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J. Biol. Chem., Vol. 280, Issue 8, 6834-6839, February 25, 2005

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Substitution of a Single Residue, Asp⁵⁷⁵, Renders the NCKX2 K⁺-dependent Na⁺/Ca²⁺ Exchanger Independent of K^+*

Kyeong-Jin Kang, Yoshiyuki Shibukawa¶, Robert T. Szerencsei, and Paul P. M. Schnetkamp, Scientist of the Alberta Heritage Foundation for Medical Research||

From the Department of Physiology & Biophysics, Faculty of Medicine, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Received for publication, November 16, 2004 , and in revised form, December 9, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

 
The Na⁺/Ca²⁺-K⁺ exchanger (NCKX) is a polytopic membrane protein that uses both the inward Na⁺ gradient and the outward K⁺ gradient to drive Ca²⁺ extrusion across the plasma membrane. NCKX1 is found in retinal rod photoreceptors, while NCKX2 is found in retinal cone photoreceptors and is also widely expressed in the brain. Here, we have identified a single residue (out of >100 tested) for which substitution removed the K⁺ dependence of NCKX-mediated Ca²⁺ transport. Charge-removing replacement of Asp⁵⁷⁵ by either asparagine or cysteine rendered the mutant NCKX2 proteins independent of K⁺, whereas the charge-conservative substitution of Asp⁵⁷⁵ to glutamate resulted in a nonfunctional mutant NCKX2 protein, accentuating the critical nature of this residue. Asp⁵⁷⁵ is conserved in the NCKX1–5 genes, while an asparagine is found in this position in the three NCX genes, coding for the K⁺-independent Na⁺/Ca²⁺ exchanger.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

 
The SLC8 and SLC24 gene families encode plasma membrane proteins that are expressed in many tissues of the body and carry out K⁺-independent Na⁺/Ca²⁺ exchange (NCX)¹ and K⁺-dependent Na⁺/Ca²⁺ exchange (NCKX), respectively (1, 2). For example, NCX1 plays a critical role in muscle contraction in the heart, whereas NCKX1 plays an important role in phototransduction in the outer segments of retinal rod photoreceptors. Both NCX and NCKX are bidirectional transporters that can mediate Ca²⁺ influx or Ca²⁺ efflux from cells, depending on the transmembrane Na⁺ gradient (NCX) or the transmembrane Na⁺ and K⁺ gradients (NCKX). The ion transport pathways in both NCX and NCKX are thought to be associated with two sets of membrane-spanning segments (TMs) that are separated by a large cytosolic loop. The two TMs contain the so-called 1 and 2 repeats, respectively, thought to have arisen from an early gene duplication event (3). The repeats are the only regions of sequence conservation between members of the NCX and NCKX gene families, and are thought to be in antiparallel orientation for both NCX1 (4, 5) and NCKX2 (6). Individual substitution of most of the residues conserved between NCX and NCKX results in a large decrease in or even loss of cation transport function (7, 8), but little is known about residues that directly contribute to cation binding. The key distinguishing feature between NCX and NCKX is that the latter requires and transports K⁺ and a stoichiometry of 4Na⁺: 1Ca²⁺ + 1K⁺ has been reported for NCKX1 and NCKX2 (9–11). Most studies have reported a 3:1 coupling ratio for the NCX1 Na⁺/Ca²⁺ exchanger, although in a few studies a 4:1 ratio has been advocated as well (reviewed in Refs. 12 and 13). In a previous study we identified two acidic residues (Asp²⁵⁸ and Asp⁵⁷⁵) thought to be located deep within membrane-spanning helices and important for NCKX function, which do not appear to have similarly placed counterparts in the NCX topology (8). Here, we show that mutagenesis of the codon for Asp⁵⁷⁵ in the human NCKX2 cDNA results in mutant NCKX2 proteins that were no longer dependent on K⁺ as judged by their ability to carry out K⁺-independent Ca²⁺ influx representing reverse Na⁺/Ca²⁺ exchange.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

 
Mutagenesis and Expression Systems—Mutagenesis of the short splice variant of the Myc-tagged human retinal cone NCKX2 cDNA (AAF25811 [GenBank] was carried out as described before (8). The codon of the Asp⁵⁷⁵ residue was replaced by codons for either asparagine, cysteine, or glutamic acid. The mutant NCKX2 cDNAs were placed in either the pEIA vector for expression in an insect cell line (High Five cells) or in the pCDNA3.1 vector for expression in a mammalian cell line (HEK293 cells). Cell culture and transfection protocols were as described before (8, 14).

