Clinical practice with steroid therapy for Duchenne muscular dystrophy: An expert survey in Asia and Oceania.

BACKGROUND: Several studies on clinical practice for Duchenne muscular dystrophy (DMD) have been conducted in Western countries. However, there have been only a few similar studies in Asia and Oceania. Here, we investigate the steroid therapy-related clinical practice for DMD among the local experts. In 2015, we conducted a DMD expert survey in Asia and Oceania to acquire information regarding patients with DMD and to assess current clinical practice with the cooperation of Asian and Oceanian Myology Centre, a neuromuscular disease research network. RESULTS: We obtained survey responses from 87 out of 148 clinicians (62%) from 13 countries and regions. In China, 1385 DMD patients were followed-up by 5 respondent neurologists, and 84% were between 0 and 9 years of age (15% were 10-19 years, 1% > 19 years). While in Japan, 1032 patients were followed-up by 20 clinicians, and the age distribution was similar between the 3 groups (27% were 0-9 years, 35% were 10-19 years, 38% were >19 years). Most respondent clinicians (91%) were aware of DMD standard of care recommendations. Daily prednisolone/prednisone administration was used most frequently at initiation (N = 45, 64%). Inconsistent opinion on steroid therapy after loss of ambulation and medication for bone protection was observed. CONCLUSIONS: Rare disease research infrastructures have been underdeveloped in many of Asian and Oceanian countries. In this situation, our results show the snapshots of current medical situation and clinical practice in DMD. For further epidemiological studies, expansion of DMD registries is necessary.

The glass micropipette electrode: A history of its inventors and users to 1950.

Soon after the glass micropipette was invented as a micro-tool for manipulation of single bacteria and the microinjection and microsurgery of living cells, it was seen to hold promise as a microelectrode to stimulate individual cells electrically and to study electrical potentials in them. Initial successes and accurate mechanistic explanations of the results were achieved in giant plant cells in the 1920s. Long known surface electrical activity in nerves and muscles was only resolved at a similar cellular level in the 1930s and 1940s after the discovery of giant nerve fibers and the development of finer tipped microelectrodes for normal-sized cells.

Movement of hClC-1 C-termini during common gating and limits on their cytoplasmic location.

Functionally, the dimeric human skeletal muscle chloride channel hClC-1 is characterized by two distinctive gating processes, fast (protopore) gating and slow (common) gating. Of these, common gating is poorly understood, but extensive conformational rearrangement is suspected. To examine this possibility, we used FRET (fluorescence resonance energy transfer) and assessed the effects of manipulating the common-gating process. Closure of the common gate was accompanied by a separation of the C-termini, whereas, with opening, the C-termini approached each other more closely. These movements were considerably smaller than those seen in ClC-0. To estimate the C-terminus depth within the cytoplasm we constructed a pair of split hClC-1 fragments tagged extracellularly and intracellularly respectively. These not only combined appropriately to rescue channel function, but we detected positive FRET between them. This restricts the C-termini of hClC-1 to a position close to its membrane-resident domain. From mutants in which fast or common gating were affected, FRET revealed a close linkage between the two gating processes with the carboxyl group of Glu²³² apparently acting as the final effector for both.

Inter-subunit communication and fast gate integrity are important for common gating in hClC-1.

Proteins of the CLC family are comprised of two subunits, each with its own fast-gated protopore, both of these being regulated simultaneously by a slower common gate. Based on the X-ray crystal structure of a bacterial CLC, the carboxyl side chain of glutamate residue E232 has been proposed as the fast gate of hClC-1, swinging into each pore to close it and competing with chloride. We now show, using hClC-1 mutants expressed in whole-cell patch-clamped HEK293 cells, that elimination of this side chain in the E232Q mutation prevents fast gate closure at all voltages but common gating is also eliminated suggesting that E232 could be the final effector of both fast and common gating. We hypothesise that the conformational information essential for common gating flows between the two E232 protopore residues across the intra-membrane interface, rather than via any cytoplasmic carboxyl-tail interface, to drive common gating. Informed by in silico modelling, we have produced five site-directed mutants that increase the volumes of residues which might be involved in allosteric transfer (A272V, A272L, S289L, V292L and T293L) and assessed them for effects on gating. These mutations could be expected to increase molecular forces between, or torques around, the intimate L287-L287 and I290-I290 contacts that form the pseudo-asymmetric axis of the hClC-1 dimer. Common gating is practically eliminated in V292L and open probability is shifted to more depolarised potentials in A272V, S289L and T293L mainly by altering the voltage dependence of common gating.

