Elena Babini

Mutation in the neuronal voltage-gated sodium channel SCN1A in familial hemiplegic migraine

BACKGROUNDnFamilial hemiplegic migraine is an autosomal dominant severe subtype of migraine with aura characterised by some degree of hemiparesis during the attacks. So far, mutations in two genes regulating ion translocation-CACNA1A and ATP1A2-have been identified in pedigrees with this disease.nnnMETHODSnTo identify additional genes for familial hemiplegic migraine, we did a genome-wide linkage analysis of two disease pedigrees without mutations in CACNA1A and ATP1A2. Ion channel genes in the candidate interval were analysed for mutations, and the functional consequences of the recorded sequence alteration were determined.nnnFINDINGSnWe identified a novel locus for familial hemiplegic migraine on chromosome 2q24. Sequencing of candidate genes in this region revealed a heterozygous missense mutation (Gln1489Lys) in the neuronal voltage-gated sodium channel gene SCN1A, mutations of which have been associated with epilepsy. This same mutation was present in three families with familial hemiplegic migraine. It results in a charge-altering aminoacid exchange in the so-called hinged-lid domain of the protein, which is critical for fast inactivation of the channel. Whole-cell recordings in transiently transfected tsA201 cells expressing the highly homologous SCN5A sodium channel showed that the mutation induces a two-fold to four-fold accelerated recovery from fast inactivation without altering any of the other channel parameters investigated.nnnINTERPRETATIONnDysfunction of the neuronal sodium channel SCN1A can cause familial hemiplegic migraine. Our findings have implications for the understanding of migraine aura. Moreover, our study reinforces the molecular links between migraine and epilepsy, two common paroxysmal disorders.

Systematic analysis of three FHM genes in 39 sporadic patients with hemiplegic migraine

Background: Familial (FHM) and sporadic (SHM) hemiplegic migraine are severe subtypes of migraine associated with transient hemiparesis. For FHM, three genes have been identified encoding subunits of a calcium channel (CACNA1A), a sodium–potassium pump (ATP1A2), and a sodium channel (SCN1A). Their role in SHM is unknown. Establishing a genetic basis for SHM may further the understanding of its pathophysiology and relationship with common types of migraine. It will also facilitate the often difficult differential diagnosis from other causes of transient hemiparesis. Methods: We systematically scanned 39 well-characterized patients with SHM without associated neurologic features for mutations in the three FHM genes. Functional assays were performed for all new sequence variants. Results: Sequence variants were identified in seven SHM patients: one CACNA1A mutation, five ATP1A2 mutations, and one SCN1A polymorphism. All six mutations caused functional changes in cellular assays. One SHM patient later changed to FHM because another family member developed FHM attacks. Conclusion: We show that FHM genes are involved in at least a proportion of SHM patients without associated neurologic symptoms. Screening of ATP1A2 offers the highest likelihood of success. Because FHM gene mutations were also found in family members with “nonhemiplegic” typical migraine with and without aura, our findings reinforce the hypothesis that FHM, SHM, and “normal” migraine are part of a disease spectrum with shared pathogenetic mechanisms.

Activation and Inhibition of Kidney CLC-K Chloride Channels by Fenamates

CLC-K Cl– channels are selectively expressed in kidney and ear, where they are pivotal for salt homeostasis, and loss-of-function mutations of CLC-Kb produce Bartters syndrome type III. The only ligand known for CLC-K channels is a derivative of the 2-p-chlorophenoxypropionic acid (CPP), 3-phenyl-CPP, which blocks CLC-Ka, but not CLC-Kb. Here we show that in addition to this blocking site, CLC-K channels bear an activating binding site that controls channel opening. Using the voltage-clamp technique on channels expressed in Xenopus laevis oocytes, we found that niflumic acid (NFA) increases CLC-Ka and CLC-Kb currents in the 10 to 1000 μM range. Flufenamic acid (FFA) derivatives or high doses of NFA produced instead an inhibitory effect on CLC-Ka, but not on CLC-Kb, and on blocker-insensitive CLC-Ka mutants, indicating that the activating binding site is distinct from the blocker site. Evaluation of the sensitivity of CLC-Ka to derivatives of NFA and FFA together with a modeling study of these ligands allow us to conclude that one major characteristic of activating compounds is the coplanarity of the two rings of the molecules, whereas block requires a noncoplanar configuration. These molecules provide a starting point for identification of diuretics or drugs useful in the treatment of Bartters syndrome.

