Amphotericin B (AmB) is a membrane-active antibiotic which has been shown to increase ion and small molecule permeability in a variety of model and biological membrane systems. A major mechanistic model, based on BLM systems, proposes that amphotericin forms barrellike pores with cholesterol which are cation selective when added to one side of the membrane and anion selective when added to both sides. We have tested this hypothesis on small and reverse-phase large unilamellar vesicles (SUV and REV) with and without cholesterol. The method used to measure K+, Cl-, and net ion currents is based on ion/H+ exchange detected by the entrapped pH probe pyranine. We find that AmB forms channels which have net selectivity for K+ over Cl- regardless of sidedness or sterol content in SUV. REV with 10% cholesterol also show net K+ selectivity with double-sided addition. Differences are noted between cholesterol- and non-sterol-containing vesicles consistent with at least two separate modes of action: (1) cholesterol-containing SUV form some larger diameter pores which allow the passage of larger ions especially when added to both sides; (2) SUV without sterol form pores which are still K+ over Cl- selective, but larger ions do not pass. The latter mode of action precludes a sterol/pore type of model but not necessarily a barrellike model consisting only of amphotericin molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Currents through the human skeletal muscle chloride channel hClC-1 can be blocked by external application of 1 mM Zn 2؉ or the histidine-reactive compound diethyl pyrocarbonate (DEPC). The current block by Zn 2؉ strongly depends on the external pH (pK a near 6.9), whereas the block by DEPC is rather independent of the pH in the range of 5.5 to 8.5. To identify the target sites of these reagents, we constructed a total of twelve cysteine-and/or histidine-replacement mutants, transfected tsA201 cells with them, and investigated the resulting whole-cell chloride currents. The majority of the mutants exhibited a similar sensitivity toward Zn 2؉ or DEPC as wild type (WT) channels. Block by 1 mM Zn 2؉was nearly absent only with the mutant C546A. Four mutants (C242A, C254A, H180A, and H451A) were slightly less sensitive to Zn 2؉ than WT. Tests with double, triple, and quadruple mutants yielded that, in addition to C546, C242 and C254 are also most likely participating in Zn 2؉ -binding.The main chloride channel of human skeletal muscle, hClC-1, 1 is a member of the ClC chloride channel family that is unrelated to any other known ion channels (1). The first membrane topology model of ClC proteins was derived from hydropathy analysis (2); several times it had to be revised on the grounds of firmer experimental evidence (3-5). Certain mutations in the human ClC-1 gene (reviewed in Ref. 6) lead to myotonia, a disease characterized by muscle stiffness. The study of such myotonia-causing mutations provided first insights into the relationships between the primary sequence and the functions of this channel (7-10). Strong evidence suggests regions between transmembrane segments D3 and the end of D5 to participate in the pore-forming structure (11). However, these results are not in agreement with the most recently postulated topology model of ClC-1 (5). Furthermore, studies of ClC-0 (reviewed in Ref. 1), a homologous chloride channel in the electric organ of Torpedo, led to the suggestion of additional protein parts being involved in forming the ion conducting pathway.ClC channels most likely consist of two subunits (12), and in the case of ClC-0, each subunit was proposed to contain a single pore (4,13,14). Recent evidence speaks against this "doublebarreled" channel model, at least in the case of ClC-1 (15). We have found earlier that the exposure of hClC-1, stably expressed in HEK-293 cells, to 1 mM Zn 2ϩ leads to a massive reduction of the conducted chloride current (16). The results were compatible with the presence of at least two extracellularly accessible zinc-binding sites and a direct effect on ion permeation, e.g. by obstruction of the pore. The histidyl-reactive diethyl pyrocarbonate (DEPC), applied from the outside, also reduced the currents through hClC-1. The aim of this study was to identify the target residues for these blockers and, thereby, to draw inferences regarding the membrane topology and the potential location of pore-forming structures. It is known from other proteins, that Zn 2ϩ binding sites are of...

