An exchanger-like protein underlies the large Mg2+ current in Paramecium

Abstract

There are very few molecules known to transport Mg(2+) in eukaryotes. The membrane of Paramecium tetraurelia passes a large Mg(2+)-selective current and exhibits a corresponding backward swimming behavior. Both are missing in a group of mutants called eccentric. By sorting an indexed WT genomic library through microinjection into the macronucleus, we have isolated a DNA fragment that complements the eccentric mutations. The Mg(2+) currents and behavior are restored fully in the transformed cells. Surprisingly, the conceptually translated protein is not homologous to any known ion channel but instead has some similarity to K(+)-dependent Na(+)Ca(2+) exchangers. Exchangers are either electrically silent or only pass very small and slow currents compared with ion-channel currents. In light of recent ion-channel crystal structures and considering the need to have narrow ion-selective filters, we speculate on how an exchanger might evolve to show channel-like activities in special circumstances. The significance of finding the molecular basis of a Mg(2+)-specific pathway is also discussed.

Fig 1.

Complementation of the eccentric phenotypes. (A) Behavior response on transfer into the Mg²⁺ test solution of clonal descendents of various cells. Those of uninjected WT (nd) or eccentric (xntA-1) injected with the XNTA1-bearing plasmid (pXNT-WT) swim backward for ≈10 s; those of xntA-1 uninjected or injected with xntA-1-bearing (pXNT-1) or empty plasmid (vector) swim backward for only ≈2 s. Mean ± SD are shown, n = 50 = 10 × 5 clones for each entry. (B) The Mg²⁺-specific current that is best measured at the start of its tail current (arrow). Descendents of xntA-1 injected with pXNT-WT or pXNT-1 were placed under voltage clamp in Mg²⁺ solution. The membrane was held at −30 mV and then stepped for 500 ms to 10 mV or −90 mV to elicit the membrane currents shown. pXNT-WT-injected, but not the pXNT-1-injected, cells exhibit an inward tail current on returning to −30 mV, a current that has been shown previously to be Mg²⁺-specific. (Lower) The amplitude of such tail currents (I_tail) elicited by 500-ms steps to the potentials indicated (V_c): mean ± SD (n = 4 pXNT-WT-expressing cells, n = 9 pXNT-1 cells) (19).

Fig 2.

XntAp, the eccentric gene product. (A) A Kyte–Doolittle hydrophilicity plot of XntAp (7-aa window). Marked are two hydrophobic regions (boxes above plot) and a connecting hydrophilic loop, the putative TM segments (TM boxes at bottom), a PEST-like sequence (slashed), and the regions corresponding to the α1 and α2 regions seen in other NCX/NCKXs (shaded boxes). Black triangles mark the location of the mutations found in the eight independently isolated mutant alleles (Table 1). (B) The XntAp sequence. Putative TM domains are outlined, and acidic residues near the possible α1 and α2 regions are circled. (C) An unrooted 50% majority-rule consensus dendrogram showing XntAp is distantly related to NCX/NCKXs. They are consistently clustered into three groups. XntAp is only slightly more related to group A (verified NCKXs and immediate relatives) and group B (NCXs and the Arabidopsis Mg²⁺ exchanger) than group C (putative exchangers with divergent C-terminal hydrophobic regions).

Fig 3.

Suppression of the Mg²⁺-specific current in the WT by gene silencing. WT cells were injected with empty plasmid (control) or XNT-ORF and their clonal descendents were examined under voltage clamp in Mg²⁺ solution for evidence of a Mg²⁺-specific current. The membrane was held at −30 mV and then stepped for 500 ms to 10 mV or −90 mV to elicit the membrane currents including tail currents (arrows) shown. The control cells exhibited normal membrane currents that were indistinguishable from an untreated WT, but cells injected with XNT-ORF closely resembled eccentric mutant cells. (Lower) The amplitudes of tail currents (I_tail) elicited by 500-ms steps to the test potentials indicated (V_c). Current amplitudes on both depolarization and hyperpolarization are reduced by ≈75% in the silenced cells. Data represent mean ± SD; currents from six cells injected with XNT-ORF (○) or seven cells injected with control plasmid (•).