Mutations in the ClC-1 muscle chloride channel cause either recessive or dominant myotonia congenita. Using a systematic screening procedure, we have now identified four novel missense mutations in dominant (V286A, F307S) and recessive myotonia (V236L, G285E), and have analysed the effect of these and other recently described mutations (A313T, I556N) on channel properties in the Xenopus oocyte expression system. Mutations V286A, F307S and A313T displayed a 'classical' dominant phenotype: their voltage dependence was shifted towards positive potentials and displayed a dominant-negative effect by significantly imparting a voltage shift on mutant-wild-type heteromeric channels as found in heterozygous patients. In contrast, the recessive mutation V236L also shifted the voltage dependence to positive values, but co-expression with wild-type ClC-1 gave almost wild-type currents. I556N, a mutation found in patients with benign dominant myotonia, drastically shifts the voltage dependence, but only a slight shift is seen when co-expressed with wild-type ClC-1. Thus, the voltage dependence of mutant heteromeric channels is not always intermediate between those of the constituent homomeric channel subunits, a conclusion further supported by mixing different ClC-1 mutants. These complex interactions correlate clinically with various inheritance patterns, ranging from autosomal dominant with various degrees of penetrance to autosomal recessive.

CLC chloride channels form a large and conserved gene family unrelated to other channel proteins. Knowledge of the transmembrane topology of these channels is important for understanding the effects of mutations found in human myotonia and inherited hypercalciuric kidney stone diseases and for the interpretation of structure-function studies. We now systematically study the topology of human ClC-1, a prototype CLC channel that is defective in human myotonia. Using a combination of in vitro glycosylation scanning and protease protection assays, we show that both N and C termini face the cytoplasm and demonstrate the presence of 10 (or less likely 12) transmembrane spans. Difficult regions were additionally tested by inserting cysteines and probing the effect of cysteine-modifying reagents on ClC-1 currents. The results show that D3 crosses the membrane and D4 does not, and that L549 between D11 and D12 is accessible from the outside. Further, since the modification of cysteines introduced between D11 and D12 and at the extracellular end of D3 strongly affect ClC-1 currents, these regions are suggested to be important for ion permeation.Voltage-gated chloride channels of the CLC family are highly conserved during evolution and are expressed in organisms ranging from bacteria (1) and yeast (2) to plants (3) and animals (4). Their physiological functions in higher organisms include the regulation of cell volume, control of electrical excitability, and transepithelial transport.The CLC proteins were identified by expression cloning of the Torpedo chloride channel ClC-0 (5). At present, nine mammalian members are known (for review, see ref. 6). ClC-1 is nearly specific for skeletal muscle (4). It ensures its electrical stability, which is evident from ClC-1 mutations leading to myotonia congenita, a disease characterized by defective muscle relaxation. Many different ClC-1 mutations were found in human myotonia and in animal models (7-11). The importance of CLC channels is underscored by another, recently identified disease. Inactivating mutations of ClC-5 cause a syndrome that is characterized by low molecular weight proteinuria, hypercalciuria, and kidney stones (12). ClC-2 may play a role in cell volume control (13) and in setting the intracellular chloride concentration, which in turn is important for synaptic transmission in certain neurons (14). However, the function of most CLC proteins is still unclear.CLC proteins are structurally unrelated to other ion channel classes. Topological models are still mainly based on hydropathy analysis, suggesting the presence of about 12-13 transmembrane domains, originally termed D1 to D13 (5). This model has already had to be revised to accommodate new experimental findings (15,16).CLC channels function as multimers of identical (11) or homologous subunits (17). Dominant negative mutations suggested that ClC-1 channels have more than three subunits (11).However, ClC-0 is a homodimeric channel with one pore per subunit (16,18,19); this may also apply for .In structure...