Clinical, electrophysiological, and molecular findings are reported for a family with dominant myotonia congenita in which all affected members have experienced long-term fluctuations of the symptom of myotonia. In some patients myotonia is combined with myalgia. The myotoniacausing mutation in this family is in the gene encoding the muscular chloride channel, hClC-1, predicting the amino acid exchange G200R. We have constructed recombinant DNA vectors for expression of the mutant protein in tsA201 cells and investigation of the properties of the mutant channel. The most prominent alteration was a +100-mV shift of the midpoint of the activation curve. Therefore, within the physiological range the open probability of the mutant channel is markedly smaller than in wild-type. This shift is likely to be responsible for the myotonia in the patients. The fluctuating symptoms of this chloride channelopathy are discussed with respect to short-term fluctuations of myotonia in the sodium channelopathy of potassium-aggravated myotonia.

Membrane diffusion potentials induced by amphotericin B (AmB), amphotericin B methyl ester (AmE), N-fructosyl AmB (N FruAmB) and vacidin, an aromatic polyene antibiotic, in ergosterol- or cholesterol-containing egg yolk phosphatidylcholine large unilamellar vesicles (LUV), were measured in various media, in order to determine the relative selectivity of Na+, K+, Cl- and other ions in these environments. Changes in the membrane potential were followed by fluorescence changes of 3,3'-dipropylthiadicarbocyanine (diS-C3-(5)). Subtle changes in intercationic selectivity were monitored by measuring biionic potentials, using the fluorescent pH sensitive probe pyranine. In all the cases studied, the intercationic selectivity of the permeability pathways induced by the four antibiotics was weak compared to that of specific biological channels, though distinct differences were noted. With AmB the selectivity appeared to be concentration dependent. Above 5 x 10(-7) M, the sequence determined for sterol-free small unilamellar vesicles (SUV) and cholesterol-containing SUV and LUV, Na+ > K+ > Rb+ > or = Cs+ > Li+ (sulfate salts), corresponded closely to Eisenman selectivity sequence number VII. At 5 x 10(-7) M and below the selectivity switched from Na+ > K+ to K+ > Na+. In contrast, Li+ was the most permeant ion for AmB channels in the presence of ergosterol. The selectivity between Na+ or K+ vs. Cl- varied with the antibiotic. It was very strong with vacidin at concentrations below 5 x 10(-7) M, smaller with AmB, nil with AmE and N FruAmB. The selectivities observed were antibiotic, concentration and time dependent, which confirms the existence of different types of channels.

We investigated electrophysiologically the unaffected parents of patients with recessive myotonia congenita. We studied 18 families, in nine of which the diagnosis was confirmed by molecular genetics. Brief myotonic discharges were present in at least one parent in 67% of the families. Fathers were more likely than mothers to show these discharges. The difficulty in distinguishing very mildly affected parents with dominant myotonia congenita from the heterozygous carriers of recessive myotonia congenita is stressed. The recessive type of myotonia congenita (RMC), described by Becker, 2 is more common than the dominant type (Thomsen's disease).2 In RMC, myotonia has a later onset (4-12 years) and usually commences in the legs, spreading after a variable interval (sometimes of years) to the arms and then to the face: it is more severe and is usually associated with transient weakness.2 Not every RMC patient conforms to this pattern and it is sometimes difficult to decide clinically whether a patient has the recessive or the dominant form. The single factor by which RMC can be clinically differentiated with relative certainty from dominant myotonia congenita is the absence of clinical myotonia in the parents. The presence of myotonic discharges on needle electromyography (electrical myotonia) of the parents, however, is compatible with the diagnosis of RMC. Although such an electromyographic finding in the parents is generally considered to be a manifestation of heterozygosity, 3 the possibility of its pointing to the presence of dominant type of inheritance has not been completely excluded. 9The aim of this study was to search for electrical myotonia in the parents of patients with RMC. In nine families, the diagnosis was confirmed by determination of mutations in one or two alleles of the muscle chloride channel gene in the patient. MATERIAL AND METHODSThe electrophysiological work was done at the Department of Neurology, University of Istanbul and the molecular biology at the Department of Physiology, University of Ulm. Consent was obtained from the Review Committees of both institutions. Eighteen families of 21 patients were studied; sibling pairs were present in three families. Seventeen patients (81%) were male. Consanquinity was present in 11 families (61%). None of the parents had myotonia by history or clinical examination.Three muscles, abductor pollicis brevis, flexor pollicis longus, and tibialis anterior, were tested in each of the parents. In each muscle, a minimum of eight sectors were examined at different depths by needle electromyography. RESULTS Myotonic Discharges.Myotonic discharges were present in at least one parent in 12 of the 18 families Abbreviations: EMG, electromyography; RMC, recessive myotonia congenita