Alkaline-sensitive two-pore domain potassium channels form functional heteromers in pancreatic ß-cells.

Two-pore domain K+ channels (K2P channels), active as dimers, produce inhibitory currents regulated by a variety of stimuli. Among them, TWIK1-related alkalinization-activated K+ channel 1 (TALK1), TWIK1-related alkalinization-activated K+ channel 2 (TALK2), and TWIK1-related acid-sensitive K+ channel 2 (TASK2) form a subfamily of structurally related K2P channels stimulated by extracellular alkalosis. The human genes encoding these proteins are clustered at chromosomal region 6p21 and coexpressed in multiple tissues, including the pancreas. The question whether these channels form functional heteromers remained open. By analyzing single-cell transcriptomic data, we show that these channels are coexpressed in insulin-secreting pancreatic ß-cells. Using in situ proximity ligation assay and electrophysiology, we show that they form functional heterodimers both upon heterologous expression and under native conditions in human pancreatic ß-cells. We demonstrate that heteromerization of TALK2 with TALK1 or with TASK2 endows TALK2 with sensitivity to extracellular alkalosis in the physiological range. We further show that the association of TASK2 with TALK1 and TALK2 increases their unitary conductance. These results provide a new example of heteromerization in the K2P channel family expanding the range of the potential physiological and pathophysiological roles of TALK1/TALK2/TASK2 channels, not only in insulin-secreting cells but also in the many other tissues in which they are coexpressed.

Physiological roles of heteromerization: focus on the two-pore domain potassium channels.

Potassium channels form the largest family of ion channels with more than 80 members involved in cell excitability and signalling. Most of them exist as homomeric channels, whereas specific conditions are required to obtain heteromeric channels. It is well established that heteromerization of voltage-gated and inward rectifier potassium channels affects their function, increasing the diversity of the native potassium currents. For potassium channels with two pore domains (K2P ), homomerization has long been considered the rule, their polymodal regulation by a wide diversity of physical and chemical stimuli being responsible for the adaptation of the leak potassium currents to cellular needs. This view has recently evolved with the accumulation of evidence of heteromerization between different K2P subunits. Several functional intragroup and intergroup heteromers have recently been identified, which contribute to the functional heterogeneity of this family. K2P heteromerization is involved in the modulation of channel expression and trafficking, promoting functional and signalling diversity. As illustrated in the Abstract Figure, heteromerization of TREK1 and TRAAK provides the cell with more possibilities of regulation. It is becoming increasingly evident that K2P heteromers contribute to important physiological functions including neuronal and cardiac excitability. Since heteromerization also affects the pharmacology of K2P channels, this understanding helps to establish K2P heteromers as new therapeutic targets for physiopathological conditions.

Lack of p11 expression facilitates acidity-sensing function of TASK1 channels in mouse adrenal medullary cells.

External acidity induces catecholamine secretion by inhibiting TASK1-like channels in rat adrenal medullary (AM) cells. TASK channels can function as a heteromer or homomer in the TASK subfamily. In this study, we elucidate the molecular identity of TASK1-like channels in mouse AM cells using gene knockout. Genetic deletion of TASK1, but not TASK3, abolished the depolarizing inward current and catecholamine secretion in response to acidity, whereas it did not affect the resting current level. Immunocytochemistry revealed that AM cells exhibited predominantly TASK1-like and little TASK3-like immunoreactivity. A proximity ligation assay showed that TASK1/3 heteromeric channels were not formed in AM cells or PC12 cells. However, the exogenous expression of p11 in PC12 cells resulted in the heteromeric formation of TASK isoforms, which were mainly located in the cytoplasm, and p11 was not expressed in rat adrenal medullae or PC12 cells. In AM cells, genetic deletion of TASK1 resulted in enhancement of the immunoreactivity of the TALK2 channel, but not TASK3. The results indicate that TASK1 homomeric channels function as acidity sensors in AM cells, and that function is facilitated by the lack of p11 expression.-Inoue, M., Matsuoka, H., Lesage, F., Harada, K. Lack of p11 expression facilitates acidity-sensing function of TASK1 channels in mouse adrenal medullary cells.

Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties.