Molecular switch for CLC-K Cl− channel block/activation: Optimal pharmacophoric requirements towards high-affinity ligands

ClC-Ka and ClC-Kb Cl− channels are pivotal for renal salt reabsorption and water balance. There is growing interest in identifying ligands that allow pharmacological interventions aimed to modulate their activity. Starting from available ligands, we followed a rational chemical strategy, accompanied by computational modeling and electrophysiological techniques, to identify the molecular requisites for binding to a blocking or to an activating binding site on ClC-Ka. The major molecular determinant that distinguishes activators from blockers is the level of planarity of the aromatic portions of the molecules: only molecules with perfectly coplanar aromatic groups display potentiating activity. Combining several molecular features of various CLC-K ligands, we discovered that phenyl-benzofuran carboxylic acid derivatives yield the most potent ClC-Ka inhibitors so far described (affinity <10 μM). The increase in affinity compared with 3-phenyl-2-p-chlorophenoxy-propionic acid (3-phenyl-CPP) stems primarily from the conformational constraint provided by the phenyl-benzofuran ring. Several other key structural elements for high blocking potency were identified through a detailed structure–activity relationship study. Surprisingly, some benzofuran-based drugs inhibit ClC-Kb with a similar affinity of <10 μM, thus representing the first inhibitors for this CLC-K isoform identified so far. Based on our data, we established a pharmacophore model that will be useful for the development of drugs targeting CLC-K channels.

Niflumic acid inhibits chloride conductance of rat skeletal muscle by directly inhibiting the CLC‐1 channel and by increasing intracellular calcium

Given the crucial role of the skeletal muscle chloride conductance (gCl), supported by the voltage‐gated chloride channel CLC‐1, in controlling muscle excitability, the availability of ligands modulating CLC‐1 are of potential medical as well as toxicological importance. Here, we focused our attention on niflumic acid (NFA), a molecule belonging to the fenamates group of non‐steroidal anti‐inflammatory drugs (NSAID).

A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels

The two human CLC Cl− channels, ClC-Ka and ClC-Kb, are almost exclusively expressed in kidney and inner ear epithelia. Mutations in the genes coding for ClC-Kb and barttin, an essential CLC-K channel β subunit, lead to Bartter syndrome. We performed a biophysical analysis of the modulatory effect of extracellular Ca2+ and H+ on ClC-Ka and ClC-Kb in Xenopus oocytes. Currents increased with increasing [Ca2+]ext without full saturation up to 50 mM. However, in the absence of Ca2+, ClC-Ka currents were still 20% of currents in 10 mM [Ca2+]ext, demonstrating that Ca2+ is not strictly essential for opening. Vice versa, ClC-Ka and ClC-Kb were blocked by increasing [H+]ext with a practically complete block at pH 6. Ca2+ and H+ act as gating modifiers without changing the single-channel conductance. Dose–response analysis suggested that two protons are necessary to induce block with an apparent pK of ∼7.1. A simple four-state allosteric model described the modulation by Ca2+ assuming a 13-fold higher Ca2+ affinity of the open state compared with the closed state. The quantitative analysis suggested separate binding sites for Ca2+ and H+. A mutagenic screen of a large number of extracellularly accessible amino acids identified a pair of acidic residues (E261 and D278 on the loop connecting helices I and J), which are close to each other but positioned on different subunits of the channel, as a likely candidate for forming an intersubunit Ca2+-binding site. Single mutants E261Q and D278N greatly diminished and the double mutant E261Q/D278N completely abolished modulation by Ca2+. Several mutations of a histidine residue (H497) that is homologous to a histidine that is responsible for H+ block in ClC-2 did not yield functional channels. However, the triple mutant E261Q/D278N/H497M completely eliminated H+ -induced current block. We have thus identified a protein region that is involved in binding these physiologically important ligands and that is likely undergoing conformational changes underlying the complex gating of CLC-K channels.

Channel or transporter? The CLC saga continues

It was recently shown that the putative bacterial Cl− channel, ClC‐ec1, is in reality a Cl−–H+ antiporter. Our group has now shown that this is also the case for two human CLCs, ClC‐4 and ClC‐5. We found that the flux of Cl− in one direction is stoichiometrically coupled to the movement of protons in the opposite direction, unveiling a behaviour that is typical of a transporter rather than a channel. This discovery will surely stimulate further research to elucidate the molecular elements responsible for the behaviour as a transporter. On the physiological level, the antiport activity of ClC‐4/ClC‐5 must lead to a review of the role of CLC proteins in intracellular compartments. Small organic molecules have been extremely useful tools for studying ion channels and many commercial drugs target specific ion channel proteins. Several blockers have been found to inhibit the plasma membrane‐localized CLC channels ClC‐0, ClC‐1 and ClC‐Ka. These compounds include 9‐anthracene‐carboxylic acid (9‐AC), p‐chlorophenoxy‐propionic acid (CPP) and its derivatives, and 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS). Two different binding sites have been identified, one extracellular and one intracellular. However, high‐affinity ligands for most CLC proteins are still missing. Apart from being useful biophysical tools, such drugs may provide a way to modulate protein function in vivo. With these tasks to be accomplished, it is definitely an exciting time in the chloride transport field.

Identification of sites responsible for the potentiating effect of niflumic acid on ClC‐Ka kidney chloride channels

Background and purpose:u2002 ClC‐K kidney Cl– channels are important for renal and inner ear transepithelial Cl– transport, and are potentially interesting pharmacological targets. They are modulated by niflumic acid (NFA), a non‐steroidal anti‐inflammatory drug, in a biphasic way: NFA activates ClC‐Ka at low concentrations, but blocks the channel above ∼1u2003mM. We attempted to identify the amino acids involved in the activation of ClC‐Ka by NFA.