Mutations in the ClC-7/Ostm1 ion transporter lead to osteopetrosis and lysosomal storage disease. Its lysosomal localization hitherto precluded detailed functional characterization. Using a mutated ClC-7 that reaches the plasma membrane, we now show that both the aminoterminus and transmembrane span of the Ostm1 b-subunit are required for ClC-7 Cl À /H þ -exchange, whereas the Ostm1 transmembrane domain suffices for its ClC-7-dependent trafficking to lysosomes. ClC-7/Ostm1 currents were strongly outwardly rectifying owing to slow gating of ion exchange, which itself displays an intrinsically almost linear voltage dependence. Reversal potentials of tail currents revealed a 2ClSeveral disease-causing CLCN7 mutations accelerated gating. Such mutations cluster to the second cytosolic cystathionine-b-synthase domain and potential contact sites at the transmembrane segment. Our work suggests that gating underlies the rectification of all endosomal/ lysosomal CLCs and extends the concept of voltage gating beyond channels to ion exchangers.

Background: ClC-7 is a homodimeric lysosomal chloride transporter important for lysosomal function and bone degradation. Results: Altered gating kinetics of one subunit affect the kinetics of the other subunit. Conclusion: Gating of ClC-7 involves both CLC subunits and requires noncovalent binding of cytoplasmic domains. Significance: Osteopetrosis and lysosomal storage disease are associated with accelerating mutations in the ClC-7 C terminus and the contacting intramembrane part.

Docosahexaenoic acid (DHA), a polyunsaturated ω-3 fatty acid enriched in oily fish, contributes to better health by affecting multiple targets. Large-conductance Ca 2+ -and voltage-gated Slo1 BK channels are directly activated by nanomolar levels of DHA. We investigated DHA-channel interaction by manipulating both the fatty acid structure and the channel composition through the site-directed incorporation of unnatural amino acids. Electrophysiological measurements show that the para-group of a Tyr residue near the ion conduction pathway has a critical role. To robustly activate the channel, ionization must occur readily by a fatty acid for a good efficacy, and a long nonpolar acyl tail with a Z double bond present at the halfway position for a high affinity. The results suggest that DHA and the channel form an ion-dipole bond to promote opening and demonstrate the channel druggability. DHA, a marine-derived nutraceutical, represents a promising lead compound for rational drug design and discovery. -4,7,10,13,16,19-hexaenoic acid], found abundantly in oily fish, are believed to have many health-promoting effects (1). For example, select ω-3 PUFAs may decrease cardiovascular disease risk (2). Furthermore, human studies suggest that ω-3 PUFAs may lower blood pressure in some individuals (3). Thus, PUFAs such as DHA are natural nutraceuticals with great potential as lead compounds for rational drug design and discovery. To be successful, such an effort requires clear mechanistic understanding of the interactions between PUFAs and their effectors (4); however, the molecular targets of PUFAs and the mechanisms of action are only beginning to be revealed.One of the high-affinity targets of PUFAs is the large-conductance Ca 2+ -and voltage-gated K + (Slo1 BK) channel (5), whose activation in vascular smooth muscle cells facilitates vessel relaxation (6). Vascular Slo1 BK channels made of four pore-forming Slo1 and auxiliary β1 subunits (Slo1 + β1) are potently and reversibly activated by DHA with a nanomolar level of EC 50 , and this effect contributes to the hypotensive action of DHA in wild-type mice but not in mice with the gene encoding the channel (KCNMA1) disrupted (5).The function of the Slo1 BK channel can be electrophysiologically monitored with high precision to produce quantitative results amenable to mechanistic interpretations (7,8). Such in vitro studies show that DHA biases the ion conduction gate of the channel toward the open conformation without any need for activation of the channel's Ca 2+ sensors in the intracellular domain or transmembrane voltage sensor domains (VSDs) (5). Structurally, the action of DHA depends on a Tyr residue [Y318 in human Slo1 (hSlo1)] near the intracellular end of the ion conduction pathway; the mutation Y318S abolishes the activating effect of DHA (9).The exact role of Y318 in mediating the effect of DHA, however, is not clear. Some clues may be gained from interactions of other proteins with PUFAs. Extracellular DHA regulates voltagegated K + channels by interacting with ...

Background and Purpose— Familial hemiplegic migraine is characterized by recurrent migraine, hemiparesis, and ataxia. Causes may be mutations in calcium and sodium channels or in a subunit of the Na/K-ATPse. Migraine treatment with calcium channel blockers was only successful in some patients. Summary of Case— We describe a 6-year-old girl with recurrent ischemic strokes after minor head trauma associated with seizures, hemiparesis, fever, and altered consciousness. Genetic analysis revealed a spontaneous, novel dominant CACNA1A mutation (c.4046G→A, p.R1349Q) that removed a highly conserved arginine of the voltage sensing region of the P/Q-type Ca v 2.1 channel. Because a homologous mutation in the tottering-5J mouse increased open probability of the channel as well as calcium influx, we treated the patient with the calcium channel blocker verapamil during characteristic prodromi after head trauma. Treatment was instantly effective and prevented a new stroke. Conclusion— CACNA1A mutations should be considered in the diagnostic workup of childhood stroke, especially if associated with ataxia and migraine.