The tandem of pore domain in a weak inwardly rectifying K(+) channel (Twik)-related acid-arachidonic activated K(+) channel (TRAAK) and Twik-related K(+) channels (TREK) 1 and TREK2 are active as homodimers gated by stretch, fatty acids, pH, and G protein-coupled receptors. These two-pore domain potassium (K2P) channels are broadly expressed in the nervous system where they control excitability. TREK/TRAAK KO mice display altered phenotypes related to nociception, neuroprotection afforded by polyunsaturated fatty acids, learning and memory, mood control, and sensitivity to general anesthetics. These channels have emerged as promising targets for the development of new classes of anesthetics, analgesics, antidepressants, neuroprotective agents, and drugs against addiction. Here, we show that the TREK1, TREK2, and TRAAK subunits assemble and form active heterodimeric channels with electrophysiological, regulatory, and pharmacological properties different from those of homodimeric channels. Heteromerization occurs between all TREK variants produced by alternative splicing and alternative translation initiation. These results unveil a previously unexpected diversity of K2P channels that will be challenging to analyze in vivo, but which opens new perspectives for the development of clinically relevant drugs.

In cellulo phosphorylation induces pharmacological reprogramming of maurocalcin, a cell-penetrating venom peptide.

The venom peptide maurocalcin (MCa) is atypical among toxins because of its ability to rapidly translocate into cells and potently activate the intracellular calcium channel type 1 ryanodine receptor (RyR1). Therefore, MCa is potentially subjected to posttranslational modifications within recipient cells. Here, we report that MCa Thr(26) belongs to a consensus PKA phosphorylation site and can be phosphorylated by PKA both in vitro and after cell penetration in cellulo. Unexpectedly, phosphorylation converts MCa from positive to negative RyR1 allosteric modulator. Thr(26) phosphorylation leads to charge neutralization of Arg(24), a residue crucial for MCa agonist activity. The functional effect of Thr(26) phosphorylation is partially mimicked by aspartyl mutation. This represents the first case, to our knowledge, of both ex situ posttranslational modification and pharmacological reprogramming of a small natural cystine-rich peptide by target cells. So far, phosphorylated MCa is the first specific negative allosteric modulator of RyR1, to our knowledge, and represents a lead compound for further development of phosphatase-resistant analogs.

Hyperoxia treatment of TREK-1/TREK-2/TRAAK-deficient mice is associated with a reduction in surfactant proteins.

We previously proposed a role for the two-pore domain potassium (K2P) channel TREK-1 in hyperoxia (HO)-induced lung injury. To determine whether redundancy among the three TREK isoforms (TREK-1, TREK-2, and TRAAK) could protect from HO-induced injury, we now examined the effect of deletion of all three TREK isoforms in a clinically relevant scenario of prolonged HO exposure and mechanical ventilation (MV). We exposed WT and TREK-1/TREK-2/TRAAK-deficient [triple knockout (KO)] mice to either room air, 72-h HO, MV [high and low tidal volume (TV)], or a combination of HO + MV and measured quasistatic lung compliance, bronchoalveolar lavage (BAL) protein concentration, histologic lung injury scores (LIS), cellular apoptosis, and cytokine levels. We determined surfactant gene and protein expression and attempted to prevent HO-induced lung injury by prophylactically administering an exogenous surfactant (Curosurf). HO treatment increased lung injury in triple KO but not WT mice, including an elevated LIS, BAL protein concentration, and markers of apoptosis, decreased lung compliance, and a more proinflammatory cytokine phenotype. MV alone had no effect on lung injury markers. Exposure to HO + MV (low TV) further decreased lung compliance in triple KO but not WT mice, and HO + MV (high TV) was lethal for triple KO mice. In triple KO mice, the HO-induced lung injury was associated with decreased surfactant protein (SP) A and SPC but not SPB and SPD expression. However, these changes could not be explained by alterations in the transcription factors nuclear factor-1 (NF-1), NKX2.1/thyroid transcription factor-1 (TTF-1) or c-jun, or lamellar body levels. Prophylactic Curosurf administration did not improve lung injury scores or compliance in triple KO mice.

Phospholipase D2 specifically regulates TREK potassium channels via direct interaction and local production of phosphatidic acid.