We investigated the effect of truncations on the human muscle chloride channel CLC-1 and studied the functional complementation from partial proteins. Almost complete deletion of the cytoplasmic amino terminus did not affect currents, but truncating the intracellular COOH terminus after Leu 720 abolished function. Currents were restored by coexpressing this membraneembedded part with the lacking cytoplasmic fragment that contains domain D13, the second of the two conserved cystathionine ␤-synthase (CBS) motifs present in all eukaryotic CLC proteins. However, if the cut was after Gln 597 before the first CBS domain, no functional complementation was seen.Complementation was also obtained with channels "split" between transmembrane domains D7 and D8 or domains D8 and D9, but not when split between D10 and D11. Specificity of currents was tested by inserting point mutations in NH 2 -terminal (G188A and G230E) or COOH-terminal (K585E) fragments. In contrast to G188A and K585E, split channels did not tolerate the D136G mutation, suggesting that it may impede association from nonlinked fragments. Duplication, but not a lack of domain D8 was tolerated in "split" channels. Membrane domains D9 -D12 can insert into the membrane without adding a preceding signal peptide to ensure the extracellular amino terminus of D9. Eventually, we succeeded in reconstituting CLC-1 channels from three separate polypeptides: the amino-terminal part up to D8, D9 through CBS1, and the remainder of the cytoplasmic carboxyl terminus.In summary, several regions of CLC channels behave autonomously regarding membrane insertion and folding and mediate protein-protein interactions strong enough to yield functional channels without a direct covalent link. CLC1 chloride channels, originally identified by the expression cloning of CLC-0 from Torpedo electric organ (1), form a large gene family with at least nine members in mammals and conservation down to organisms like Escherichia coli and yeast (for review, see Ref.2). Their importance is underscored by human inherited diseases; mutations in the muscle channel CLC-1 lead to myotonia (3, 4), and those in CLC-5 lead to proteinuria and kidney stones (5).CLC proteins are structurally unrelated to other channels, including Cl Ϫ channels like ␥-aminobutyric acid and glycine receptors or the cystic fibrosis transmembrane regulator CFTR. Hydrophobicity analysis indicated 13 hydrophobic domains (D1-D13 (1)). However, newer experimental findings (6 -8) suggest the presence of only 10 (or 12) transmembrane domains ( Fig. 1, top). The topology of the D9 -D12 region, a long hydrophobic stretch interrupted only once by a short hydrophilic segment, still poses problems. Both the amino-and the carboxyl terminus reside in the cytoplasm, and the loop between D8 and D9 is glycosylated.CLC-0 forms homodimers with one pore per subunit (7, 9, 10), and this may also apply for CLC-1 (11). This one-proteinone-pore architecture distinguishes the CLC channels from voltage-gated cation channels. In shaker K channels, four hom...

Dandruff is a chronic scalp disorder characterized by scaling and itching. A successful anti-dandruff shampoo not only has to provide superior anti-dandruff relief to ensure patient compliance. It also needs to offer excellent cosmetic and hair conditioning benefits at the same time. In this study, the efficacy of a shampoo containing 0.5% piroctone olamine and 0.45% climbazole (shampoo 1) was compared with a widely available commercial shampoo containing 1% zinc pyrithione (shampoo 2). In vitro studies investigating the anti-mycotic efficacy of a combination of 0.5% piroctone olamine and 0.45% climbazole as well as 1% zinc pyrithione were performed. To study substantivity, pig skin punches were used as a model system and a test of wet combability was performed to characterize combing ease. In vivo home-in-use studies were carried out to determine the efficacy of both shampoos to improve scalp condition and reduce itching in subjects suffering from moderate to severe dandruff. Results demonstrated a comparable anti-fungal effectiveness for 0.5% piroctone olamine plus 0.45% climbazole and 1% zinc pyrithione, respectively. Shampoo 1 showed a significantly higher anti-mycotics substantivity compared to shampoo 2. After treatment with shampoo 1, the wet combing force was significantly reduced compared with shampoo 2, suggesting a better combability following the use of shampoo 1. In an in vivo split head design study, shampoo 1 was shown to be equally effective in reducing the amount of dandruff on the scalp compared with shampoo 2. The approval rate of volunteers regarding the question 'The use of this shampoo decreases the itching of my scalp?' after a 4-week treatment with shampoo 1 equaled 90%. Overall, the shampoo formulation with 0.5% piroctone olamine and 0.45% climbazole effectively reduces the amount of dandruff and, at the same time, provides hair conditioning advantages.