HEK293 cells were used for functional studies using electrophysiological methods and using the fluorescent Ca²⁺-indicating dye Fluo-3, whereas High Five cells were used for functional studies using a ⁴⁵Ca uptake assay. High Five cells can be grown in roller bottles and showed highly uniform protein expression levels for wild-type NCKX2 and NCKX2 mutants compared with HEK293 cells (8, 14). High Five cells were less suited for the fluorescence measurements because of fast leakage of Fluo-3 from the cells. High Five cells were less appropriate for electrophysiological recordings because they showed a propensity for quickly developing large leak currents that limited the time available for useful measurements and reduced the number of high resistance seals successfully obtained.

Electrophysiology—HEK293 cells transfected with either wild-type NCKX2 cDNA or with Asp⁵⁷⁵ NCKX2 mutant cDNAs were collected in a medium containing 150 mM NaCl, 5 mM KCl, 10 mM glucose, 2 mM CaCl₂, and 20 mM Hepes (pH adjusted to 7.4 with arginine). The cells (0.5 ml volume) were placed in the recording chamber and allowed to settle for 45 min on a poly-D-lysine-coated glass coverslip at the bottom of the chamber. The recording chamber was placed on the stage of an inverted microscope (Axiovert 25, Zeiss, Jena, Germany) and viewed under phase-contrast optics. Na⁺/Ca²⁺-K⁺ exchange currents were recorded using the whole cell configuration of the patch clamp technique. Pipettes were prepared from glass capillaries (Sutter Instrument, Novato, CA) using a Flaming/Brown micropipette puller (Model P87/PC, Sutter Instrument); pipettes had a resistance of 3–7 megohm when back-filled with pipette solution. The seal resistance ranged between 5 and 10 Gohm. Currents were recorded with an L/M-EPC-7 amplifier (List-Medical, Darmstadt, Germany). Reverse Na⁺/Ca²⁺-K⁺ exchange currents were recorded in conventional whole cell configuration using a holding potential of 0 mV. Current traces were displayed using Axotape software (Axon Instruments, Foster City, CA), after digitizing the analogue signals at 0.5 kHz (DigiData 1200, Axon Instruments). Current records were filtered at 10 Hz. Data were analyzed off-line using pCLAMP (Axon Instruments), and a technical graphics/analysis program (ORIGIN, MicroCal Software, Northampton, MA). An eight-channel perfusion system (ALA Scientific Instruments Inc, Westbury, NY) was used to apply test solutions to the selected cell. Solution changes were completed in 20 ms. All experiments were carried out at room temperature (20–22 °C). Changes in current were normalized with respect to cell size as judged from cell capacitance; the average capacitance of 70 cells examined in this study was 8.2 ± 0.9 pF (average ± S.E.).

At the start of each experiment, the HEK cells in the recording chamber were in a solution containing 150 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 20 mM Hepes (pH adjusted to 7.4 with arginine), and 10 mM glucose. After the Gohm seal was formed, the recording chamber was superfused with a medium containing 150 mM LiCl, 10 mM sucrose, 1 mM EDTA, and 20 mM Hepes (pH adjusted to 7.4 with Tris) at a flow rate of 1–2 ml/min. To activate reverse exchange currents, the bath solution containing 150 mM LiCl, 10 mM sucrose, 1 mM EDTA, and 20 mM Hepes (pH adjusted to 7.4 with Tris) was rapidly changed for one containing 0.2 mM CaCl₂ (replacing 1 mM EDTA) and selected concentrations (0–150 mM) of KCl, maintaining osmotic strength by replacing LiCl with KCl. In some experiments for reverse exchange current recordings, selected concentrations of free Ca²⁺ were obtained with the use mixtures of CaHEDTA and HEDTA (diluted from 200 mM stock solutions adjusted to pH 7.4 with arginine). The pipette filling solution contained 130 mM sodium glutamate, 20 mM NaCl, 2 mM MgATP, 20 mM tetraethylammonium chloride, 10 mM EGTA, and 20 mM Hepes (pH adjusted to 7.2 with Tris).

