ClC proteins are a family of chloride channels and transporters that are found in a wide variety of prokaryotic and eukaryotic cell types. The mammalian voltage-gated chloride channel ClC-1 is important for controlling the electrical excitability of skeletal muscle. Reduced excitability of muscle cells during metabolic stress can protect cells from metabolic exhaustion and is thought to be a major factor in fatigue. Here we identify a novel mechanism linking excitability to metabolic state by showing that ClC-1 channels are modulated by ATP. The high concentration of ATP in resting muscle effectively inhibits ClC-1 activity by shifting the voltage gating to more positive potentials. ADP and AMP had similar effects to ATP, but IMP had no effect, indicating that the inhibition of ClC-1 would only be relieved under anaerobic conditions such as intense muscle activity or ischemia, when depleted ATP accumulates as IMP. The resulting increase in ClC-1 activity under these conditions would reduce muscle excitability, thus contributing to fatigue. We show further that the modulation by ATP is mediated by cystathionine ␤-synthase-related domains in the cytoplasmic C terminus of ClC-1. This defines a function for these domains as gating-modulatory domains sensitive to intracellular ligands, such as nucleotides, a function that is likely to be conserved in other ClC proteins.Skeletal muscle has a high and variable demand for energy, in the form of ATP, and has elaborate systems to maintain the ATP supply. During intense exercise, however, ATP supply may not keep up with demand, and ATP concentration can decrease rapidly. In fast twitch fibers ATP can drop to below 25% of resting concentration within 25 s (1), a rate of ATP consumption that, if it continued, would deplete all ATP within a further 10 s. As the majority of ATP is consumed by the sarcoplasmic reticulum (SR) 5 Ca 2ϩ -ATPase pumping Ca 2ϩ back into the SR after each Ca 2ϩ -activated contraction (2), complete ATP depletion would lead to a rise in cytoplasmic calcium, rigor, and calcium-dependent damage (3, 4). This does not normally occur because force generation and ATP consumption decrease during exercise, compromising short term function but protecting cells from complete metabolic exhaustion. This process is well known as fatigue, but the factors contributing to fatigue remain controversial. A direct reduction in force generation by the contractile apparatus is thought to be a factor early in fatigue (3, 5), but a significant reduction in ATP consumption only occurs with a reduction in SR Ca 2ϩ release (and consequent reuptake) that occurs late in fatigue, correlating with ATP depletion (3). Indeed, ATP depletion and the concomitant increase in cytoplasmic Mg 2ϩ

Ginkgolides are potent blockers of the glycine receptor Cl -channel (GlyR) pore. We sought to identify their binding sites by comparing the effects of ginkgolides A, B and C and bilobalide on a1, a2, a1b and a2b GlyRs. Bilobalide sensitivity was drastically reduced by incorporation of the b subunit. In contrast, the sensitivities to ginkgolides B and C were enhanced by b subunit expression. However, ginkgolide A sensitivity was increased in the a2b GlyR relative to the a2 GlyR but not in the a1b GlyR relative to the a1 GlyR. We hypothesised that the subunit-specific differences were mediated by residue differences at the second transmembrane domain 2¢ and 6¢ pore-lining positions. The increased ginkgolide A sensitivity of the a2b GlyR was transferred to the a1b GlyR by the G2¢A (a1 to a2 subunit) substitution. In addition, the a1 subunit T6¢F mutation abolished inhibition by all ginkgolides. As the ginkgolides share closely related structures, their molecular interactions with pore-lining residues were amenable to mutant cycle analysis. This identified an interaction between the variable R2 position of the ginkgolides and the 2¢ residues of both a1 and b subunits. These findings provide strong evidence for ginkgolides binding at the 2¢ pore-lining position.

Fish-like calcitonins (CTs), such as salmon CT (sCT), are widely used clinically in the treatment of bone-related disorders; however, the molecular basis for CT binding to its receptor, a class II G protein-coupled receptor, is not well defined. In this study we have used photoaffinity labeling to identify proximity sites between CT and its receptor. Calcitonins (CTs)1 are 32-amino acid peptide hormones with a wide spectrum of biological activity. The most recognized action is the inhibition of osteoclast-mediated bone resorption, which forms the basis for its primary clinical use in the treatment of bone-related disorders such as Paget disease, osteoporosis, and hypercalcemia of malignancy (1-3). CT, however, also has activity that includes modulation of renal ion excretion (4 -7), analgesia (8), inhibition of appetite (9), and gastric acid secretion (10 -12), as well as effects on reproduction via effects on embryological implantation and sperm function (13-15).Calcitonin receptors (CTRs) belong to the class II subfamily of G protein-coupled receptors, which also includes receptors for other peptides such as parathyroid hormone (PTH) and PTH-related peptide, secretin, vasoactive intestinal peptide, glucagon, glucagon-like peptide-1, growth hormone-releasing hormone, calcitonin gene-related peptide, and corticotropinreleasing factor. These peptide hormone class II G proteincoupled receptors share 30 -50% amino acid identity as well as a number of conserved structural motifs and are thought to interact with their ligands in a similar manner (16 -18).Alternative RNA splicing yields multiple CTR mRNA isoforms. In man, at least six potential variants exist (19 -26); however, the most common hCTR isoforms differ by the presence (hCTR b ) or absence (hCTR a ) of a 16-amino acid insert between amino acids 174 and 175, within the first intracellular loop of the receptor (23). Of these, the hCTR a is the major human receptor isoform and is expressed in essentially all tissues known to express the CTR.CTs from different species can be subdivided into three major classes: human/rodent, artiodactyl, and teleost/avian. Of these, the members of the teleost/avian group are generally the most potent, although relative potency varies in a species-and isoform-specific manner (19,(27)(28)(29)(30)(31). The higher potency combined with a longer in vivo half-life has led to fish-like CTs, exemplified by salmon CT (sCT), being the principle form of CT used for the clinical treatment of bone disorders (32, 33).However, the usefulness of CT is limited by the development of clinical resistance. This can be due to the development of circulating antibodies against non-human CT (34 -38), but it also occurs from loss of responsiveness to CT, presumably via receptor down-regulation and inhibition of new receptor synthesis (39 -41). The optimal use of CTs remains unresolved, a situation that stems in part from lack of understanding of the bimolecular interaction between CT and its receptor and how

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