Schmieder S et al. (2002), Cloning and characterisation of amphibian ClC-3...

XB-ART-6232

Biochim Biophys Acta 2002 Nov 13;15661-2:55-66. doi: 10.1016/s0005-2736(02)00594-1.

Show Gene links Show Anatomy links

Cloning and characterisation of amphibian ClC-3 and ClC-5 chloride channels.

Schmieder S , Lindenthal S , Ehrenfeld J .

???displayArticle.abstract???
Amphibians have provided important model systems to study transepithelial transport, acid-base balance and cell volume regulation. Several families of chloride channels and transporters are involved in these functions. The purpose of this review is to report briefly on some of the characteristics of the chloride channels so far reported in amphibian epithelia, and to focus on recently cloned members of the ClC family and their possible physiological roles. The electrophysiological characterisation, distribution, localisation and possible functions are reviewed and compared to their mammalian orthologs.

???displayArticle.pubmedLink??? 12421537
???displayArticle.link??? Biochim Biophys Acta

Species referenced: Xenopus laevis
Genes referenced: adm clcn3 clcn5 clcnkb

???attribute.lit??? ???displayArticles.show???

	Fig. 1. V-ATPase in MR cells energises ion transport in frog skin. Upper part: V-ATPase immunoreactivity of MR cells in R. esculenta skin epithelium viewed by epifluorescence illumination (a), the same view by differential interference contrast is shown in (b) (from Ref. [11]). The frog skin epithelium presents different cell layers (stratum corneum, stratum granulosum, stratum spinosum, and basal lamina). Two MR cells are clearly distinguishable in the outermost cell layer (c). Lower part: (A) Model of ion reabsorption from low salt containing media. The electrogenic proton pump (V-ATPase) in MR cells energizes the Na+ entry (through amiloride sensitive sodium channels present in granular (GR) and MR cells) and the Cl/HCO 3 exchanger present in the same MR cells [11,35]. (B) Model of ion reabsorption in isotonic saline. A large chloride conductance is present in high salt containing solutions; the relative contribution of the cellular pathway through MR cells [11] and a paracellular pathway [12] is not solved. For clarity, the Cl/HCO 3 exchanger (A) is not represented here; its contribution to chloride absorption is smaller than that of the chloride conductance, but it is nevertheless significant [9,11].
	Fig. 2. Electrophysiological properties of IxClC-3 and IxClC-5. Representative traces of currents of (A) water-, (B) xClC-3 cRNA- and (C) xClC-5 cRNA-injected oocytes. The oocytes were investigated by voltage-clamp 4 days after injection of 5 ng cRNA/oocyte or water. Oocytes were sequentially clamped from a holding potential of 50 mV to voltages between 100 and + 80 mV for 800 ms in steps of 20 mV. (D) Mean current– voltage (I/V) relationships of waterinjected oocytes (y, n = 17), xClC-3 cRNA-injected oocytes (n, n = 17) and xClC-5 cRNA-injected oocytes (E, n = 15). Data from Ref. [66] and our unpublished data. Methods are as described in Ref. [66].
	Fig. 3. Western blot analysis of xClC-3 from cRNA-injected Xenopus oocytes and A6 cells, and enzymatic deglycosylations. Membrane preparations obtained from xClC-3 cRNA-injected oocytes (A) and A6 cells (B) were incubated at 37 jC for 2 h without (‘‘control’’, lane a) and with endoglycosidase H (‘‘ + EndoH’’, lane b) or endoglycosidase F/N-glycosidase F mixture (‘‘ + EndoF/N-GlycoF’’, lane c) prior to separation on SDS/PAGE and immunoblotting. Endoglycosidase H digestion had no effect on xClC-3 from injected oocytes and A6 cells. For both samples, digestion with the endoglycosidase F/Nglycosidase F enzyme mixture led to a shift of the recognised band from 105 to 85 kDa corresponding to the calculated molecular mass of unglycosylated xClC-3 protein. Methods are as described in Ref. [77]. Expression of xClC-3 was carried out according to the methods described for expression of xClC-5 [66].
	Fig. 4. xClC-5 current is inhibited by the tyrosine kinase inhibitor genistein. (A) Current/voltage relationships of oocytes expressing xClC-5 under control conditions, after 5 min perfusion with 100 AM genistein, and after 5 min of wash-out of the inhibitor. An inhibition of 41F2% was achieved after 5 min of perfusion with genistein. Only 50% of this inhibition could be recovered after a 5 min wash-out period. Daidzein as a control was ineffective (not shown). (B) Inhibition of xClC-5 by 10, 25 and 100 AM genistein. Represented are the percentage of inhibition of the currents measured at + 80 mV (our unpublished data). Methods for cRNA injection and electrical recordings are as described in Ref. [66].
	Fig. 5. Tissue distribution and localisation of xClC-5. (A) The tissue distribution of xClC-5 was examined in Xenopus with anti-ClC-5 antibodies described in Ref. [81]. Bands of different molecular weights could be detected in xClC-5 cRNA-injected oocytes, kidney, intestine, and brain. The highest band (130 kDa, arrowhead) was predominant in cRNA-injected oocytes and kidney and is likely to correspond to a highly glycosylated form of xClC-5. A faint band was seen in heart. Brain and intestine presented a predominant lower band (at about 85 kDa, arrowhead). The same pattern of bands was observed with our antibody (antibody described in Ref. [66]). The 50 kDa band observed in oocytes corresponds probably to a nonspecific band as it can also be observed in noninjected control oocytes (data not shown). Methods are as described in Ref. [66]. Briefly, 50 Ag of Xenopus oocytes and 150 Ag of Xenopus tissues were separated by SDS-PAGE, electrotransfered onto nitrocellulose membrane, and incubated overnight with the anti-ClC-5 antibodies, kindly provided by T. Jentsch. (Our unpublished data.) (B) Immunolocalisation of xClC-5 in A6 cells grown on coverslips. Cells were fixed in 2% paraformaldehyde. Primary anti-ClC-5 antibodies were described previously [66] and used at 1:50. Secondary antibodies were FITC-conjugated, and were used at 1:300. Bar: 10 Am. (Our unpublished data.) (C) Functional model for ClC-5 (model modified from Ref. [104]). ClC-5 co-localises with proton pumps in early endosomes in proximal tubule cells [104]. Parallel functioning of the proton pumps and ClC-5 channels allows the acidification of early endosomes involved in recycling and degradation of apical receptors and reabsorption of low molecular weight proteins.