Functional Analyses: Fluo-3 Measurements in HEK293 Cells and ⁴⁵Ca Uptake in High Five Cells—In addition to electrophysiological characterization of the Asp⁵⁷⁵ NCKX2 mutants, we used two other functional assays developed in our laboratory to measure NCKX function. NCKX-mediated changes in cytosolic-free [Ca²⁺] were measured with the fluorescent Ca²⁺-indicating dye Fluo-3 in HEK293 cells transfected with cDNA encoding wild-type NCKX2 or NCKX2 mutants as described in detail before (14–16).

K⁺-dependent ⁴⁵Ca uptake in Na⁺-loaded High Five cells was measured essentially as described before (17). Briefly, High Five cells were Na⁺-loaded with the ionophore monensin and the ionophore was removed through washing with bovine serum albumin-containing solutions. ⁴⁵Ca was measured with liquid scintillation counting after ⁴⁵Ca taken up the by High Five cells was separated from ⁴⁵Ca remaining in the medium by a rapid filtration method using borosilicate glass fiber filters.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

 
Members of the NCX and NCKX gene families have been shown to carry out either Ca²⁺ efflux or Ca²⁺ influx depending on the direction of the transmembrane Na⁺ gradient (NCX) or the transmembrane Na⁺ and K⁺ gradients (NCKX) (1, 2). Ca²⁺ influx in exchange for intracellular Na⁺ is readily observed upon removal of extracellular Na⁺ and is referred to as reverse exchange. Reverse exchange has proven to be a very convenient way to carry out functional analyses of heterologously expressed NCX and NCKX proteins. For example, the extracellular Ca²⁺ and K⁺ dependences of NCKX-mediated transport are readily determined from reverse exchange (10, 11, 18, 19), and the strict dependence of reverse Na⁺/Ca²⁺ exchange on extracellular K⁺ has become a standard assay to distinguish NCKX from NCX. We have previously characterized functional consequences of individual substitutions of a large number of residues (>100) in the human NCKX2 protein (8, 20). These mutant NCKX2 proteins were all tested for extracellular K⁺ dependence of reverse Na⁺/Ca²⁺ exchange using a simple ⁴⁵Ca uptake assay (17) (shown here in Fig. 4). For all but one of the NCKX2 mutants examined so far, the strict requirement for extracellular K⁺ was retained although maximal transport rates (8) and K⁺ concentration dependence (16) could differ from that observed for wild-type NCKX2. The human NCKX2 sequence contains only four acidic residues thought to be located well within membrane-spanning helices and conserved among the NCKX1–5 genes. The position of these residues is shown in Fig. 1A. Glu¹⁸⁸ and Asp⁵⁴⁸ are also conserved in the three NCX genes, while Asp²⁵⁸ does not have a similarly placed counterpart in the NCX sequence. The NCKX2 Asp⁵⁷⁵ residue is adjacent to a short stretch of residues conserved between NCX and NCKX, but the three NCX genes contain the codon for asparagine at this position (Fig. 1B). Here, we describe that substitution of the Asp⁵⁷⁵ residue renders the heterologously expressed mutant NCKX2 protein K⁺ independent as determined by three independent assays.

View larger version (24K): [in this window] [in a new window]   FIG. 4. K+-independent 45Ca uptake by the D575N, D575C NCKX2 mutant proteins. 45Ca uptake was measured in Na+-loaded High Five cells as described under "Experimental Procedures." The uptake medium contained 150 mM choline chloride, 80 µM sucrose, 20 mM Hepes (pH adjusted to 7.4 with arginine), and 1 mM carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP); uptake was initiated at time 0 by addition of 1 µCi of 45Ca and CaCl2 to a final concentration of 36 µM. The indicated final K+ concentrations were obtained by isotonic substitution of choline chloride by KCl (filled circles, 10 mM KCl; inverted triangles, 1 mM KCl; squares, 0 mM KCl). In all cases, the Ca2+ uptake values were corrected for uptake observed in a medium where 150 mM NaCl replaced choline chloride. In the presence of 150 mM NaCl, NCKX-mediated uptake was completely inhibited at the Ca2+ concentration of 36 µM used, and uptake was the same as that observed in cells transfected with empty vector under this condition. Nonspecific Ca2+ uptake amounted to 0.30 nmol of Ca2+/mg of protein. The bottom right-hand panel shows the average K+ dependence (± S.E.) obtained for the three Asp575 mutants in five (D575C, circles; D575N, squares; control with empty vector, inverted triangles) or three (D575E, open triangles) separate transfection experiments. Transport activities were defined as the average Ca2+ uptake observed for the 60, 90, and 120 s time points. Temperature: 25 °C.

