 core protein. Introduction of individual transmembrane segments of Kv6.3 in Kv2.1 was tolerated, with the exception of S6. Indeed, introduction of the S6 domain of Kv6.3 in Kv2.1 was enough to cause ER retention, which was due to the C-terminal section of S6. The S4 segment of Kv6.3 could act as a voltage sensor in the Kv2.1 context, albeit with a major hyperpolarizing shift in the voltage dependence of activation and inactivation, apparently caused by the presence of a tyrosine in Kv6.3 instead of a conserved arginine. This study suggests that the silent behaviour of Kv6.3 is largely caused by the C-terminal part of its sixth transmembrane domain that causes ER retention of the subunit.

Voltage-gated K+ channels play a central role in the modulation of excitability. In these channels, the voltage-dependent movement of the voltage sensor (primarily S4) is coupled to the (S6) gate that opens the permeation pathway. Because of the tetrameric structure, such coupling could occur within each subunit or between adjacent subunits. To discriminate between these possibilities, we analyzed various combinations of a S4 mutation (R401N) and a S6 mutation (P511G) in hKv1.5, incorporated into tandem constructs to constrain subunit stoichiometry. R401N shifted the voltage dependence of activation to negative potentials while P511G did the opposite. When both mutations were introduced in the same α-subunit of the tandem, the positive shift of P511G was compensated by the negative shift of R401N. With each mutation in a separate subunit of a tandem, this compensation did not occur. This suggests that for Kv channels, the coupling between voltage sensing and gating reflects primarily an intrasubunit interaction.

The beta-subunits of the KChIP family modulate properties and expression level of Kv4 channels. We report the cloning of the first splice variant of KChIP1 (KChIP1b) which contains an extra exon, rich in aromatic residues, in the amino terminus. Both splice variants interacted equally well with Kv4.2 subunits based on confocal imaging and upregulation of current density (more than five-fold). No effects on the voltage dependence of activation or inactivation were noted. However, the effects on the kinetics of recovery from inactivation were opposite: KChIP1b induced a slow component in the recovery (tau approximately 1.2 s), in contrast to the increased recovery rate (tau = 125 ms) with KChIP1a. Accordingly, frequency-dependent accumulation of inactivation was enhanced by KChIP1b but reduced by KChIP1a. Since Kv4.2 channels are involved in protection against back propagating action potentials in dendritic spines, a differential expression of either splice variant could shape the dendritic function.

Guanidine, creatinine (CTN), methylguanidine (MG) and guanidinosuccinic acid (GSA) are four endogenous guanidino compounds with proven neuroexcitatory actions, and putative pathophysiological significance as uraemic toxins. The effects of these uraemic guanidino compounds, were studied on whole-cell current evoked by gamma-amino butyric acid (GABA) on mouse spinal cord neurones in vitro. CTN, MG and GSA concentration dependently blocked GABA-evoked current with calculated IC50 values (+/-SE) of 9.6 +/- 0.9, 9.7 +/- 1.5 and 5.1 +/- 0.4 mM, respectively. CTN, MG and GSA were shown to block inward and outward currents to the same extent, demonstrating voltage independent block of GABA-evoked current by these compounds. Guanidine, however, evoked inward whole-cell currents, which were almost completely blocked by strychnine, indicating that the guanidine-evoked current might have been due to glycine receptor activation.