Functional study of cytoplasmic loops of human skeletal muscle chloride channel, hClC-1.

The membrane-resident domain of chloride channels and transporters of the CLC family is composed of 18 alpha-helices (designated A to R) connected sequentially by extracellular and intracellular loops, whose functional characteristics are generally unclear. To study the relevance of the intracellular loops linking helices D and E, F and G, H and I and J and K, alanine-exchange mutagenesis, split channel strategy, GST (glutathione transferase)-pull-down methods and whole-cell patch-clamp recordings were used. We investigated the possible roles of these loops in binding to the cytoplasmic, carboxyl tail (C-tail) of the protein, as well as their physiological roles in channel function. Although other interacting positions are conceivable, our results indicate that there is unlikely to be significant binding between the C-tail and any one of these individual cytoplasmic loops. Particular loops might, however, be essential for some channel characteristics. For example, alanine-exchange mutation of the loop linking helix D to E eliminated channel currents; of the loop linking helix H to I caused a significant shift of the open probability of fast gating (P(o)(f)), towards more positive voltages; and of the loop linking helix J to K locked the common gating of hClC-1 open. Therefore, the gating mechanisms of hClC-1 might not only involve the helices and the C-tail, but also involve some of the loops.

Analysis of carboxyl tail function in the skeletal muscle Cl- channel hClC-1.

Human ClC-1 (skeletal muscle Cl- channel) has a long cytoplasmic C-tail (carboxyl tail), containing two CBS (cystathionine beta-synthase) domains, which is very important for channel function. We have now investigated its significance further, using deletion and alanine-scanning mutagenesis, split channels, GST (glutathione transferase)-pull-down and whole-cell patch-clamping. In tagged split-channel experiments, we have demonstrated strong binding between an N-terminal membrane-resident fragment (terminating mid-C-tail at Ser(720) and containing CBS1) and its complement (containing CBS2). This interaction is not affected by deletion of some sequences, suggested previously to be important, particularly in channel gating. Contact between CBS1 and CBS2, however, may make a major contribution to assembly of functional channels from such co-expressed complements, although the possibility that C-tail fragments could, in addition, bind to other parts of the membrane-resident component has not been eliminated. We now show such an interaction between a membrane-resident component terminating at Ser(720) (but with CBS1 deleted) and a complete C-tail beginning at Leu(598). Channel function is rescued in patch-clamped HEK-293T (human embryonic kidney) cells co-expressing these same fragments. From our own results and those of others, we conclude that the CBS1-CBS2 interaction is not sufficient, in itself, for channel assembly, but rather that this might normally assist in bringing some part of the CBS2/C-tail region into appropriate proximity with the membrane-resident portion of the protein. Previously conflicting and anomalous results can now be explained by an hypothesis that, for split channels to be functional, at least one membrane-resident component must include a plasma membrane trafficking signal between Leu(665) and Lys(680).

Stem cell treatment of dystrophic dogs.

Human muscular dystrophies are devastating and incurable inherited diseases. Hopes of progress towards therapy of muscular dystrophies were aroused when Sampaolesi et al. reported "extensive recovery of dystrophin expression, normal muscle...function", and "remarkable clinical amelioration" in golden retriever muscular dystrophy dogs treated with 'mesoangioblasts'. Here I re-examine their results, showing how their assessments might be flawed and their conclusions overstated. Further studies will be required to evaluate fully the clinical potential of this work.