View larger version (32K): [in this window] [in a new window]   FIG. 1. Topology of NCKX2 and location of transmembrane acidic residues. A, NCKX2 sequence contains eleven hydrophobic segments (1–11) as well as a hydrophobic segment at the N terminus (0), that functions as a cleavable signal peptide (14) as indicated here by the break. The arrangement of hydrophobic segments contains two clusters of five transmembrane spanning helices each (6). The position of the four acidic residues discussed in the text is indicated. The shaded parts in helices 2 and 8 indicate the 1 and 2 repeats, respectively. B, alignment of short sequence elements in dog NCX1 and human NCKX2 containing the NCKX2 residues Glu188, Asp548, and Asp575 (large font). Conserved residues are shaded gray. The arrowhead indicates the critical Asp575 residue.

Cells expressing either the D575C or the D575N NCKX2 mutant proteins showed significant Ca²⁺-activated reverse exchange currents in the absence of any extracellular K⁺ (Fig. 2). Increasing extracellular K⁺ did not result in a clear increase in reverse exchange current. In contrast, cells expressing wild-type NCKX2 showed a strong dependence of reverse exchange current on extracellular K⁺ as reported before (11, 19). No K⁺-independent and Ca²⁺-activated reverse exchange current was observed in cells expressing wild-type NCKX2. Cells expressing wild-type NCKX2 showed a transient current profile in high KCl medium with an initial peak current followed by a sag to a much smaller current, and this pattern was more pronounced as the initial NCKX2 current was larger. The time course of the decline in current became faster as the EGTA concentration in the pipette-filling solution was lowered, suggesting that NCKX-mediated current rapidly (seconds) changed submembrane Ca²⁺ concentration and reduced the driving force (data not shown). Clearly, the reverse exchange currents observed in cells expressing either the D575N or the D575C NCKX2 mutant were significantly smaller than currents observed in cells expressing wild-type NCKX2. The average current densities observed were: cells expressing wild-type NCKX2: 9.9 ± 4.0 pA/pF (n = 11); cells expressing the D575N mutant: 1.4 ± 0.4 pA/pF (n = 10); cells expressing the D575C mutant: 1.6 ± 0.5 pA/pF (n = 10) (average current density ± S.E. for n cells; the average cell capacitance was 8 pF, see "Experimental Procedures").

View larger version (26K): [in this window] [in a new window]   FIG. 2. K+-independent Na+/Ca2+ exchange currents carried by the D575N and D575C NCKX2 mutant proteins. Patch clamp recordings of reverse Na+/Ca2+ exchange currents were made in HEK293 cells transfected with the indicated NCKX cDNA using the whole cell configuration as described in detail under "Experimental Procedures." A, typical current recordings are shown of a cell expressing either wild-type NCKX2 (top trace), the D575N NCKX2 mutant (middle trace), or the D575C NCKX2 mutant (bottom trace) when the indicated protocol of solution switches was applied. B, the average current densities are shown normalized to the current densities observed in the 150 mM KCl, 0.2 mM CaCl2 medium. The number of cells is indicated in parentheses, and error bars represent S.E. The maximal reverse exchange current densities observed were: 9.9 ± 4.0 pA/pF (11) (cells expressing wild-type NCKX2); 1.6 ± 0.5 pA/pF (10) (cells expressing D575C mutant NCKX2); 1.4 ± 0.4 pA/pF (10) (cells expressing D575N mutant NCKX2) (average values ± S.E. with the number of cells in parentheses).