The effects of CGP 37849 [DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoate] and its ethylester CGP 39551 on whole-cell currents evoked by the endogenous excitatory amino acids, L-glutamate and L-aspartate, were studied in cultured mouse spinal cord neurones. Although CGP 37849 was the more potent compound, both antagonists inhibited 20 microM L-aspartate or 2 microM L-glutamate currents concentration-dependently and reversibly. We calculated IC50 values of 370 +/- 180 nM for CGP 37849 and 2200 +/- 140 nM for CGP 39551 (inhibition of L-aspartate current), and 210 +/- 25 nM for CGP 37849 and 6000 +/- 4700 nM for CGP 39551 (inhibition of L-glutamate current). Both CGP 37849 and CGP 39551 selectively blocked N-methyl-D-aspartate (NMDA)-evoked inward current. Current evoked by 5 microM kainate or 5 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) was unaffected by 10 microM CGP 39551. Current evoked by NMDA was concentration-dependently blocked by CGP 39551 with an IC50 of 2100 +/- 220 nM. After application of 10 microM CGP 37849, 17 +/- 6% of the current evoked by 5 microM L-glutamate remained. This residual current was due to non-NMDA receptor activation since application of 25 microM 2-amino-5-phosphonovalerate (APV) together with CGP 37849 did not significantly alter the residual current, whereas application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) with CGP 37849 did significantly inhibit this current. Clamping cells at potentials ranging from -80 to +60 mV showed a linear potential--current relationship for the 20 microM L-aspartate-evoked current with reversal potential around 0 mV. The proportion of the L-aspartate current antagonized by CGP 37849 or CGP 39551 appeared to be independent of clamping potential. The concentration--current relationship of L-aspartate in the absence of the antagonists showed an EC50 of 49 +/- 14 microM. Upon application of 1 microM CGP 37849 and 10 microM CGP 39551, the L-aspartate concentration--current curve shifted to higher concentrations, and resulted in a 5- and 13-fold increase in the EC50 of L-aspartate, respectively, whereas Imax was not changed by application of the antagonists. Thus, the potent NMDA antagonists CGP 37849 and CGP 39551 were shown to inhibit excitatory amino acid responses specifically by competitive binding to the neurotransmitter recognition site of the NMDA receptor. Selective, competitive antagonism of L-glutamate- and L-aspartate-evoked NMDA receptor responses probably underlies the effects of CGP 37849 and CGP 39551 such as their anticonvulsant, neuroprotectant and antidepressant actions.

1 The sinus node inhibitors UL–FS 49 and DK–AH 269 reduce heart rate by slowing diastolic depolarization rate in the sino-atrial (SA) node, which might originate from the use-dependent blockade of a hyperpolarization-activated current If. A hyperpolarization-activated current Ih, which is present in many types of neurons, is similar to If. We studied the effects of these drugs on Ih in cultured mouse dorsal root ganglion (DRG) neurons. 2 With the whole-cell patch-clamp technique use-dependent block of Ih was observed. The steady-state block following a voltage-clamp pulse train (1-s steps from −38 to −108 mV applied at 0.5 Hz) was dependent on drug concentration and showed an apparent Kd of 0.1 and 0.79 μM with DK–AH 269 and UL–FS 49 respectively. 3 The rate of block increased linearly with drug concentration. The rate of recovery from block was, however, much slower compared to cardiac tissue. 4 There was no significant effect of UL–FS 49 on the activation curve. 5 At high concentrations of UL–FS 49 a clear association of the drug with the open channel was observed. 6 When the cell was stimulated at a frequency of 3 Hz, a distinct hyperpolarization was observed in the presence of extracellular Cs+ or when Ih was blocked with UL–FS 49, but not in the absence of Cs+ and UL–FS 49. 7 These results indicate that Ih protects the cell against hyperpolarizations and subsequent inexcitability. The action of the drugs on the hyperpolarization-activated current in cardiac and neuronal tissue show some similarities; however, some pronounced differences indicate that different subtypes of the channel might exist. British Journal of Pharmacology (1998) 125, 741–750; doi:10.1038/sj.bjp.0702153

