Ionic transporting systems of skeletal muscle in relation with innervation and their involvement in myotonic diseases

Excitation-contraction coupling describes the series of events that begins with propagated action potential on the muscle fiber surface membrane and leads to the twitch contraction of the fiber. The generation of an action potential during excitation requires rapid sequential changes in membrane conductances of Na⁺, Ca²⁺, and K⁺ ions that depend upon the opening and closing of the respective channels. Myotonic disorders are inherited diseases whose clinical manifestations include electrophysiological signs such as increased excitability and delayed relaxation of the muscles after voluntary contraction. All these disorders appears to be due to an abnormality of the muscle itself since they persist after section or blocking of the motor nerve after curarization. Most experimental and clinical data suggest that human myotonia arises from genetically-induced structural and functional alterations of the muscle membrane. The purpose of this article is to focus on the more recent developments in the molecular and pharmacological analysis of cation transporting systems such as ionic channels and (Na⁺, K⁺) ATPase in myotonic disorders.Special issue dedicated to Dr. Lawrence Austin.     

Detoxification of xenobiotics by plant cells: characterisation of vacuolar amphiphilic organic anion transporters

The transport activities of two primary ATP-dependent organic-anion transporters in the tonoplast of isolated barley (Hordeum vulgare L. cv. Klaxon) vacuoles have been characterised with N-ethylmaleimide glutathione (NEM-SG) and taurocholate as substrates. The transporters showed different sensitivities to organic anions and a variety of transport inhibitors and drugs. The vacuolar uptake of NEM-SG was inhibited by carbonylcyanide 4-trifluoromethoxyphenylhydrazone, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), S-(2,4-dinitrophenyl)glutathione, alkyl-S-glutathione derivatives and taurocholate but stimulated by probenecid. The uptake of taurocholate was inhibited by vinblastine, DIDS and probenecid. Both transporters were unaffected by verapamil. The kinetic properties of the transporters indicate a general preference for amphiphilic anions with some substrate overlap. These characteristics of the transporters are similar to those displayed by the multidrug resistance protein of mammalian drug-resistant cells. We suggest that these vacuolar transporters be described as plant multispecific organic anion transporters (pMOATs).Abbreviations Bm-S bimane S-glutathione - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - DNP-SG S-(2,4-dinitrophenyl)glutathione - FCCP carbonylcyanide 4-trifluoromethoxyphenylhydrazone - LTC₄ cysteinyl leukotriene - MDR multidrug transporter - MRP multidrug resistance protein - NEM-SG N-ethylmaleimide glutathione We thank Prof E. Martinoia for technical advice on the uptake experiments and Prof J. Palmer for helpful discussions and suggestions. M.B.-K. was partially sponsored by a grant from Stichting VSB Fonds, The Netherlands. IACR receives grant-aided support from the Biotechnology and Biological Science Research Council of the United Kingdom     

Sequence and structural homology among membrane-associated domains of CFTR and certain transporter proteins

A sequence comparison of the two membrane-associated (MA) domains of the cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance transporter (MDR), and -factor pheromone export system (STE6) proteins, each of which are believed to contain a total of 12 transmembrane (TM) segments, reveals significant amino acid homology and length conservation in the loop regions that connect individual TM sequences. Similar structural homology is observed between these proteins, hemolysin B (HLYB) and the major histocompatibility-linked peptide transporter, HAM1, the latter two which contain a single MA domain composed of six TM segments. In addition, there are specific sequences that are conserved within the TM segments of the five different membrane proteins. This observation suggests that the folding topologies of the MA domains of MDR, STE6, and CFTR in the plasma membrane are likely to be very similar. The sequence analysis also reveals that there are three characteristic motifs (a pair of aromatic residues, LTLXXXXXXP and GXXL) that are conserved in MDR, STE6, HLYB, HAM1, but not in CFTR. We propose that although CFTR may be evolutionarily related to these other membrane proteins, it belongs to a separate subclass.     

Overexpression of Slc30a7/ZnT7 increases the mitochondrial matrix levels of labile Zn2+ and modifies histone modification in hyperinsulinemic cardiomyoblasts