In this study, it was investigated how estrogens (17-beta-estradiol, E2) affect the estrogen receptor (ER) expression and gene regulation of male versus female human scalp hair follicles in vitro. Anagen VI follicles from frontotemporal scalp skin were microdissected and organ-cultured for up to 9 d in the presence of E2 (1-100 nm). Immunohistochemistry was performed for ERbeta-expression, known to be predominant in human scalp hair follicles, and for TGF-beta2-expression (as negative key hair growth modulator), and E2-responsive genes in organ-cultured human scalp hair follicles (48 h, 10 nM) were explored by cDNA microarray, using a commercial skin focus chip (Memorec, Cologne, Germany). The distribution pattern of ERbeta and TGF-beta2-immunoreactivity differed between male and female hair follicles after 48 h culture. Of 1300 genes tested, several genes were regulated sex-dependent differently. The study reveals substantial sex-dependent differences in the response of frontotemporal human scalp hair follicles to E2. Recognition and systematic dissection of the E2-dependent gene regulation will be crucial for the development of more effective, gender-tailored management strategies for female versus male pattern balding.

Our results show that the treatment of human sebocytes with L-carnitine significantly augments β-oxidation and significantly decreases intracellular lipid content in human sebocytes. Topically applied L-carnitine is bioavailable and leads to a significant sebum reduction in vivo. In conclusion, L-carnitine represents a valuable compound, produced naturally within the body, for the topical treatment of oily skin in humans.

We have previously shown that precursors of odorous components characteristic of axillary sweat are hardly detectable or undetectable in individuals carrying the 538G > A SNP in the ABCC11 transporter gene. However, it is unclear, whether ABCC11 is directly involved in the transport of these compounds. To approach this question, transport of peptide-conjugated potential precursors of 3-methyl-3-sulfanylhexanol (3M3SH), a key determinant of axillary malodour, was measured using membrane vesicles of Sf9 insect cells overexpressing human ABCC11. Whilst no ABCC11-mediated transport was detected for the dipeptide precursor Cys-Gly-3M3SH, the glutathione conjugate of 3M3SH (SG-3M3SH) was robustly taken up by ABCC11 at a transport rate of 0.47 pmol/mg/min. Collectively, these results illuminate SG-3M3SH as a putative precursor of 3M3SH, which then may undergo intra-vesicular maturation to generate Cys-Gly-3M3SH. Critically, the apocrine sweat gland was demonstrated to express γ-glutamyl transferase 1 (GGT1) protein, which is known to catalyse the deglutamylation of glutathionyl conjugates. Additionally, we provide evidence that recombinant and isolated hepatic human GGT1 is capable of transforming SG-3M3SH to Cys-Gly-3M3SH in vitro. To sum up, we demonstrate that the functionality of ABCC11 is likely to play an important role in the generation of axillary malodour. Furthermore, we identify GGT1 as a key enzyme involved in the biosynthesis of Cys-Gly-3M3SH.

Mouse models show that experimental stress mimicking prolonged life-stress exposure enhances neurogenic inflammation, induces adaptive immunity cytokine-imbalance characterized by a shift to Type 1 T-helper cell cytokines and increases apoptosis of epithelial cells. This affects hair growth in otherwise healthy animals. In this study, we investigate whether a prolonged naturalistic life-stress exposure affects cytokine balance and hair parameters in healthy humans. 33 (18 exam, 15 comparison) female medical students with comparable sociobiological status were analyzed during a stressful final examination period, at three points in time (T) 12 weeks apart. T1 was before start of the learning period, T2 between the three-day written exam and an oral examination, and T3 after a 12 week rest and recovery from the stress of the examination period. Assessments included: self-reported distress and coping strategies (Perceived Stress Questionnaire [PSQ], Trier Inventory for the Assessment of Chronic Stress [TICS]), COPE), cytokines in supernatants of stimulated peripheral blood mononucleocytes (PBMCs), and trichogram (hair cycle and pigmentation analysis). Comparison between students participating in the final medical exam at T2 and non-exam students, revealed significantly higher stress perception in exam students. Time-wise comparison revealed that stress level, TH1/TH2 cytokine balance and hair parameters changed significantly from T1 to T2 in the exam group, but not the control. However, no group differences were found for cytokine balance or hair parameters at T2. The study concludes that in humans, naturalistic stress, as perceived during participation in a major medical exam, has the potential to shift the immune response to TH1 and transiently hamper hair growth, but these changes stay within a physiological range. Findings are instructive for patients suffering from hair loss in times of high stress. Replication in larger and more diverse sample populations is required, to assess suitability of trichogram analysis as biological outcome for stress studies.