Membrane lipids serve as second messengers and docking sites for proteins and play central roles in cell signaling. A major question about lipid signaling is whether diffusible lipids can selectively target specific proteins. One family of lipid-regulated membrane proteins is the TWIK-related K channel (TREK) subfamily of K2P channels: TREK1, TREK2, and TWIK-related arachdonic acid stimulated K(+) channel (TRAAK). We investigated the regulation of TREK channels by phosphatidic acid (PA), which is generated by phospholipase D (PLD) via hydrolysis of phosphatidylcholine. Even though all three of the channels are sensitive to PA, we found that only TREK1 and TREK2 are potentiated by PLD2 and that none of these channels is modulated by PLD1, indicating surprising selectivity. We found that PLD2, but not PLD1, directly binds to the C terminus of TREK1 and TREK2, but not to TRAAK. The results have led to a model for selective lipid regulation by localization of phospholipid enzymes to specific effector proteins. Finally, we show that regulation of TREK channels by PLD2 occurs natively in hippocampal neurons.

The two-pore domain potassium channel, TWIK-1, has a role in the regulation of heart rate and atrial size.

The two-pore domain potassium (K(+)) channel TWIK-1 (or K2P1.1) contributes to background K(+) conductance in diverse cell types. TWIK-1, encoded by the KCNK1 gene, is present in the human heart with robust expression in the atria, however its physiological significance is unknown. To evaluate the cardiac effects of TWIK-1 deficiency, we studied zebrafish embryos after knockdown of the two KCNK1 orthologues, kcnk1a and kcnk1b. Knockdown of kcnk1a or kcnk1b individually caused bradycardia and atrial dilation (p<0.001 vs. controls), while ventricular stroke volume was preserved. Combined knockdown of both kcnk1a and kcnk1b resulted in a more severe phenotype, which was partially reversed by co-injection of wild-type human KCNK1 mRNA, but not by a dominant negative variant of human KCNK1 mRNA. To determine whether genetic variants in KCNK1 might cause atrial fibrillation (AF), we sequenced protein-coding regions in two independent cohorts of patients (373 subjects) and identified three non-synonymous variants, p.R171H, p.I198M and p.G236S, that were all located in highly conserved amino acid residues. In transfected mammalian cells, zebrafish and wild-type human TWIK-1 channels had a similar cellular distribution with predominant localization in the endosomal compartment. Two-electrode voltage-clamp experiments using Xenopus oocytes showed that both zebrafish and wild-type human TWIK-1 channels produced K(+) currents that are sensitive to external K(+) concentration as well as acidic pH. There were no effects of the three KCNK1 variants on cellular localization, current amplitude or reversal potential at pH7.4 or pH6. Our data indicate that TWIK-1 has a highly conserved role in cardiac function and is required for normal heart rate and atrial morphology. Despite the functional importance of TWIK-1 in the atrium, genetic variation in KCNK1 is not a common primary cause of human AF.

Cacnb4 directly couples electrical activity to gene expression, a process defective in juvenile epilepsy.

Calcium current through voltage-gated calcium channels (VGCC) controls gene expression. Here, we describe a novel signalling pathway in which the VGCC Cacnb4 subunit directly couples neuronal excitability to transcription. Electrical activity induces Cacnb4 association to Ppp2r5d, a regulatory subunit of PP2A phosphatase, followed by (i) nuclear translocation of Cacnb4/Ppp2r5d/PP2A, (ii) association with the tyrosine hydroxylase (TH) gene promoter through the nuclear transcription factor thyroid hormone receptor alpha (TRα), and (iii) histone binding through association of Cacnb4 with HP1γ concomitantly with Ser(10) histone H3 dephosphorylation by PP2A. This signalling cascade leads to TH gene repression by Cacnb4 and is controlled by the state of interaction between the SH3 and guanylate kinase (GK) modules of Cacnb4. The human R482X CACNB4 mutation, responsible for a form of juvenile myoclonic epilepsy, prevents association with Ppp2r5 and nuclear targeting of the complex by altering Cacnb4 conformation. These findings demonstrate that an intact VGCC subunit acts as a repressor recruiting platform to control neuronal gene expression.

Activation of neurotensin receptor 1 facilitates neuronal excitability and spatial learning and memory in the entorhinal cortex: beneficial actions in an Alzheimer's disease model.

Neurotensin (NT) is a tridecapeptide distributed in the CNS, including the entorhinal cortex (EC), a structure that is crucial for learning and memory and undergoes the earliest pathological alterations in Alzheimer's disease (AD). Whereas NT has been implicated in modulating cognition, the cellular and molecular mechanisms by which NT modifies cognitive processes and the potential therapeutic roles of NT in AD have not been determined. Here we examined the effects of NT on neuronal excitability and spatial learning in the EC, which expresses high density of NT receptors. Brief application of NT induced persistent increases in action potential firing frequency, which could last for at least 1 h. NT-induced facilitation of neuronal excitability was mediated by downregulation of TREK-2 K(+) channels and required the functions of NTS1, phospholipase C, and protein kinase C. Microinjection of NT or NTS1 agonist, PD149163, into the EC increased spatial learning as assessed by the Barnes Maze Test. Activation of NTS1 receptors also induced persistent increases in action potential firing frequency and significantly improved the memory status in APP/PS1 mice, an animal model of AD. Our study identifies a cellular substrate underlying learning and memory and suggests that NTS1 agonists may exert beneficial actions in an animal model of AD.

