Functional characterization and determination of the physiological role of a calcium-dependent potassium channel from cyanobacteria

Abstract

Despite the important achievement of the high-resolution structures of several prokaryotic channels, current understanding of their physiological roles in bacteria themselves is still far from complete. We have identified a putative two transmembrane domain-containing channel, SynCaK, in the genome of the freshwater cyanobacterium Synechocystis sp. PCC 6803, a model photosynthetic organism. SynCaK displays significant sequence homology to MthK, a calcium-dependent potassium channel isolated from Methanobacterium thermoautotrophicum. Expression of SynCaK in fusion with enhanced GFP in mammalian Chinese hamster ovary cells' plasma membrane gave rise to a calcium-activated, potassium-selective activity in patch clamp experiments. In cyanobacteria, Western blotting of isolated membrane fractions located SynCaK mainly to the plasma membrane. To understand its physiological function, a SynCaK-deficient mutant of Synechocystis sp. PCC 6803, ΔSynCaK, has been obtained. Although the potassium content in the mutant organisms was comparable to that observed in the wild type, ΔSynCaK was characterized by a depolarized resting membrane potential, as determined by a potential-sensitive fluorescent probe. Growth of the mutant under various conditions revealed that lack of SynCaK does not impair growth under osmotic or salt stress and that SynCaK is not involved in the regulation of photosynthesis. Instead, its lack conferred an increased resistance to the heavy metal zinc, an environmental pollutant. A similar result was obtained using barium, a general potassium channel inhibitor that also caused depolarization. Our findings thus indicate that SynCaK is a functional channel and identify the physiological consequences of its deletion in cyanobacteria.

Figure 1.

Predicted primary structure of SynCaK. ClustalW alignment of SynCaK amino acid sequence (accession no. NP_440478) with that of potassium channel protein MthK of M. thermoautotrophicum (accession no. O27564). The highly conserved selectivity filter of potassium channels (TXXTGFGE) is highlighted with a box. Different functional regions can be distinguished in the primary sequence: transmembrane domains TM1 and TM2, the pore region, TrkA-N (and within this the Glycine domain) and Trk-C domains. All these domains are highlighted with boxes. [See online article for color version of this figure.]

Figure 2.

SynCaK functions as a potassium channel when expressed in CHO cells. A, Representative current traces recorded under symmetrical ionic conditions (150 mm potassium gluconate on both sides plus 2 mm CaCl₂ in the bath) in the inside-out excised patch configuration. The voltage ramp protocol shown (top) was used to elicit channel activity. Two representative, consecutive current traces are shown. The bottom section shows activity recorded at –170 mV on an extended time scale. Under these conditions, reversal potential is at 0 mV. B, Representative current trace recorded under asymmetrical ionic conditions (150 mm potassium gluconate solution with 2 mm CaCl₂ in the bath and 150 mm sodium gluconate solution in the pipette; top). Current recording on extended time scale from the indicated part of the top section (middle). Representative trace recorded under the same ionic condition from CHO cells transfected with mutant SynCaK (bottom). No activity can be recorded; the observed current is due to leak. C, Current trace recorded under the same ionic conditions except that bath, i.e. the intracellular side, contained 10 mm CaCl₂ (top). Traces in bottom section show activity on extended time scale from the indicated region and at +100 mV. [See online article for color version of this figure.]

Figure 3.

Localization of SynCaK in cyanobacteria. A PM, soluble (S), thylakoid membrane (TH), and outer membrane (OM) fractions were isolated from Synechocystis sp. PCC 6803 (2 μg of proteins per lane). The channel protein was detected using α-SynCaK antibody at the expected molecular weight of 41 kD. B, The purity of PM and TH fractions were checked by using antibodies against the PM marker NrtA and the thylakoid marker CP43 (2 μg of proteins per lane).

Figure 4.

