The functioning of mammalian ClC-2 chloride channel in Saccharomyces cerevisiae cells requires an increased level of Kha1p

Abstract

The mammalian chloride channel ClC-2 is a member of the CLC voltage-gated chloride channels family. This broadly expressed protein shows diverse cellular locations and despite numerous studies, its precise function is poorly understood. Disruption of ClC-2-encoding gene in mouse leads to retinal and testicular degeneration and mutations in CLC2 (gene encoding the ClC-2 channel) are associated with idiopathic generalized epilepsies. ClC-2 may also be responsible for Cl- transport in mouse salivary glands. The only CLC homologue of the yeast Saccharomyces cerevisiae, Gef1p, exhibits CLC activity. We expressed the mammalian ClC-2 protein in S. cerevisiae devoid of Gef1p in an attempt to identify yeast proteins influencing the functioning of ClC-2. The presence of such proteins in yeast could indicate the existence of their homologues in mammalian cells and would greatly aid their identification. Expression of ClC-2 in yeast required optimization of the sequence context of the AUG translation initiation codon. After obtaining an efficient translation, we found that rat ClC-2 cannot directly substitute for yeast Gef1p. Functional substitution for Gef1p was, however, achieved in the presence of an increased level of intact or C-terminally truncated yeast Kha1 protein. Based on the deduced amino acid sequence, the Kha1 protein can be classified as a Na+/H+ transporter since it has a large N-terminal domain similar to the family of NHEs (Na+/H+ exchangers). This suggests that the Kha1p may take part in the regulation of intracellular cation homoeostasis and pH control. We have established that Kha1p is localized in the same cellular compartment as Gef1p and yeast-expressed ClC-2: the Golgi apparatus. We propose that Kha1p may aid ClC-2-dependent suppression of the Deltagef1-associated growth defects by keeping the Golgi apparatus pH in a range suitable for ClC-2 activity. The approach employed in the present study may be of general applicability to the characterization of poorly understood proteins by their functional expression in yeast.

Identification and Characterization of Vnx1p, a Novel Type of Vacuolar Monovalent Cation/H+ Antiporter of Saccharomyces cerevisiae

“…A pre-vacuolar-bound Na ϩ /H ϩ antiporter (Nhx1p) mediates cation/H ϩ exchange in prevacuoles and possibly in other intracellular compartments (12,16). More recently, a K ϩ /H ϩ exchanger (Kha1p) at the Golgi was reported, and its role in pH regulation was postulated (10,11). Although in S. cerevisiae, vacuolar Na ϩ (and K ϩ ) transport has been attributed mainly to Nhx1p (14), our data and other reports (18,19) Our results clearly indicated that Vnx1 mediated Na ϩ , K ϩ , and Li ϩ transport into the vacuole and that the lack of Vnx1p increased the salt sensitivity of the cells.…”

“…Kha1p has been described as a putative K ϩ /H ϩ antiporter, and its deletion induced a growth defect at high external pH and hygromycin (10). Kha1p co-localized with Mntp1p, a Golgi-specific marker (11). Although Kha1p displays high amino acid sequence similarity to other Na ϩ /H ϩ antiporters, its ability to mediate K ϩ /H ϩ exchange has not been yet demonstrated.…”

Potassium and the K+/H+ Exchanger Kha1p Promote Binding of Copper to ApoFet3p Multi-copper Ferroxidase

“…Although this family of proteins has been identified from many organisms ranging from bacteria to humans (13,(15)(16)46), the physiological role of Kha1p has not been defined. Previous studies suggested that Kha1p might serve as a K ϩ efflux transporter (47), which localized to the Golgi (48,49) or mitochondria (50). Cells expressing Kha1p fused with RFP (Kha1p-RFP) display fluorescence associated with vesicle-like compartments (Fig.…”

Alkali Metal Cation Transport and Homeostasis in Yeasts

“…Among the three yeast Na ϩ /H ϩ antiporters, Kha1 (encoded by YJL094c) has the highest level of similarity to bacterial sodium or potassium/proton antiporters (218,227). Although for some time it was believed to be a plasma membrane transporter (227), later it was shown to reside in the membrane of the Golgi apparatus (78,153). Deletion of KHA1 results in a growth defect in media with high pH or containing hygromycin B.…”

“…There is no direct evidence regarding the substrate specificity of Kha1, but potassium is most probably its substrate, as the inability of the kha1⌬ mutant to grow at higher pH can be suppressed by the addition of moderate concentrations of KCl (78,153). Similar to the case for the other yeast intracellular alkali metal cation/H ϩ antiporters, Kha1 is believed to be involved in the regulation of intraorganellar potassium and pH homeostasis and most probably acts in collaboration with the Gef1 anion channel, as high cellular levels of Kha1 are required for the functional expression of a mammalian ClC-2 channel in S. cerevisiae and for complementation of the gef1⌬ phenotype (78).…”