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Comparative Study
. 1996 Nov 12;93(23):13256-61.
doi: 10.1073/pnas.93.23.13256.

ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae

S A Goldstein  1 , L A Price, D N Rosenthal, M H Pausch
Affiliations
Comparative Study

ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae

S A Goldstein et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A 1999 Jan 5;96(1):318

Abstract

A K+ channel gene has been cloned from Drosophila melanogaster by complementation in Saccharomyces cerevisiae cells defective for K+ uptake. Naturally expressed in the neuromuscular tissues of adult flies, this gene confers K+ transport capacity on yeast cells when heterologously expressed. In Xenopus laevis oocytes, expression yields an ungated K(+)-selective current whose attributes resemble the "leak" conductance thought to mediate the resting potential of vertebrate myelinated neurons but whose molecular nature has long remained elusive. The predicted protein has two pore (P) domains and four membrane-spanning helices and is a member of a newly recognized K+ channel family. Expression of the channel in flies and yeast cells makes feasible studies of structure and in vivo function using genetic approaches that are not possible in higher animals.

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Figures

Figure 1
Figure 1
Molecular cloning of ORK1 by complementation. ORK1 expression allows growth of yeast cells deficient in K+ transport; CY162 yeast cells were transformed with the designated plasmids, streaked on plates of uracil-deficient synthetic complete medium (SC), or SC containing 100 mM KCl and incubated for 40 hr at 30°C.
Figure 2
Figure 2
Nucleotide and deduced amino acid sequence of ORK1. (a) The 2.4-kb cDNA insert encoding ORK1 contains a single long ORF. Segments corresponding to putative pore-forming P domains and transmembrane segments (M1–M4) are underlined. The National Center for Biotechnology Information accession number for the nucleotide and amino acid sequence of ORK1 is U55321U55321. (b) Kyte–Doolittle hydrophilicity analysis of the ORK1 ORF with a window of 20 residues. (c) Alignment of the P domains of ORK1, Tok1, TWIK, and C24A3.6, a two P domain ORF from the Caenorhabditis elegans sequence data base (residues identical in 2 or more sequences are in boldface type); the probability that 8 random sequences would have the amino acid identities shown here is less than 10−50 as calculated by the method of Jan and Jan (30). While the overall similarity of ORK1 and Tok1 is low, the region extending from the first P domain to 20 residues past the second yields a 56% similarity and 23% identity with an quality score of 79 by the method of Needleman and Wunsch (26). Sequential randomizations and alignments of this region produce an average quality score of only 51 ± 3, suggesting that the channels share a single common ancestor; sequence data from other species are needed to establish this thesis. ORK1 and TWIK (14) show an overall amino acid identity of 21% and similarity of 48% with a quality score of 214. (d) In vitro translation of ORK1 with (+) or without (−) dog pancreatic microsomes resolved in an SDS/12.5% polyacrylamide gel and visualized by autoradiography. (e) Predicted membrane topology of ORK1.
Figure 3
Figure 3
ORK1 is expressed in the excitable tissues of adult D. melanogaster. (a) Northern blot analysis of ORK1 mRNA. D. melanogaster mRNA (5 μg, adult, Clontech) was resolved in a formaldehyde/agarose gel, blotted to nitrocellulose, and probed with 32P-labeled 2.4-kb ORK1 XhoI fragment overnight at 65°C. The blot was exposed to x-ray film for 46 hr at −70°C with an intensifying screen. (b) In situ hybridization to polytene chromosomes identifies a band on 1–10A1-2 by the method of Todd Laverty using biotin-labeled probes produced by nick-translation and peroxidase-based visualization (Enzo Diagnostics, New York). (c) In situ hybridization in whole adult fly preparations reveals expression of ORK1 mRNA in excitable tissues in the head and thorax (Upper) and abdomen (Lower) with antisense but not control cRNA. Digoxigenin-labeled antisense RNA (to the terminal 300 nucleotides of the coding region and 400 nucleotides of the 3′ untranslated region) or a kit-supplied control RNA were hybridized to 18-μm sections of quick-frozen, OCT-embedded, D. melanogaster CS overnight at 65°C and visualized by an alkaline phosphatase-catalyzed reaction (Boehringer Mannheim).