Construction of a SynCaK-deficient Synechocystis sp. PCC 6803 strain. A, Schematic diagram of construction of the mutant organism. ΔSynCaK was obtained by inserting a Kan^R into the Synechocystis sp. PCC 6803 genome. Forward and reverse primers, introducing mutagenic sites, were used to generate a PCR product containing the SynCaK gene in the central position and two flanking regions (see “Materials and Methods” for details). B, PCR analyses indicated lack of wild-type gene and correct insertion of the Kan^R gene in the mutant organism. Agarose gel electrophoresis of analytical PCR amplifications performed on genomic DNAs from kanamycin-resistant control and ΔSynCaK strains: molecular mass marker (1-kb ladder, Promega; 1); wild-type DNA amplified with VC9 and CV10 primers (2); ΔSynCaK DNA amplified with VC9 primers (3); wild-type DNA amplified with VC9 and DISP2 primers (4); ΔSynCaK DNA with VC9 and DISP2 primers (5); wild-type DNA with DISP3 and CV10 primers (6); ΔSynCaK DNA with DISP3 and CV10 primers (7); wild-type DNA amplified with DISP2 and DISP3 primers (8); and ΔSynCaK DNA with DISP2 and DISP3 primers (9). C, Western blotting of protein extracts (optical density at 730 nm = 0.3) using the SynCaK antibody, showing no detection of SynCaK channel (41 kD, arrow) in the mutant strain. Unspecific recognition of two other bands indicates equal loading. WT, Wild type.

Figure 5.

Wild-type and mutant organisms do not differ in potassium content, and their growth is comparable at various light intensities and under osmotic and salt stress. A, The potassium concentration was determined by atomic absorption spectroscopy. The amount of K⁺ was normalized to the chlorophyll concentration of the respective cultures (P > 0.05). B, Wild-type and SynCaK-less cells were grown on solid BG11 medium supplemented with 5 mm Glc (except when indicated without Glc [No Glc]) at the indicated light intensities. Photos were taken at day 4. The experiment was repeated two times giving similar results. C, Cells were grown as in B under the indicated conditions at 20 µE m^–2 s^–1 light intensity. WT, Wild type. [See online article for color version of this figure.]

Figure 6.

Membrane potential analysis in wild-type and SynCaK-deficient Synechocystis sp. PCC 6803 strain. The JC-1 cationic carbocyanine dye has been used to highlight membrane potential differences between wild-type and mutant Synechocystis sp. PCC 6803 strains. A, Confocal microscope analysis of the JC-1 fluorescence. The green fluorescence belongs to the monomer form of the dye; the red fluorescence is due to the J-aggregates that form at high dye concentration, i.e. high membrane potential. Chlorophyll is pseudocolored in blue. Two series of images are shown for each strain. B, Green/red fluorescence ratio of the dye in mutants or in the wild type cultured with 1 mm barium is higher than in wild-type bacteria (three independent experiments: n = 257 [wild type], 229 [mutant], and 285 [wild type + barium]). Differences with respect to the wild type are statistically significant (P < 0.05). MUT, Mutant; WT, wild type.

Figure 7.

SynCaK-deficient Synechocystis sp. PCC 6803 strain is more resistant to zinc than the wild type. A, Growth of wild-type (W) and mutant (M) cells on agar BG11 medium with 5 mm Glc at the indicated concentrations of ZnCl₂. Duplicates are shown. Photos were taken at day 5. Optical densities at 730 nm at 0 time point are indicated. At the two highest concentrations of zinc (corresponding to 22 and 44 µm zinc, respectively), spots obtained at 0.01 starting optical density were omitted due to lack of spots under these conditions characterized by lack of growth. Spot tests repeated three times gave the same results. B, Optical density of liquid culture measured after 24 h of growth in the absence or presence of 2 µm or 4 µm Zn²⁺ added to BG11 in the absence or presence of 1 mm Ba²⁺. Values are normalized to optical density under control condition and are from three independent experiments. Differences are statistically significant for the wild type in the presence of zinc. WT, Wild type. [See online article for color version of this figure.]