CGP 40116 is the active (R)-enantiomer of the most potent N-methyl-d-aspartic acid (NMDA) receptor antagonist presently available: 2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 37849). In this study, we describe the effect of CGP 40116 on whole-cell currents induced by excitatory amino acids in cultured mouse spinal cord cells by use of the whole-cell patch-clamp technique. We found that application of CGP 40116 in the nm range, concentration-dependently inhibited whole-cell current evoked by 20 μm NMDA in mouse cultured spinal neurones (IC50±s.e.mean 48±8 nm CGP 40116). The compound appeared to be highly selective for the NMDA current. At concentrations as high 1 μm, currents evoked by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid were not affected by CGP 40116. The threshold concentration for antagonism of NMDA-induced responses was 10 nm suggesting a selectivity ratio of 100 fold for NMDA receptors versus AMPA or kainate receptors. CGP 40116 produced a parallel rightward displacement of the NMDA log concentration-current curve indicating competitive antagonism at the transmitter recognition site of the NMDA receptor complex. An apparent dissociation constant for the antagonist was calculated from the displacement of the agonist concentration-current curve: 117±53 nm CGP 40116 (estimated Kd±s.e.). Like other competitive NMDA antagonists, CGP 40116 blocked NMDA-evoked current in a voltage-independent manner. British Journal of Pharmacology (1997) 120, 211–214; doi:10.1038/sj.bjp.0700886

The striatum is the main input structure to the basal ganglia and consists mainly out of medium spiny neurons. The numerous spines on their dendrites render them capable of integrating cortical glutamatergic inputs with a motivational dopaminergic signal that originates in the midbrain. This integrative function is thought to underly attribution of incentive salience, a process that is severely disrupted in schizophrenic patients. Phosphodiesterase 10A (PDE10A) is located mainly to the striatal medium spiny neurons and hydrolyses cAMP and cGMP, key determinants of MSN signaling. We show here that genetic depletion of PDE10A critically mediates attribution of salience to reward-predicting cues, evident in impaired performance in PDE10A knockout mice in an instrumentally conditioned reinforcement task. We furthermore report modest impairment of latent inhibition in PDE10A knockout mice, and unaltered prepulse inhibition. We suggest that the lack of effect on PPI is due to the pre-attentional nature of this task. Finally, we performed whole-cell patch clamp recordings and confirm suggested changes in intrinsic membrane excitability. A decrease in spontaneous firing in striatal medium spiny neurons was found. These data show that PDE10A plays a pivotal role in striatal signaling and striatum-mediated salience attribution.

In vivo, KCNQ1 α-subunits associate with the β-subunit KCNE1 to generate the slowly activating cardiac potassium current (I(Ks)). Structurally, they share their topology with other Kv channels and consist out of six transmembrane helices (S1-S6) with the S1-S4 segments forming the voltage-sensing domain (VSD). The opening or closure of the intracellular channel gate, which localizes at the bottom of the S6 segment, is directly controlled by the movement of the VSD via an electromechanical coupling. In other Kv channels, this electromechanical coupling is realized by an interaction between the S4-S5 linker (S4S5(L)) and the C-terminal end of S6 (S6(T)). Previously we reported that substitutions for Leu(353) in S6(T) resulted in channels that failed to close completely. Closure could be incomplete because Leu(353) itself is the pore-occluding residue of the channel gate or because of a distorted electromechanical coupling. To resolve this and to address the role of S4S5(L) in KCNQ1 channel gating, we performed an alanine/tryptophan substitution scan of S4S5(L). The residues with a "high impact" on channel gating (when mutated) clustered on one side of the S4S5(L) α-helix. Hence, this side of S4S5(L) most likely contributes to the electromechanical coupling and finds its residue counterparts in S6(T). Accordingly, substitutions for Val(254) resulted in channels that were partially constitutively open and the ability to close completely was rescued by combination with substitutions for Leu(353) in S6(T). Double mutant cycle analysis supported this cross-talk indicating that both residues come in close contact and stabilize the closed state of the channel.

BACKGROUND Zatebradine (UL-FS 49) is a drug with a specific bradycardiac electrophysiological profile. It reduces heart rate by lengthening the duration of diastolic depolarization in the sinoatrial (SA) node. The ionic basis of this action, however, is not clarified. METHODS AND RESULTS We used the whole-cell patch-clamp technique to study the effects of zatebradine on ionic currents underlying diastolic depolarization of isolated rabbit SA node cells. Low concentrations of zatebradine simultaneously reduced diastolic depolarization rate and the pacemaker current I(f). The drug blocked the pacemaker current, I(f), in a use-dependent manner without causing a shift of its activation curve. At hyperpolarized potentials, unblock of I(f) occurred. Clinically relevant concentrations of the drug have little effect on the L-type calcium current or delayed rectifier potassium current. CONCLUSIONS This use-dependent block of the If channel can account for most of the pharmacological characteristics of zatebradine and is probably the mechanism of heart rate reduction caused by this agent. Thus, the sinus node inhibitor zatebradine belongs to a new class of "I(f) blockers" with possible advantages over currently available drugs for the treatment of ischemic heart disease.