Figure 4
Figure 4
ORK1-dependent yeast cell growth and K+ uptake are inhibited barium. (a) Inhibition of ORK1 or KAT1-dependent yeast growth by three common K+ channel blockers. CY162 cells (105) expressing the designated channels were plated in RPD medium with 1 mM KCl. Sterile filter disks were placed on the surface of the agar and saturated with 20 μl of solutions containing chloride salts of barium (5 μmol), TEA (20 μmol), or cesium (2 μmol). The plates were incubated for 36 hr at 30°C. (b) Uptake of rubidium by CY162 cells carrying pRAD-ORK1 (•) or pRAD (○) at various concentrations of unlabeled rubidium chloride. (c) Inhibition of rubidium uptake by barium (○) or TEA (•) at 50 μM rubidium chloride. The solid curve is fitted to the data according to (1 + [blocker]/Ki)−1, where Ki is the equilibrium dissociation constant; under these conditions 0.15 mM external barium decreases flux ≈50%.
Figure 5
Figure 5
ORK1 currents in X. laevis oocytes. (a) ORK1 currents in physiological levels of [K]o are outwardly rectifying. Currents were assessed in oocytes injected with 1 ng of cRNA (+ ORK1) or water (control) by two-electrode voltage clamp under constant perfusion with 5 mM KCl solution. Oocytes were pulsed from −150 to 60 mV in 15-mV steps for 75 ms followed by a 15-ms step to −150 mV before returning to the holding potential of −80 mV; a 1-s interpulse interval was employed. Currents are displayed without leak subtraction. Scale bars represent 2 μA and 15 ms. (b) ORK1 current–voltage relation in 5 mM KCl solution at 10 ms into the test pulse normalized to current at 60 mV by the protocol in a (mean ± SEM, n = 4 cells). (c) ORK1 currents are K+ selective. The reversal potential of currents was studied with 5, 10, 20, 50, or 100 mM KCl solutions by the protocol in a (mean ± SEM, n = 4 cells). Linear regression gives a shift of 55 ± 2 mV per 10-fold change in KCl concentration. (d) ORK1 currents flow inward at hyperpolarized voltages under constant perfusion with 100 mM KCl solution; protocol and scale bars as in a. (e) ORK1 current–voltage relation in 100 mM KCl solution as in b (mean ± SEM, n = 4 cells). (f) ORK1 current–voltage relation for one oocyte studied in 5, 10, 20, 50, and 100 mM KCl solutions as in b. (g) Theoretical current–voltage relations under the conditions used to study ORK1 in e according to Goldman (17) and Hodgkin and Katz (18): formula image where Ps is the permeability of K+, [S] refers to K+ concentration, and z, V, F, R, and T have their usual meanings, and assuming an internal K+ concentration of 90 mM, as reported previously (35).
Figure 6
Figure 6
ORK1 currents in oocytes are blocked by barium but not TEA. (a) Current–voltage relationship with constant perfusion of 20 mM KCl solution containing 0 mM (▿), 0.3 mM (○), or 1 mM (•) barium chloride by the protocol in Fig. 4a (mean ± SEM, n = 3 cells). (b) Current–voltage relationship with constant perfusion of 20 mM KCl solution with 0 mM (▿), 2.5 mM (○), or 25 mM (•) TEA chloride isotonically substituted for NaCl (mean ± SEM, n = 3 cells). (c) ORK1-induced currents in an oocyte perfused with 20 mM KCl solution in the absence (control) or presence of 1 mM barium chloride by the protocol in Fig. 4a. Scale bars represent 2 μA and 15 ms. (d) Voltage dependence of barium block was assessed by measurements of conductance from −150 mV to −75 mV in the presence of 0.3 mM (○) or 1 mM (•) barium chloride by the protocol in Fig. 4a. The data shown are from one oocyte but are representative. Go and G are the cord conductances measured in the absence and presence of barium, respectively. The electrical distance was calculated from Ki(V) = Ki(0)exp(zδFV/RT), where Ki(0) is the zero voltage inhibition constant, z is the valence of the blocking ion, and δ is the fraction of the applied voltage drop experienced at the binding site (36).
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