The family of K2P channels: salient structural and functional properties.

Potassium channels participate in many biological functions, from ion homeostasis to generation and modulation of the electrical membrane potential. They are involved in a large variety of diseases. In the human genome, 15 genes code for K(+) channels with two pore domains (K2P ). These channels form dimers of pore-forming subunits that produce background conductances finely regulated by a range of natural and chemical effectors, including signalling lipids, temperature, pressure, pH, antidepressants and volatile anaesthetics. Since the cloning of TWIK1, the prototypical member of this family, a lot of work has been carried out on their structure and biology. These studies are still in progress, but data gathered so far show that K2P channels are central players in many processes, including ion homeostasis, hormone secretion, cell development and excitability. A growing number of studies underline their implication in physiopathological mechanisms, such as vascular and pulmonary hypertension, cardiac arrhythmias, nociception, neuroprotection and depression. This review gives a synthetic view of the most noticeable features of these channels.

Tandem pore domain halothane-inhibited K+ channel subunits THIK1 and THIK2 assemble and form active channels.

Despite a high level of sequence homology, tandem pore domain halothane-inhibited K(+) channel 1 (THIK1) produces background K(+) currents, whereas THIK2 is silent. This lack of activity is due to a unique combination of intracellular retention and weak basal activity in the plasma membrane. Here, we designed THIK subunits containing dominant negative mutations (THIK1(DN) and THIK2(DN)). THIK2(DN) mutant inhibits THIK1 currents, whereas THIK1(DN) inhibits an activated form of THIK2 (THIK2-A155P-I158D). In situ proximity ligation assays and Förster/fluorescence resonance energy transfer (FRET) experiments support a physical association between THIK1 and THIK2. Next, we expressed covalent tandems of THIK proteins to obtain expression of pure heterodimers. Td-THIK1-THIK2 (where Td indicates tandem) produces K(+) currents of amplitude similar to Td-THIK1-THIK1 but with a noticeable difference in the current kinetics. Unlike Td-THIK2-THIK2 that is mainly detected in the endoplasmic reticulum, Td-THIK1-THIK2 distributes at the plasma membrane, indicating that THIK1 can mask the endoplasmic reticulum retention/retrieval motif of THIK2. Kinetics and unitary conductance of Td-THIK1-THIK2 are intermediate between THIK1 and THIK2. Altogether, these results show that THIK1 and THIK2 form active heteromeric channels, further expanding the known repertoire of K(+) channels.

Silent but not dumb: how cellular trafficking and pore gating modulate expression of TWIK1 and THIK2.

Among K2P channels, a few of them turned out to be difficult to express in heterologous systems and were coined "silent subunits". Recent studies have shed light on the mechanisms behind this apparent lack of channel activity at the plasma membrane. For TWIK1 and THIK2 channels, silence is related to a combination of intracellular retention and low intrinsic activity. TWIK1 is constitutively endocytosed from the plasma membrane before being transported to recycling endosomes, whereas THIK2 is restricted to endoplasmic reticulum. These intracellular localizations are related to trafficking signals located in the cytoplasmic parts of the channels. When these motifs are mutated or masked, channels are redistributed at the plasma membrane and produce measurable currents. However, these currents are of modest amplitude. This weak basal activity is due to a hydrophobic barrier in the deep pore that limits water and ions in the conduction pathway. Other silent channels KCNK7, TWIK2, and TASK5 are still under study. Expression and characterization of these K2P channels pave the way for a better understanding of the mechanisms controlling intracellular trafficking of membrane proteins, ion conduction, and channel gating.

TWIK1, a unique background channel with variable ion selectivity.