BackgroundCellular free Zn²⁺ concentrations ([Zn²⁺]) are primarily coordinated by Zn²⁺-transporters, although their roles are not well established in cardiomyocytes. Since we previously showed the important contribution of a Zn²⁺-transporter ZnT7 to [Zn²⁺]_i regulation in hyperglycemic cardiomyocytes, here, we aimed to examine a possible regulatory role of ZnT7 not only on [Zn²⁺]_i but also both the mitochondrial-free Zn²⁺ and/or Ca²⁺ in cardiomyocytes, focusing on the contribution of its overexpression to the mitochondrial function.MethodsWe mimicked either hyperinsulinemia (by 50-μM palmitic acid, PA-cells, for 24-h) or overexpressed ZnT7 (ZnT7OE-cells) in H9c2 cardiomyoblasts.ResultsOpposite to PA-cells, the [Zn²⁺]_i in ZnT7OE-cells was not different from untreated H9c2-cells. An investigation of immunofluorescence imaging by confocal microscopy demonstrated a ZnT7 localization on the mitochondrial matrix. We demonstrated the ZnT7 localization on the mitochondrial matrix by using immunofluorescence imaging. Later, we determined the mitochondrial levels of [Zn²⁺]_Mit and [Ca²⁺]_Mit by using the Zn²⁺ and Ca²⁺ sensitive FRET probe and a Ca²⁺-sensitive dye Fluo4, respectively. The [Zn²⁺]_Mit was found to increase significantly in ZnT7OE-cells, similar to the PA-cells while no significant changes in the [Ca²⁺]_Mit in these cells. To examine the contribution of ZnT7 overexpression on the mitochondria function, we determined the level of reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP) in these cells in comparison to the PA-cells. There were significantly increased production of ROS and depolarization in MMP and increases in marker proteins of mitochondria-associated apoptosis and autophagy in ZnT7-OE cells, similar to the PA-cells, parallel to increases in K-acetylation. Moreover, we determined significant increases in trimethylation of histone H3 lysine27, H3K27me3, and the mono-methylation of histone H3 lysine36, H3K36 in the ZnT7OE-cells, demonstrating the role of [Zn²⁺]_Mit in epigenetic regulation of cardiomyocytes under hyperinsulinemia through histone modification.ConclusionsOverall, our data have shown an important contribution of high expression of ZnT7-OE, through its buffering and muffling capacity in cardiomyocytes, on the regulation of not only [Zn²⁺_]i but also both [Zn²⁺]_Mit and [Ca²⁺]_Mit affecting mitochondria function, in part, via histone modification.     

Differential expression of zinc transporters in functionally contrasting tissues involved in zinc homeostasis

Abstract

Zinc homeostasis is maintained by 24 tissue-specific zinc transporters which include ZnTs (ZnT1-10), ZIPs (ZIP1-14), in addition to metallothionein (MT). Current study aimed the role of zinc transporters in maintaining the basal levels of zinc in functionally contrasting tissue specific THP-1 (monocyte), RD (muscle), and Saos-2 (bone) cells. Zinc transporters expression was assessed by qRT-PCR. The mRNA levels of ZnTs (ZnT5-7 & ZnT9), ZIPs (ZIP6-10, ZIP13-14), and MT were significantly (p?<?0.05) higher in Saos-2 compared to THP-1 and RD. The present study suggests that distinct expression pattern of zinc transporters and metallothionein might be responsible for the differential zinc assimilation.     

Increased Expression of SLC2A9 Decreases Urate Excretion From the Kidney

Urate is the final metabolite of purine in humans. Renal urate handling is clinically important because under-reabsorption or underexcretion causes hypouricemia or hyperuricemia, respectively. We have identified a urate-anion exchanger, URAT1, localized at the apical side and a voltage-driven urate efflux transporter, URATv1, expressed at the basolateral side of the renal proximal tubules. URAT1 and URATv1 are vital to renal urate reabsorption because the experimental data have illustrated that functional loss of these transporter proteins affords hypouricemia. While mutations affording enhanced function via these transporter proteins on urate handling is unknown, we have constructed kidney-specific transgenic (Tg) mice for URAT1 or URATv1 to investigate this problem. In our study, each transgene was under the control of the mouse URAT1 promoter so that transgene expression was directed to the kidney. Plasma urate concentrations in URAT1 and URATv1 Tg mice were not significantly different from that in wild-type (WT) mice. Urate excretion in URAT1 Tg mice was similar to that in WT mice, while URATv1 Tg mice excreted more urate compared with WT. Our results suggest that hyperfunctioning URATv1 in the kidney can lead to increased urate reabsorption and may contribute to the development of hyperuricemia.     

F508del disturbs the dynamics of the nucleotide binding domains of CFTR before and after ATP hydrolysis

The cystic fibrosis transmembrane conductance regulator (CFTR) channel is an ion channel responsible for chloride transport in epithelia and it belongs to the class of ABC transporters. The deletion of phenylalanine 508 (F508del) in CFTR is the most common mutation responsible for cystic fibrosis. Little is known about the effect of the mutation in the isolated nucleotide binding domains (NBDs), on dimer dynamics, ATP hydrolysis and even on nucleotide binding. Using molecular dynamics simulations of the human CFTR NBD dimer, we showed that F508del increases, in the prehydrolysis state, the inter-motif distance in both ATP binding sites (ABP) when ATP is bound. Additionally, a decrease in the number of catalytically competent conformations was observed in the presence of F508del. We used the subtraction technique to study the first 300 ps after ATP hydrolysis in the catalytic competent site and found that the F508del dimer evidences lower conformational changes than the wild type. Using longer simulation times, the magnitude of the conformational changes in both forms increases. Nonetheless, the F508del dimer shows lower C-α RMS values in comparison to the wild-type, on the F508del loop, on the residues surrounding the catalytic site and the portion of NBD2 adjacent to ABP1. These results provide evidence that F508del interferes with the NBD dynamics before and after ATP hydrolysis. These findings shed a new light on the effect of F508del on NBD dynamics and reveal a novel mechanism for the influence of F508del on CFTR.