Silent voltage-gated K+ (Kv) subunits interact with Kv2 subunits and primarily modulate the voltage dependence of inactivation of these heterotetrameric channels. Both Kv2 and silent Kv subunits are expressed in the mammalian nervous system, but little is known about their expression and function in sensory neurons. This study reports the presence of Kv2.1, Kv2.2, and silent subunit Kv6.1, Kv8.1, Kv9.1, Kv9.2, and Kv9.3 mRNA in mouse dorsal root ganglia (DRG). Immunocytochemistry confirmed the protein expression of Kv2.x and Kv9.x subunits in cultured small DRG neurons. To investigate if Kv2 and silent Kv subunits are underlying the delayed rectifier K+ current ( IK) in these neurons, Kv2-mediated currents were isolated by the extracellular application of rStromatoxin-1 (ScTx) or by the intracellular application of Kv2 antibodies. Both ScTx- and anti-Kv2.1-sensitive currents displayed two components in their voltage dependence of inactivation. Together, both components accounted for approximately two-thirds of IK. A comparison with results obtained in heterologous expression systems suggests that one component reflects homotetrameric Kv2.1 channels, whereas the other component represents heterotetrameric Kv2.1/silent Kv channels. These observations support a physiological role for silent Kv subunits in small DRG neurons.

In this study, we pharmacologically characterized gambierol, a marine polycyclic ether toxin which is produced by the dinoflagellate Gambierdiscus toxicus. Besides several other polycyclic ether toxins like ciguatoxins, this scarcely studied toxin is one of the compounds that may be responsible for ciguatera fish poisoning (CFP). Unfortunately, the biological target(s) that underlies CFP is still partly unknown. Today, ciguatoxins are described to specifically activate voltage-gated sodium channels by interacting with their receptor site 5. But some dispute about the role of gambierol in the CFP story shows up: some describe voltage-gated sodium channels as the target, while others pinpoint voltage-gated potassium channels as targets. Since gambierol was never tested on isolated ion channels before, it was subjected in this work to extensive screening on a panel of 17 ion channels: nine cloned voltage-gated ion channels (mammalian Na(v)1.1-Na(v)1.8 and insect Para) and eight cloned voltage-gated potassium channels (mammalian K(v)1.1-K(v)1.6, hERG and insect ShakerIR) expressed in Xenopus laevis oocytes using two-electrode voltage-clamp technique. All tested sodium channel subtypes are insensitive to gambierol concentrations up to 10 microM. In contrast, K(v)1.2 is the most sensitive voltage-gated potassium channel subtype with almost full block (97%) and an half maximal inhibitory concentration (IC(50)) of 34.5 nM. To the best of our knowledge, this is the first study where the selectivity of gambierol is tested on isolated voltage-gated ion channels. Therefore, these results lead to a better understanding of gambierol and its possible role in CFP and they may also be useful in the development of more effective treatments.

