Cloning and characterization of a potassium-dependent sodium/calcium exchanger in Drosophila

Abstract

Sodium/calcium(-potassium) exchangers (NCX and NCKX) are critical for the rapid extrusion of calcium, which follows the stimulation of a variety of excitable cells. To further understand the mechanisms of calcium regulation in signaling, we have cloned a Drosophila sodium/calcium-potassium exchanger, Nckx30C. The overall deduced protein topology for NCKX30C is similar to that of mammalian NCKX, having five membrane-spanning domains in the NH(2) terminus separated from six at the COOH-terminal end by a large intracellular loop. We show that NCKX30C functions as a potassium-dependent sodium/calcium exchanger, and is not only expressed in adult neurons as was expected, but is also expressed during ventral nerve cord development in the embryo and in larval imaginal discs. Nckx30C is expressed in a dorsal-ventral pattern in the eye-antennal disc in a pattern that is similar to, but broader than that of wingless, suggesting that large fluxes of calcium may be occurring during imaginal disc development. Nckx30C may not only function in the removal of calcium and maintenance of calcium homeostasis during signaling in the adult, but may also play a critical role in signaling during development.

Figure 1

The phototransduction cascade in Drosophila. Absorption of a photon photoactivates rhodopsin, leading to the opening of the cation-selective channels (Light). Extracellular calcium (Ca⁺²) and sodium (Na⁺) enter the cell via the light-activated channels, causing a depolarization of the photoreceptor cells. It is estimated that calcium levels rise from 100 nM to >10 μM upon light stimulation (Hardie 1996b). Calcium entering through the light-sensitive channels is thought to play a key role in deactivation of the light response and light adaptation. Rapid removal of calcium from the photoreceptor cells is key to the recovery from the light response. Removal of calcium following light-stimulation may occur via NCKX (filled circle) and NCX (shaded circle). NCX utilizes a stoichiometry of 3Na⁺/1Ca²⁺ to extrude calcium, and NCKX uses both the inward sodium gradient and the outward potassium gradient to extrude calcium at a stoichiometry of 4Na⁺/1Ca²⁺,1K⁺. Broken arrows indicate that the cation influx through the light-sensitive channel does not occur in the dark (Dark). Rh, rhodopsin molecule; Rh*, photoexcited rhodopsin molecule. This figure is an adaptation of one drawn by Yau 1994.

Figure 2

Complementary DNA and protein sequence. Numbers for nucleotides are indicated to the left and for amino acids to the right. TM domains are underlined.

Figure 2

Complementary DNA and protein sequence. Numbers for nucleotides are indicated to the left and for amino acids to the right. TM domains are underlined.

Figure 3

Nckx30C encodes a novel Drosophila NCKX. CLUSTAL W alignment between Drosophila NCKX30C, human rod NCKX1, and rat brain NCKX2 (Tsoi et al. 1998; Tucker et al. 1998). Dark shading indicates identity and light shading indicates similarity. The 11 TM domains of NCKX30C are indicated by lines above each row.

Figure 4

Hydropathy plot of the conceptual NCKX30C protein. (A) Map of Nckx30C cDNA. The black boxes represent the TM regions of the deduced amino acid sequence. Cleavage sites for BamHI (indicated by B), EcoRI (indicated by E), Cla I (indicated by C), and EcoRV (indicated by RV) are shown. (B) Hydropathy plot of the conceptual NCKX30C protein, analyzed by the Kyte-Doolittle algorithm (Kyte and Doolittle 1982). Hydrophobic regions are designated in black. (C) Domain structure of Drosophila NCKX30C, human rod photoreceptor NCKX1, and rat brain NCKX2. Filled boxes represent the two clusters of TM segments (TM1–TM5 and TM6–TM11) that display high identity among the three sequences. Numbers represent the number of amino acids per segment.

