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P.P. Schnetkamp, T.G. Kinjo, K. Kang, R.T. Szerencsei, R.J. Winkfein; Helix packing of the retinal cone Na/Ca–K exchanger: helices that contribute to the cation transport site. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1283.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Determine helix packing and residues important for cation transport of the retinal cone Na/Ca–K exchanger. Methods: All nine cysteine residues of the wild–type human Na/Ca–K exchanger protein (NCKX2) were mutated to serine to yield a "cysless" NCKX2. Pairs of cysteine residues were introduced into this cysless background and mutant NCKX2 proteins were expressed in HEK293 or in High Five cells. Copper–phenanthroline was used to form intramolecular disulfide bonds to probe for close proximity of the two each pair of inserted cysteine residues. In addition, conservative substitutions were made for residues critical for NCKX2 transport function and their effect assessed. Results: Retinal rod and cone photoreceptors express two related, but distinct gene products of the Na/Ca–K exchanger (NCKX) gene family that utilize the energy provided by both transmembrane sodium and potassium gradients to extrude calcium that enters the rod or cone outer segments via the light–sensitive, cGMP–gated channels. Both rod and cone NCKX proteins contain two clusters of hydropobic transmembrane spanning segments (TM1 and TM2) separated by a large cytosolic loop. We recently showed that each TM contains five membrane spanning helices (Biochemistry 42: 2485–2491), and that the second helix in both TM1 and TM2 contains residues critical for cation transport (Biochemistry 42: 543–552). This suggests that these two helices are close together in the native NCKX protein albeit separated by ∼360 residues. Here, we have addressed the helix packing of the cone NCKX2 protein by the formation of intramolecular disulfide bonds requiring close proximity of the two cysteine residues involved. When expressed heterologously, the cysless NCKX2 showed wild–type protein expression and retained ∼50% of wild–type NCKX2 function. Several critical hydroxyl–containing and acidic residues located in helices 2 and 7, respectively, could be readily cross–linked indicating that they are in close proximity in the native NCKX2 protein and likely form the cation binding site. In agreement with this, conservative substitution of critical aspartate and glutamate residues found in helix 2 and 7, respectively, caused changes in both external calcium and potassium affinity measured for Na/Ca–K exchange transport. Conclusions:We identified critical residues in the cone Na/Ca–K exchanger that constitute the cation binding site(s).
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