TWIK1 belongs to the family of background K(+) channels with two pore domains. In native and transfected cells, TWIK1 is detected mainly in recycling endosomes. In principal cells in the kidney, TWIK1 gene inactivation leads to the loss of a nonselective cationic conductance, an unexpected effect that was attributed to adaptive regulation of other channels. Here, we show that TWIK1 ion selectivity is modulated by extracellular pH. Although TWIK1 is K(+) selective at neutral pH, it becomes permeable to Na(+) at the acidic pH found in endosomes. Selectivity recovery is slow after restoration of a neutral pH. Such hysteresis makes plausible a role of TWIK1 as a background channel in which selectivity and resulting inhibitory or excitatory influences on cell excitability rely on its recycling rate between internal acidic stores and the plasma membrane. TWIK1(-/-) pancreatic ß cells are more polarized than control cells, confirming a depolarizing role of TWIK1 in kidney and pancreatic cells.

TASK-2 channels contribute to pH sensitivity of retrotrapezoid nucleus chemoreceptor neurons.

Phox2b-expressing glutamatergic neurons of the retrotrapezoid nucleus (RTN) display properties expected of central respiratory chemoreceptors; they are directly activated by CO2/H(+) via an unidentified pH-sensitive background K(+) channel and, in turn, facilitate brainstem networks that control breathing. Here, we used a knock-out mouse model to examine whether TASK-2 (K2P5), an alkaline-activated background K(+) channel, contributes to RTN neuronal pH sensitivity. We made patch-clamp recordings in brainstem slices from RTN neurons that were identified by expression of GFP (directed by the Phox2b promoter) or ß-galactosidase (from the gene trap used for TASK-2 knock-out). Whereas nearly all RTN cells from control mice were pH sensitive (95%, n = 58 of 61), only 56% of GFP-expressing RTN neurons from TASK-2(-/-) mice (n = 49 of 88) could be classified as pH sensitive (>30% reduction in firing rate from pH 7.0 to pH 7.8); the remaining cells were pH insensitive (44%). Moreover, none of the recorded RTN neurons from TASK-2(-/-) mice selected based on ß-galactosidase activity (a subpopulation of GFP-expressing neurons) were pH sensitive. The alkaline-activated background K(+) currents were reduced in amplitude in RTN neurons from TASK-2(-/-) mice that retained some pH sensitivity but were absent from pH-insensitive cells. Finally, using a working heart-brainstem preparation, we found diminished inhibition of phrenic burst amplitude by alkalization in TASK-2(-/-) mice, with apneic threshold shifted to higher pH levels. In conclusion, alkaline-activated TASK-2 channels contribute to pH sensitivity in RTN neurons, with effects on respiration in situ that are particularly prominent near apneic threshold.

Silencing of the tandem pore domain halothane-inhibited K+ channel 2 (THIK2) relies on combined intracellular retention and low intrinsic activity at the plasma membrane.

The tandem pore domain halothane-inhibited K(+) channel 1 (THIK1) produces background K(+) currents. Despite 62% amino acid identity with THIK1, THIK2 is not active upon heterologous expression. Here, we show that this apparent lack of activity is due to a unique combination of retention in the endoplasmic reticulum and low intrinsic channel activity at the plasma membrane. A THIK2 mutant containing a proline residue (THIK2-A155P) in its second inner helix (M2) produces K(+)-selective currents with properties similar to THIK1, including inhibition by halothane and insensitivity to extracellular pH variations. Another mutation in the M2 helix (I158D) further increases channel activity and affects current kinetics. We also show that the cytoplasmic amino-terminal region of THIK2 (Nt-THIK2) contains an arginine-rich motif (RRSRRR) that acts as a retention/retrieval signal. Mutation of this motif in THIK2 induces a relocation of the channel to the plasma membrane, resulting in measurable currents, even in the absence of mutations in the M2 helix. Cell surface delivery of a Nt-THIK2-CD161 chimera is increased by mutating the arginines of the retention motif but also by converting the serine embedded in this motif to aspartate, suggesting a phosphorylation-dependent regulation of THIK2 trafficking.

The thermosensitive potassium channel TREK-1 contributes to coolness-evoked responses of Grueneberg ganglion neurons.