Contains fulltext : 52042.pdf (Publisher’s version ) (Closed access) The long QT syndrome (LQTS) is a multi-factorial disorder that predisposes to life-threatening arrhythmias. Both hereditary and acquired subforms have been identified. Here, we present clinical and biophysical evidence that the hERG mutation c.1039 C>T (p.Pro347Ser or P347S) is responsible for both the acquired and the congenital phenotype. In one case the genotype remained silent for years until the administration of several QT-prolonging drugs resulted into a full-blown phenotype, that was reversible upon cessation of these compounds. On the other hand the mutation was responsible for a symptomatic congenital LQTS in a Dutch family, displaying a substantial heterogeneity of the clinical symptoms. Biophysical characterization of the p.Pro347Ser potassium channels using whole-cell patch clamp experiments revealed a novel pathogenic mechanism of reciprocal changes in the inactivation kinetics combined with a dominant-negative reduction of the functional expression in the heterozygous situation, yielding a modest genetic predisposition for LQTS. Our data show that in the context of the multi-factorial aetiology underlying LQTS a modest reduction of the repolarizing power can give rise to a spectrum of phenotypes originating from one mutation. This observation increases the complexity of genotype-phenotype correlations in more lenient manifestations of the disease and underscores the difficulty of predicting the expressivity of the LQTS especially for mutations with a more subtle impact such as p.Pro347Ser.

Noise-induced hearing loss (NIHL) is one of the most important occupational diseases and, after presbyacusis, the most frequent cause of hearing loss. NIHL is a complex disease caused by an interaction between environmental and genetic factors. The various environmental factors involved in NIHL have been relatively extensively studied. On the other hand, little research has been performed on the genetic factors responsible for NIHL. To test whether the variation in genes involved in coupling of cells and potassium recycling in the inner ear might partly explain the variability in susceptibility to noise, we performed a case-control association study using 35 SNPs selected in 10 candidate genes on a total of 218 samples selected from a population of 1,261 Swedish male noise-exposed workers. We have obtained significant differences between susceptible and resistant individuals for the allele, genotype, and haplotype frequencies for three SNPs of the KCNE1 gene, and for the allele frequencies for one SNP of KCNQ1 and one SNP of KCNQ4. Patch-clamp experiments in high K+-concentrations using a Chinese hamster ovary (CHO) cell model were performed to investigate the possibility that the KCNE1-p.85N variant (NT_011512.10:g.21483550G>A; NP_00210.2:p.Asp85Asn) was causative for high noise susceptibility. The normalized current density generated by KCNQ1/KCNE1-p.85N channels, thus containing the susceptibility variant, differed significantly from that from wild-type channels. Furthermore, the midpoint potential of KCNQ1/KCNE1-p.85N channels (i.e., the voltage at which 50% of the channels are open) differed from that of wild-type channels. Further genetic and physiological studies will be necessary to confirm these findings.

Objective Long QT syndrome (LQTS) is an inherited disorder of ventricular repolarization caused by mutations in cardiac ion channel genes, including KCNQ1. In this study the electrophysiological properties of a LQTS-associated mutation in KCNQ1 (Q357R) were characterized. This mutation is located near the C-terminus of S6, a region that is important for the gate structure. Methods and results Co-assembly of KCNE1 with the mutant Q357R elicited a current displaying slower activation compared to the wild-type KCNQ1/KCNE1 channels. The voltage dependence of activation of Q357R was shifted to more positive potentials. Moreover, a strong reduction in current density was observed that was partially attributed to the altered voltage dependence and kinetics of activation. The reduced current amplitude was also caused by intracellular retention of Q357R/KCNE1 channels as was shown by confocal microscopy. It indicated that the Q357R mutation disturbed protein expression by a trafficking or assembly problem of the Q357R/KCNE1 complex. To mimic the patient status KCNQ1, Q357R and KCNE1 were co-expressed, which revealed a dominant negative effect on current density and activation kinetics. Conclusion The effects of the Q357R mutation on the activation of the channel together with a reduced expression at the membrane would lead to a reduction in I Ks and thus in “repolarization reserve” under physiological circumstances. As such it explains the long QT syndrome observed in these patients.