Fluorometric Measurement of Na⁺/Ca²⁺ Exchange in HEK Cells Expressing the Asp⁵⁷⁵ Mutants—The functional activity of the three Asp⁵⁷⁵ mutants in HEK293 cells was also measured with a fluorometric assay after loading cells with the intracellular fluorescent Ca²⁺-indicating dye Fluo-3 as detailed elsewhere (16, 20). HEK293 cells, loaded with Fluo-3, were placed in a cuvette containing a buffered 150 mM LiCl solution with EDTA and HEDTA present. The fluorescence of the cells was stable under these conditions indicating that no changes in cytosolic free [Ca²⁺] occurred in this medium. Next, reverse Na⁺/Ca²⁺ exchange (Ca²⁺ influx) was initiated by raising free [Ca²⁺] in the cuvette to 10.8 µM and the ensuing increase in Fluo-3 fluorescence was measured either in the LiCl medium or in the LiCl medium to which 20 mM KCl was added. For cells expressing wild-type NCKX2 the Ca²⁺ addition only resulted in a large Fluo-3 signal in the presence of 20 mM KCl, whereas a small and fast signal (i.e. within the mixing time of the Ca²⁺ addition) was observed in the LiCl medium without KCl (Fig. 3). The large Fluo-3 signal observed in the presence of KCl represented a rapid increase in cytosolic free [Ca²⁺] due to K⁺-dependent reverse Na⁺/Ca²⁺ exchange. The small signal observed in LiCl medium without KCl was caused by the small amount of Fluo-3 leaked from the HEK cells into the external medium. As a separate control, the experiment was repeated in high NaCl medium, a standard condition to inhibit reverse Na⁺/Ca²⁺ exchange (17). As observed in the LiCl medium, Ca²⁺ addition to cells in the NaCl medium only led to the small instantaneous signal caused by the leaked dye. The signals representing dye leakage varied somewhat between different batches of Fluo-loaded cells and increased over time. The Ca²⁺-induced amplitude of the Fluo-3 signals observed in cells transfected with wild-type NCKX2 were normalized with respect to the maximal fluorescence signal observed after the cell membrane was permeabilized by addition of saponin (0.02%) in the presence of 5 mM CaCl₂ to saturate Fluo-3 fluorescence. This value (about 42% in the experiment illustrated in Fig. 3) ranged between 30 and 50% in different transfection experiments, similar to the percentage of GFP-positive cells observed in the electrophysiological recordings (see above), which indicates the percentage of successfully transfected cells. Resting free [Ca²⁺] in the HEK293 cells prior to activation of reverse exchange was quite low and close to minimal fluorescence as obtained by addition of saponin in the presence of 0.1 mM EDTA.

View larger version (22K): [in this window] [in a new window]   FIG. 3. K+-independent Na+/Ca2+ exchange by the D575N and D575C NCKX2 mutants. Wild-type NCKX2 and the D575C, D575N, and D575E NCKX2 mutants were expressed in HEK293 cells and transport function was examined with the Fluo-3 assay described under "Experimental Procedures." 50 ml of Fluo-3-loaded HEK293 cells in 150 mM NaCl, 2 mM CaCl2, 6 mM D-glucose, 0.25 mM sulfinpyrazone, and 20 mM HEPES pH 7.4, were added to a cuvette containing 1.95 ml of the media described below. Sulfinpyrazone is an anion transporter inhibitor, which may reduce leakage of Fluo-3 from the cells. Assay media contained either 150 mM LiCl (black trace), 150 mM LiCl plus 20 mM KCl (green trace), or 150 mM NaCl (red trace) in addition to 20 mM Hepes (pH adjusted to 7.4 with arginine), 6 mM D-glucose, 0.25 mM sulfinpyrazone, 0.1 mM EDTA, and 1 mM HEDTA. At 10 s, CaHEDTA was added to a final concentration of 8 mM to initiate reverse Na+/Ca2+ exchange. HEDTA and CaHEDTA were added from 200 mM stock solutions adjusted to pH 7.4 with arginine. The amplitude of the Ca2+-induced Fluo-3 signals were normalized with respect to the fluorescence signal observed when 0.02% saponin was added in the presence of 5 mM CaCl2. Saponin caused the release of Fluo-3 into the external medium and saturated Fluo-3 fluorescence by the high [Ca2+] present. Temperature: 25 °C.