Neurons of the Grueneberg ganglion (GG) residing in the vestibule of the murine nose are activated by cool ambient temperatures. Activation of thermosensory neurons is usually mediated by thermosensitive ion channels of the transient receptor potential (TRP) family. However, there is no evidence for the expression of thermo-TRPs in the GG, suggesting that GG neurons utilize distinct mechanisms for their responsiveness to cool temperatures. In search for proteins that render GG neurons responsive to coolness, we have investigated whether TREK/TRAAK channels may play a role; in heterologous expression systems, these potassium channels have been previously found to close upon exposure to coolness, leading to a membrane depolarization. The results of the present study indicate that the thermosensitive potassium channel TREK-1 is expressed in those GG neurons that are responsive to cool temperatures. Studies analyzing TREK-deficient mice revealed that coolness-evoked responses of GG neurons were clearly attenuated in these animals compared with wild-type conspecifics. These data suggest that TREK-1 channels significantly contribute to the responsiveness of GG neurons to cool temperatures, further supporting the concept that TREK channels serve as thermoreceptors in sensory cells. Moreover, the present findings provide the first evidence of how thermosensory GG neurons are activated by given temperature stimuli in the absence of thermo-TRPs.

Mechanistic basis of the dynamic response of TWIK1 ionic selectivity to pH.

Highly selective for K+ at neutral pH, the TWIK1 channel becomes permeable to Na+ upon acidification. Using molecular dynamics simulations, we identify a network of residues involved in this unique property. Between the open and closed states previously observed by electron microscopy, molecular dynamics simulations show that the channel undergoes conformational changes between pH 7.5-6 involving residues His122, Glu235, Lys246 and Phe109. A complex network of interactions surrounding the selectivity filter at high pH transforms into a simple set of stronger interactions at low pH. In particular, His122 protonated by acidification moves away from Lys246 and engages in a salt bridge with Glu235. In addition, stacking interactions between Phe109 and His122, which stabilize the selectivity filter in its K+-selective state at high pH, disappear upon acidification. This leads to dissociation of the Phe109 aromatic side chain from this network, resulting in the Na+-permeable conformation of the channel.

Task2 potassium channels set central respiratory CO2 and O2 sensitivity.

Task2 K(+) channel expression in the central nervous system is surprisingly restricted to a few brainstem nuclei, including the retrotrapezoid (RTN) region. All Task2-positive RTN neurons were lost in mice bearing a Phox2b mutation that causes the human congenital central hypoventilation syndrome. In plethysmography, Task2(-/-) mice showed disturbed chemosensory function with hypersensitivity to low CO(2) concentrations, leading to hyperventilation. Task2 probably is needed to stabilize the membrane potential of chemoreceptive cells. In addition, Task2(-/-) mice lost the long-term hypoxia-induced respiratory decrease whereas the acute carotid-body-mediated increase was maintained. The lack of anoxia-induced respiratory depression in the isolated brainstem-spinal cord preparation suggested a central origin of the phenotype. Task2 activation by reactive oxygen species generated during hypoxia could silence RTN neurons, thus contributing to respiratory depression. These data identify Task2 as a determinant of central O(2) chemoreception and demonstrate that this phenomenon is due to the activity of a small number of neurons located at the ventral medullary surface.

A need for exhaustive and standardized characterization of ion channels activity. The case of KV11.1.

hERG, the pore-forming subunit of the rapid component of the delayed rectifier K+ current, plays a key role in ventricular repolarization. Mutations in the KCNH2 gene encoding hERG are associated with several cardiac rhythmic disorders, mainly the Long QT syndrome (LQTS) characterized by prolonged ventricular repolarization, leading to ventricular tachyarrhythmias, sometimes progressing to ventricular fibrillation and sudden death. Over the past few years, the emergence of next-generation sequencing has revealed an increasing number of genetic variants including KCNH2 variants. However, the potential pathogenicity of the majority of the variants remains unknown, thus classifying them as variants of uncertain significance or VUS. With diseases such as LQTS being associated with sudden death, identifying patients at risk by determining the variant pathogenicity, is crucial. The purpose of this review is to describe, on the basis of an exhaustive examination of the 1322 missense variants, the nature of the functional assays undertaken so far and their limitations. A detailed analysis of 38 hERG missense variants identified in Long QT French patients and studied in electrophysiology also underlies the incomplete characterization of the biophysical properties for each variant. These analyses lead to two conclusions: first, the function of many hERG variants has never been looked at and, second, the functional studies done so far are excessively heterogeneous regarding the stimulation protocols, cellular models, experimental temperatures, homozygous and/or the heterozygous condition under study, a context that may lead to conflicting conclusions. The state of the literature emphasizes how necessary and important it is to perform an exhaustive functional characterization of hERG variants and to standardize this effort for meaningful comparison among variants. The review ends with suggestions to create a unique homogeneous protocol that could be shared and adopted among scientists and that would facilitate cardiologists and geneticists in patient counseling and management.