Objective: Mutations in the KCNH2 (hERG, human ether-a-go-go related gene) gene may cause a reduction of the delayed rectifier current I-Kr, thereby leading to the long QT syndrome (LQTS). The reduced I-Kr delays the repolarisation of cardiac cells and renders patients vulnerable to ventricular arrhythmias and sudden death. We identified a novel mutation in a LQTS family and investigated its functional consequences using molecular and microscopic techniques. Methods and results: Genetic screening in the LQTS family revealed a heterozygous frameshift mutation p.Pro872fs located in the C-terminus of the KCNH2 gene. The mutation leads to a premature truncation of the C-terminus of the hERG protein. p.Pro872fs channels lack 282 amino acids at the C-terminus and possess an extra 4-amino acid tail. Both the kinetic and biochemical properties of the p.Pro872fs and p.Pro872fs/WT channels were studied in HEK293 cells and resulted in a novel proof of concept for heterozygous LQTS mutations: homotetrameric p.Pro872fs channels displayed near-normal expression, trafficking, and channel kinetics. Unexpectedly, upon co-expression of p.Pro872fs and WT channels, the repolarising power (the proportion of hERG current contributing to the action potential as the percentage of the total current available) was substantially higher during action potential clamp experiments as compared to WT channels alone. This would lead to a shorter rather than a prolonged QT interval. However, at the same time, heterotetramerisation of p.Pro872fs and WT channels also caused a dominant negative effect on trafficking by an increase in ER retention of these heterotetrameric channels, which surpassed the former gain in repolarising power. Conclusion: The LQTS phenotype in the studied family is caused by a mutation with novel properties. We demonstrate that a KCNH2 mutation that clinically leads to long QT syndrome causes at the cellular level both a "gain" and a "loss" of HERG channel function due to a kinetic increase in repolarising power and a decrease in trafficking efficiency of heteromultimeric channels. (C) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved

The recent crystallization of a voltage-gated K+ channel has given insight into the structure of these channels but has not resolved the issues of the location and the operation of the gate. The conserved PXP motif in the S6 segment of Shaker channels has been proposed to contribute to the intracellular gating structure. To investigate the role of this motif in the destabilization of the alpha-helix, both prolines were replaced to promote an alpha-helix (alanine) or to allow a flexible configuration (glycine). These substitutions were nonfunctional or resulted in drastically altered channel gating, highlighting an important role of these prolines. Combining these mutations with a proline substitution scan demonstrated that proline residues in the midsection of S6 are required for functionality, but not necessarily at the positions conserved throughout evolution. These results indicate that the destabilization or bending of the S6 alpha-helix caused by the PXP motif apparently creates a flexible "hinge" that allows movement of the lower S6 segment during channel gating and opening.

A novel mutation (T65P) in the PAS domain of the human potassium channel HERG results in the long QT syndrome by trafficking deficiency.Paulussen A, Raes A, Matthijs G, Snyders DJ, Cohen N, Aerssens J.Department of Pharmacogenomics, Johnson & Johnson Pharmaceutical Research and Development, Beerse B-2340, Belgium. paulussen.aimee@advalvas.beThe congenital long QT syndrome is a cardiac disease characterized by an increased susceptibility to ventricular arrhythmias. The clinical hallmark is a prolongation of the QT interval, which reflects a delay in repolarization caused by mutations in cardiac ion channel genes. Mutations in the HERG (human ether-a-go-go-related gene KCNH2 can cause a reduction in I(Kr), one of the currents responsible for cardiac repolarization. We describe the identification and characterization of a novel missense mutation T65P in the PAS (Per-Arnt-Sim) domain of HERG, resulting in defective trafficking of the protein to the cell membrane. Defective folding of the mutant protein could be restored by decreased cell incubation temperature and pharmacologically by cisapride and E-4031. When trafficking was restored by growing cells at 27 degrees C, the kinetics of the mutated channel resembled that of wild-type channels although the rate of activation, deactivation, and recovery from inactivation were accelerated. No positive evidence for the formation of heterotetramers was obtained by co-expression of wild-type with mutant subunits at 37 degrees C. As a consequence the clinical symptoms may be explained rather by haploinsufficiency than by dominant negative effects. This study is the first to relate a PAS domain mutation in HERG to a trafficking deficiency at body temperature, apart from effects on channel deactivation