K⁺-independent ⁴⁵Ca Uptake in Insect Cells Expressing the Asp⁵⁷⁵ NCKX2 Mutants—A small component of K⁺-independent reverse Na⁺/Ca²⁺ exchange has previously been reported for in situ bovine NCKX1 (21) as well as for heterologously expressed wild-type human NCKX2 (18) when the assay medium contained either sucrose or choline chloride as the major component. Addition of alkali cations such as Na⁺ or Li⁺ to the external solution suppressed the K⁺-independent component, and, therefore, NCKX transport assays are routinely carried out in LiCl media. The K⁺-independent component of reverse Na⁺/Ca²⁺ exchange was observed when ⁴⁵Ca uptake was measured in Na⁺-loaded High Five insect cells transfected with NCKX cDNAs, a simple quantitative system to assay NCKX function (17). Cells transfected with wild-type NCKX2 showed the typical K⁺-dependent ⁴⁵Ca uptake, but a small K⁺-independent component was present as well when the assay medium contained choline chloride (Fig. 4, top left-hand panel). In contrast, both the D575N and D575C mutant NCKX2 proteins mediated K⁺-independent ⁴⁵Ca uptake and addition of K⁺ to the external medium resulted if anything in a slight inhibition of ⁴⁵Ca uptake (Fig. 4, right-hand panels). This suggests that K⁺ may still interact with NCKX2 but is no longer required for Ca²⁺ transport and is presumably no longer transported itself. When this experiment was repeated in LiCl rather than choline chloride medium, the K⁺-independent ⁴⁵Ca uptake disappeared in cells expressing wild-type NCKX2 (as described before, Ref. 18), but the results obtained with cells expressing the Asp⁵⁷⁵ NCKX2 mutants were essentially the same as observed here in choline chloride medium (not shown). This demonstrates that reverse exchange mediated by the D575N or D575C NCKX2 mutants was independent of K⁺ in the assay medium. Consistent with results obtained with the Fluo-3 assay in HEK293 cells (Fig. 3), the D575E NCKX2 mutant showed no function as assessed by the ⁴⁵Ca uptake assay in High Five cells regardless of the composition of the medium. The results obtained with cells expressing the D575E mutant NCKX2 were identical to those obtained with control High Five cells transfected with empty vector.

The ⁴⁵Ca uptake levels observed for the three Asp⁵⁷⁵ NCKX2 mutants and for control cells were averaged for five experiments (Fig. 4, bottom panel). For comparison, in five experiments the average K⁺-independent ⁴⁵Ca uptake observed for wild-type NCKX2 in the choline medium was 0.17 ± 0.04 nmol of Ca²⁺/mg of protein (average of last three time points ± S.E.).

Mutagenesis of the Asn⁸⁴² Residue in NCX1—Sequence alignment illustrated in Fig. 1B shows that members of the NCX gene family have an asparagine residue in the position of the Asp⁵⁷⁵ residue found in NCKX. Therefore, we examined substitution of this residue to aspartate or to cysteine. The converse mutation in NCX1 (N842D) yielded a nonfunctional mutant NCX1 protein, while the N842C NCX1 mutant showed greatly reduced transport activity (8.3% ± 0.6 in LiCl medium, 9.7% ±1.2% in LiCl + 10 KCl medium; average activity with respect to wild-type NCX1 of four experiments ± S.E.) as assessed with the ⁴⁵Ca uptake assay in High Five cells. The N842C NCX1 mutant was previously reported to have significantly reduced activity with wild-type affinity for Ca²⁺ (22). These results suggest that N842 is an important residue for NCX1 transport function, but do not allow any further conclusion about cation dependence of transport. In a second effort, we made two different NCX1-NCKX2 chimeras in which different parts of the 2 repeat from NCKX2 were placed into NCX1 replacing its 2 repeat, either stopping short of the stretch containing the Asp⁵⁷⁵ residue or including Asp⁵⁷⁵. These chimeras were nonfunctional when expressed in either HEK293 cells or in High Five cells.

	CONCLUSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

 
Asp⁵⁷⁵ Is Critical for the K⁺ Dependence of NCKX2—The novel finding from this study is the identification of a single residue, Asp⁵⁷⁵, that is critical for the K⁺ dependence of NCKX2. The D575N and D575C NCKX2 mutant proteins had completely lost the K⁺ dependence of reverse Na⁺/Ca²⁺ exchange as determined here with three independent assays for NCKX function (Figs. 2, 3, 4). The D575N and D575C NCKX2 mutant proteins also showed reduced maximal transport capacity (15–30% with respect to wild-type NCKX2), whereas the D575E mutant NCKX2 was nonfunctional. The protein expression level of the D575E mutant was not different from that of the D575C and D575N mutant NCKX2 proteins (not shown). We propose that Asp⁵⁷⁵ plays an important role in K⁺ binding to and transport via the NCKX cation binding pocket. Loss of K⁺ dependence in the D575N and D575C NCKX2 mutants suggests that the ion coupling ratio in these mutants has been altered from 4Na⁺:1Ca²⁺ + 1K⁺ to either 4Na⁺:1Ca²⁺ or 3Na⁺:1Ca²⁺. In the results reported here the reverse exchange currents observed for the D575N and D575C NCKX2 mutants were about 16% of the current observed for wild-type NCKX2 (Fig. 2), while the Ca²⁺ flux measurements in cells expressing the D575N and D575C NCKX2 mutants showed 20–27% of wild-type NCKX2 activity (Figs. 3 and 4). This would be more consistent with a coupling ratio of 3Na⁺:1Ca²⁺ as the amount of current carried per transport cycle is doubled for an exchanger with a 4Na⁺:1Ca²⁺ coupling ratio compared with exchangers having coupling ratios of either 3Na⁺:1Ca²⁺ or 4Na⁺: 1Ca²⁺ + 1K⁺. Together with the results from another recent study (16), our data suggest that Asp⁵⁷⁵ together with Glu¹⁸⁸ and Asp⁵⁴⁸ are key residues lining a single Ca²⁺ and K⁺ pocket of NCKX2 situated in the middle of the phospholipid bilayer. The critical nature of the Asp⁵⁷⁵ residue is accentuated by the lack of function observed for the charge-conservative D575E substitution, suggesting considerable steric constraints at this residue position where a mere lengthening of the side chain by one methylene group eliminated transport function. Asp⁵⁷⁵ is conserved in the mammalian NCKX1–5 isoforms and also in several NCKX cloned from lower organisms, while members of the NCX gene family have an asparagine at this position close to a short stretch of residues conserved between both gene families (Fig. 1B). Recently, a putative sixth member of the NCKX gene family was cloned, but two studies report opposite results concerning the K⁺ dependence of NCKX6 (23, 24). NCKX6 has an asparagine at the position equivalent to Asp⁵⁷⁵ in NCKX2, but it should be pointed out that several other residues that are thought to be important for NCKX function and are conserved between members of the NCX and NCKX gene families (16) are not conserved in NCKX6 (23).

	FOOTNOTES

 
^* This work was supported by an operating grant from the Canadian Institutes for Health Research (to P. P. M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

Recipient of a studentship from the Foundation Fighting Blindness Canada.

^¶ Supported in part by the Lions Sight Foundation.

^|| To whom correspondence should be addressed: Dept. of Physiology & Biophysics, Faculty of Medicine, University of Calgary, 3330 Hospital Dr., N. W. Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-5448; Fax: 403-283-8731; E-mail: pschnetk{at}ucalgary.ca.

¹ The abbreviations used are: NCX, Na⁺/Ca²⁺ exchanger; NCKX, Na⁺/Ca²⁺-K⁺ exchanger; GFP, green fluorescent protein; HEDTA, N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid; TM, transmembrane; HEK, human embryonic kidney; F, farad.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

 