Figure 5

NCKX30C is an NCKX. Reverse exchange was measured as Na_inside-dependent ⁴⁵Ca uptake in High Five cells transformed with Nckx30C in the presence of external KCl (A–C, filled circles). Mean ± SEM results are shown in A–C (filled circles). (A) Reverse sodium/calcium exchange requires intracellular sodium. ⁴⁵Ca uptake was measured in KCl medium with 20 μM monensin present (shaded squares) or without monensin (filled circles). Monensin was added 30 s before addition of ⁴⁵Ca, and causes the release intracellular sodium to the external medium. Average values ± SEM are shown for 13 experiments conducted in KCl medium, and 4 experiments conducted in media containing KCl plus monensin. (B) Reverse sodium/calcium exchange is inhibited by extracellular sodium. ⁴⁵Ca uptake was measured in KCl medium (filled circles) or NaCl medium lacking potassium (triangles). Average values ± SEM are shown for 13 experiments conducted in KCl medium, and 10 experiments conducted in media containing NaCl. (C) Reverse sodium/calcium exchange requires extracellular potassium. ⁴⁵Ca uptake was measured in KCl medium (filled circles) or LiCl medium lacking potassium (diamonds). Average values ± SEM are shown for 13 experiments conducted in KCl medium, and 8 experiments conducted in media containing LiCl.

Figure 6

Nckx30C encodes 8-kb and 10-kb transcripts. Northern blot analysis reveals two transcripts of 8 kb and 10 kb expressed in adult heads (H) and adult bodies (B). Mutant flies lacking eyes (eya) also express both transcripts, whereas only the lower 8-kb transcript is detected in embryos (E) and larvae (L). We loaded 10 μg of polyA⁺ RNA in each lane. Identical results were obtained with four different radiolabeled riboprobes corresponding to various regions of the exchanger. Specifically, we used a 1.1-kbp BamHI-BamHI and 500 nucleotide BamHI-ClaI, ClaI-ClaI loop region, and ClaI-EcoRV (see Fig. 4 A).

Figure 7

Nckx30C and Calx are expressed in the adult eye and the brain of Drosophila. (A) Shown are in situ hybridizations to 14-μm cryostat sections of adult heads hybridized with digoxigenin-labeled riboprobes. (A) Antisense riboprobe for Nckx30C; (B) sense probe for Nckx30C; (C) antisense riboprobe for Calx; and (D) sense riboprobe for Calx. E, eye; L, lamina; M, medulla; and Br, brain.

Figure 9

Nckx30C is expressed in the third instar imaginal discs of larvae. Shown are in situ hybridizations to imaginal discs from wild-type larvae with digoxigenin-labeled riboprobes for Nckx30C and chaoptin. (A) Eye-antennal disc, antisense riboprobe for chaoptin; (B) eye-antennal disc, antisense riboprobe for Nckx30C; (C) eye-antennal disc, control sense riboprobe for Nckx30C; (D) wing disc, antisense riboprobe for Nckx30C; (E) wing disc, sense riboprobe for Nckx30C; (F) haltere disc, antisense riboprobe for Nckx30C; and (G) leg disc, antisense riboprobe for Nckx30C. Note that control hybridizations with sense probes did not produce signals. The eye discs are oriented posterior to the left and dorsal up. The wing and haltere discs are orientated with posterior to the left and ventral up. The leg disc is oriented ventral down.

Figure 8

Nckx30C and Calx are expressed in the ventral nerve cord of the Drosophila embryo. Shown are in situ hybridizations to whole-mount wild-type embryos (Canton S strain) hybridized with digoxigenin-labeled antisense riboprobes for Nckx30C and Calx. (A) Lateral view, preblastoderm, Nckx30C; (B) lateral view, preblastoderm, Calx; (C) lateral view, blastoderm, Nckx30C; (D) lateral view, blastoderm, Calx; (E) ventral view, stage 13-14, Nckx30C; (F) lateral view, stage 11, Calx; (G) ventrolateral view, stage 15, Nckx30C; (H) ventral view, stage 12, Calx; (I) ventrolateral view, stage 16, Nckx30C; and (J) ventrolateral view, stage 16, Calx. Note the labeling of the developing ventral nerve cord. Control hybridizations with sense probes did not produce signals (data not shown). All embryos are orientated anterior to the left. In lateral views, all embryos are orientated dorsal side up.