Functional complementation of truncated human skeletal-muscle chloride channel (hClC-1) using carboxyl tail fragments.

Crystal structures of bacterial CLC (voltage-gated chloride channel family) proteins suggest the arrangement of permeation pores and possible gates in the transmembrane region of eukaryotic CLC channels. For the extensive cytoplasmic tails of eukaryotic CLC family members, however, there are no equivalent structural predictions. Truncations of cytoplasmic tails in different places or point mutations result in loss of function or altered gating of several members of the CLC family, suggesting functional importance. In the present study, we show that deletion of the terminal 100 amino acids (N889X) in human ClC-1 (skeletal-muscle chloride channel) has minor consequences, whereas truncation by 110 or more amino acids (from Q879X) destroys channel function. Use of the split channel strategy, co-injecting mRNAs and expressing various complementary constructs in Xenopus oocytes, confirms the importance of the Gln879-Arg888 sequence. A split between the two CBS (cystathionine b-synthase) domains (CBS1 and CBS2) gives normal function (e.g. G721X plus its complement), whereas a partial complementation, eliminating the CBS1 domain, eliminates function. Surprisingly, function is retained even when the region Gly721-Ala862 (between CBS1 and CBS2, and including most of the CBS2 domain) is omitted from the complementation. Furthermore, even shorter peptides from the CBS2-immediate post-CBS2 region are sufficient for functional complementation. We have found that just 26 amino acids from Leu863 to Arg888 are necessary since channel function is restored by co-expressing this peptide with the otherwise inactive truncation, G721X.

Zinc inhibits human ClC-1 muscle chloride channel by interacting with its common gating mechanism.

Transition metals block the muscle Cl- channel ClC-1, which belongs to a large family of double-barreled Cl- channels and transporters. In the Torpedo Cl- channel ClC-0, Zn2+ block is closely related to the common gating mechanism that opens and closes both pores of the channel simultaneously, and the mutation C212S, which locks the common gate open, also eliminates the block. In ClC-1, however, previous results suggested that Zn2+ block is independent of gating, and that the cysteine residues involved in Zn2+ binding are in different positions to those that confer Zn2+ sensitivity on ClC-0. In this work, we show that Zn2+ block of ClC-1 is faster at hyperpolarized potentials where the channel is more likely to be in the closed state. Mutation C277S, equivalent to C212S in ClC-0, which locks the common gate in ClC-1 open, virtually eliminates Zn2+ block. A mutation, V321A, which reduces open probability of the common gate, facilitated Zn2+ block. These results demonstrate that Zn2+ block is state dependent, acting on the common gate. The extent of the block, however, is not a simple function of the open probability of the common gate. The Q10 of approximately 13 of the time course of Zn2+ block, which is significantly higher than the Q10 of common gating transitions in WT ClC-1, suggests that Zn2+ binds to a very high temperature-dependent low-probability closed substate of the common gate, which has not yet been characterized in this channel.

Characterization of three myotonia-associated mutations of the CLCN1 chloride channel gene via heterologous expression.

Two novel mutations of the human CLCN1 chloride channel gene, c.592C>G (p.L198V) and c.2255A>G (p.K752R), are described, occurring coincidentally in the one myotonic patient. These individual mutations and a construct with both mutations in the one cDNA were transcribed and expressed in Xenopus oocytes where channel gating parameters were extracted from chloride currents recorded under voltage clamp. We found that the p.L198V mutation has its major effects on the common (or slow) gate of the chloride channel, as do other dominant ClC-1 mutations, and may therefore be causative of the patient's symptoms (when co-expressed with wild-type human ClC-1, the p.L198V mutation exerts a dominant negative effect on common gating) but the p.K752R mutation appears to be innocuous and may be a benign polymorphism. A third mutant, the recently described c.2795C>T (p.P932L), was expressed in HEK 293 cells. Despite the severity of the disease associated with this mutation, chloride currents in cells expressing p.P932L were not significantly different from those of cells expressing wild-type ClC-1.