1. Quednau, B. D., Nicoll, D. A., and Philipson, K. D. (2004) Eur. J. Physiol. 447, 543-548[CrossRef][Medline] [Order article via Infotrieve]
2. Schnetkamp, P. P. M. (2004) Eur. J. Physiol. 447, 683-688[CrossRef][Medline] [Order article via Infotrieve]
3. Schwarz, E. M., and Benzer, S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10249-10254[Abstract/Free Full Text]
4. Nicoll, D. A., Ottolia, M., Lu, L., Lu, Y., and Philipson, K. D. (1999) J. Biol. Chem. 274, 910-917[Abstract/Free Full Text]
5. Iwamoto, T., Nakamura, T. Y., Pan, Y., Uehara, A., Imanaga, I., and Shigekawa, M. (1999) FEBS Lett. 446, 264-268[CrossRef][Medline] [Order article via Infotrieve]
6. Kinjo, T. G., Szerencsei, R. T., Winkfein, R. J., Kang, K.-J., and Schnetkamp, P. P. M. (2003) Biochemistry 42, 2485-2491[CrossRef][Medline] [Order article via Infotrieve]
7. Nicoll, D. A., Hryshko, L. V., Matsuoka, S., Frank, J. S., and Philipson, K. D. (1996) J. Biol. Chem. 271, 13385-13391[Abstract/Free Full Text]
8. Winkfein, R. J., Szerencsei, R. T., Kinjo, T. G., Kang, K.-J., Perizzolo, M., Eisner, L., and Schnetkamp, P. P. M. (2003) Biochemistry 42, 543-552[CrossRef][Medline] [Order article via Infotrieve]
9. Schnetkamp, P. P. M., Basu, D. K., and Szerencsei, R. T. (1989) Am. J. Physiol. (Cell Physiol.) 257, C153-C157[Abstract/Free Full Text]
10. Szerencsei, R. T., Prinsen, C. F. M., and Schnetkamp, P. P. M. (2001) Biochemistry 40, 6009-6015[CrossRef][Medline] [Order article via Infotrieve]
11. Dong, H., Light, P. E., French, R. J., and Lytton, J. (2001) J. Biol. Chem. 276, 25919-25928[Abstract/Free Full Text]
12. Blaustein, M. P., and Lederer, W. J. (1999) Physiol. Rev. 79, 763-854[Abstract/Free Full Text]
13. Kang, T. M., and Hilgemann, D. W. (2004) Nature 427, 544-548[CrossRef][Medline] [Order article via Infotrieve]
14. Kang, K.-J., and Schnetkamp, P. P. M. (2003) Biochemistry 42, 9438-9445[CrossRef][Medline] [Order article via Infotrieve]
15. Cooper, C. B., Szerencsei, R. T., and Schnetkamp, P. P. M. (2000) Methods Enzymol. 315, 847-864[Medline] [Order article via Infotrieve]
16. Kang, K.-J., Kinjo, T. G., Szerencsei, R. T., and Schnetkamp, P. P. M. (2005) J. Biol. Chem. 280, 6823-6833[Abstract/Free Full Text]
17. Szerencsei, R. T., Tucker, J. E., Cooper, C. B., Winkfein, R. J., Farrell, P. J., Iatrou, K., and Schnetkamp, P. P. M. (2000) J. Biol. Chem. 275, 669-676[Abstract/Free Full Text]
18. Prinsen, C. F. M., Szerencsei, R. T., and Schnetkamp, P. P. M. (2000) J. Neurosci. 20, 1424-1434[Abstract/Free Full Text]
19. Sheng, J.-Z., Prinsen, C. F. M., Clark, R. B., Giles, W. R., and Schnetkamp, P. P. M. (2000) Biophys. J. 79, 1945-1953[Abstract/Free Full Text]
20. Kinjo, T. G., Szerencsei, R. T., Winkfein, R. J., and Schnetkamp, P. P. M. (2004) Biochemistry 43, 7940-7947[CrossRef][Medline] [Order article via Infotrieve]
21. Schnetkamp, P. P. M., Li, X. B., Basu, D. K., and Szerencsei, R. T. (1991) J. Biol. Chem. 266, 22975-22982[Abstract/Free Full Text]
22. Iwamoto, T., Uehara, A., Imanaga, I., and Shigekawa, M. (2000) J. Biol. Chem. 275, 38571-38580[Abstract/Free Full Text]
23. Cai, X., and Lytton, J. (2004) J. Biol. Chem. 279, 5867-5876[Abstract/Free Full Text]
24. Palty, R., Ohana, E., Hershfinkel, M., Volokita, M., Elgazar, V., Beharier, O., Silverman, W. F., Argaman, M., and Sekler, I. (2004) J. Biol. Chem. 279, 25234-25240[Abstract/Free Full Text]

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