Voltage-gated K + channels control excitability in neuronal and various other tissues. We identified three unique α-subunits of voltage-gated K + -channels in the human genome. Analysis of the full-length sequences indicated that one represents a previously uncharacterized member of the Kv6 subfamily, Kv6.3, whereas the others are the first members of two unique subfamilies, Kv10.1 and Kv11.1. Although they have all of the hallmarks of voltage-gated K + channel subunits, they did not produce K + currents when expressed in mammalian cells. Confocal microscopy showed that Kv6.3, Kv10.1, and Kv11.1 alone did not reach the plasma membrane, but were retained in the endoplasmic reticulum. Yeast two-hybrid experiments failed to show homotetrameric interactions, but showed interactions with Kv2.1, Kv3.1, and Kv5.1. Co-expression of each of the previously uncharacterized subunits with Kv2.1 resulted in plasma membrane localization with currents that differed from typical Kv2.1 currents. This heteromerization was confirmed by co-immunoprecipitation. The Kv2 subfamily consists of only two members and uses interaction with “silent subunits” to diversify its function. Including the subunits described here, the “silent subunits” represent one-third of all Kv subunits, suggesting that obligatory heterotetramer formation is more widespread than previously thought.

We have cloned HERG USO, a C-terminal splice variant of the human ether-à-go-go-related gene (HERG), the gene encoding the rapid component of the delayed rectifier (IKr), from human heart, and we find that its mRNA is approximately 2-fold more abundant than that for HERG1 (the originally described cDNA). After transfection of HERG USO in Ltk- cells, no current was observed. However, coexpression of HERG USO with HERG1 modified IKr by decreasing its amplitude, accelerating its activation, and shifting the voltage dependence of activation 8.8 mV negative. As with HERG USO, HERGDeltaC (a HERG1 construct lacking the C-terminal 462 amino acids) also produced no current in transfected cells. However, IKr was rescued by ligation of 104 amino acids from the C terminus of HERG1 to the C terminus of HERGDeltaC, indicating that the C terminus of HERG1 includes a domain (/=104 amino acids) that is critical for faithful recapitulation of IKr. The lack of this C-terminal domain not only explains the finding that HERG USO does not generate IKr but also indicates a similar mechanism for hitherto-uncharacterized long QT syndrome HERG mutations that disrupt the splice site or the C-terminal. We suggest that the amplitude and gating of cardiac IKr depends on expression of both HERG1 and HERG USO.

In mouse dorsal root ganglion (DRG) neurons the activation curve of the hyperpolarization-activated current (Ih) shifted towards depolarized potentials when cAMP was present in the pipette. The relation between the midpoint potential and cAMP concentration could be described by a Hill function with a half-maximal concentration of 0.55 microM cAMP, reflecting a direct action of cAMP on the channel. With 5 mM ATP and a saturating concentration of cAMP an additional shift of the midpoint potential is observed which can be explained by phosphorylation. Application of Rp-cAMPS and Sp-cAMPS support the hypothesis of both a phosphorylation pathway and a direct effect exhibited by these molecules. The bell-shaped curves, relating the time constants for the slow and fast current components to the voltage, shifted towards positive membrane potentials when cAMP and ATP were in the pipette. The fully activated Ih/voltage relation and the reversal potential were not dependent on the presence of cAMP or ATP in the pipette. The mean resting membrane potential of -59 mV, using the perforated-patch configuration, hyperpolarized in the presence of extracellular CsCl. In the whole-cell configuration the resting membrane potential was significantly more negative at 0 microM cAMP (-61 mV) than at 100 microM cAMP (-57 mV). Thus, the activation of Ih, regulated by both the intracellular cAMP and the ATP concentration, may influence the excitability of DRG neurons.

Levels of methylguanidine (MG) and guanidinosuccinate (GSA) are known to be highly increased in uraemic patients. In the present work, the effects of these uraemic guanidino compounds on the excitatory amino acid system were investigated in vivo and in vitro. It was found that convulsions induced by intracerebroventricular GSA injection in mice were antagonized by N-methyl-D-aspartate (NMDA) receptor blockade, whereas those induced by MG were not significantly altered. Application of GSA (between 25 and 10,000 microM) to mouse spinal cord neurones in primary dissociated cell cultures, evoked depolarizing, inward whole-cell currents in a dose-dependent fashion and with reversal potential at 0 mV; MG did not produce such effects. GSA-induced whole-cell currents were caused by NMDA receptor activation since NMDA receptor antagonists (2-amino-5-phosphonovalerate, Mg2+ and ketamine) blocked GSA-evoked whole-cell currents completely and reversibly, whereas co-application of a non-NMDA receptor antagonist (6-cyano-7-nitroquinoxaline-2,3-dione) did not affect GSA-induced current. Evoked field potentials in CA1 region of rat hippocampal slices were completely abolished by GSA, and this effect was antagonized by NMDA receptor blockade. All data were consistent with selective agonist action of GSA upon the NMDA-type glutamate receptor. In view of the results presented here, it should be examined whether NMDA receptors contribute to the neurological complications of renal failure through GSA-induced inappropriate or excessive activation of NMDA receptors.

Voltage-gated potassium (Kv) channels are tetramers of α-subunits, each composed out of six membrane-spanning helices (S1-S6) with a pore loop between S5 and S6 that forms the channel's selectivity filter. The activation gate that seals off the ion conducting pore in a closed channel is controlled by the transmembrane potential. After channel activation most Kv channels display C-type inactivation, a process that is believed to involve reorientations of the selectivity filter and results in a non-conducting channel although the channel gate is open. hKv1.5 (a Shaker-type Kv channel) displays such C-type inactivation. Here we report that an alanine substitution for residue T480 that is located at the end of the pore-helix prevents hKv1.5 channels from entering the inactivated state. The mutant T480A had an isochronal activation curve similar to Wild Type hKv1.5 when determined with 250ms depolarizing steps. Longer depolarizations (∼5 seconds) caused WT channels to inactivate (∼58%) displaying an inactivation curve with a midpoint of −23.2 ± 1.2 mV and a slope factor of about 4. However, T480A did not inactivate and such long depolarizations caused an additional (slow) activation at more negative potentials thus generating an isochronal activation curve with properties that were similar to the inactivation process in WT channels. To investigate the structural changes that underlie the unusual behaviour of this mutant, we performed a series of MD simulations of the pore domain of WT hKv1.5 and T480A. Analysis of the trajectories shows that T480A affects the stability and flexibility of the filter region and the surrounding pore loop. These results show that residue T480 (located outside the pore region that determines the integrity of the selectivity filter) affects the stability of the filter and influences C-type inactivation.

KCNQ1 α-subunits are composed out of six transmembrane segments (S1-S6) that tetramerize into a functional channel. In vivo, KCNQ1 α-subunits associate with the β-subunit KCNE1 to generate the slowly activating cardiac IKs and consequently mutations in KCNQ1 are linked to the congenital LQT1 syndrome. Similar to other Kv channels, the S1-S4 segments form the voltage sensing domain that senses the membrane potential and that controls the opening or closure of the channel gate through an electromechanical coupling. In other channels a direct interaction between the S4-S5 linker and bottom part of S6 has been shown to constitute this electromechanical coupling. We previously identified residues in the C-terminal part of S6 that are critical for KCNQ1 gating. To investigate if these residues interact with the S4-S5 linker, we performed an alanine/tryptophan substitution scan of the S4-S5 linker sequence. Based on their impact on channel gating, we categorized these substitutions as either “high” or “low impact”. The pattern of “high impact” positions was consistent with an α-helical configuration and clustered on one side of the S4-S5 linker. Since substitutions at these positions markedly impaired channel gating, they are good candidates to contact residues in the bottom part of S6. Indeed, replacing valine 254 in the S4-S5 linker by a leucine resulted in channels that were partially constitutively open but channel closure could be rescued by combining V254L with the S6 mutation L353A that by itself displayed a similar phenotype as V254L. The observation that all known LQT1 mutations in the S4-S5 linker map on the “high impact” side further strengthens the proposal that this face of the S4-S5 linker contacts the C-terminal S6 segment and constitutes part of the electromechanical coupling in KCNQ1 channels.