Our knowledge of the crystal structures of two related ligand binding domains (
One approach that has been used successfully with CNG channels, and many other types of proteins, is that of perturbing function by altering amino acid side chains. Through site-directed mutagenesis, any individual amino acid or group of amino acids can be replaced with natural or even unnatural amino acids. In CNG channels, this method has led to the elucidation of the mechanism of calmodulin modulation (
Two papers in this issue combine old and new technology to probe the energetic coupling between ligand binding and the allosteric conformational change in CNG channels (
To examine the interaction between the energetics of channel activation and the effects of UV modification, the authors altered channel function in three ways: altering the primary sequence (CNG2 channels compared with CNG1 channels), using two agonists with different efficacies (cAMP compared with cGMP), and applying a potentiator of CNG1 channel activation (the divalent transition metal Ni2+). These experiments revealed an inherently different energetic cost for modifying the tryptophan targets in CNG2 than in CNG1. Finally, three types of models of activation were examined: an independent Hodgkin-Huxley (HH) model (
Modifying tryptophan residues with UV light has several advantages. As the most highly conserved amino acid, efforts to substitute another amino acid for tryptophan using site-directed mutagenesis often results in nonfunctional proteins. This, in fact, was the case for every combination of two tryptophans that Middendorf and colleagues attempted to alter in the CNG channel sequence. Thus, using UV light can be a way to trick a protein into substituting something else for a tryptophan, with the caveat that only a small number of substitutions are possible and the experimenter cannot control which one will result. Their highly conserved nature makes tryptophans excellent subjects for this type of analysisif they are so important to channel function, altering their structure is almost sure to perturb channel function.
As a general approach, the utility of using UV light to modify tryptophans in ion channels is limited by a few technical issues. One issue is that the Xenopus oocytes used for expression in this study contained a conductance that was activated by UV light. This slightly voltage-dependent, cation-selective conductance increased exponentially with cumulative UV light dose, and did not saturate within the range of the amplifier used. This is not likely to be a limitation unique to the oocyte expression system; similar UV-activated conductances have been reported in several mammalian cell lines (
UV light can be an important tool in our quest for understanding the structural basis for ion channel function. As with any technique, it has its own strengths and weaknesses. By taking advantage of its specificity (wavelength-dependent affect) and unique ability to target mutagenesis-resistant residues, photomodification could be used to gain insights into the importance of tryptophans, in particular ion channels and other proteins.
Submitted: 7 July 2000
Revised: 7 July 2000
Accepted: 11 July 2000
![]() |
References |
---|
![]() ![]() |
---|
Brown, R.L., Haley, T.L., Snow, S.D. 2000. Irreversible activation of cyclic nucleotidegated ion channels by sulfhydryl-reactive derivatives of cyclic GMP. Biochemistry. 39:432-441[Medline].
Brown, R.L., Snow, S.D., Haley, T.L. 1998. Movement of gating machinery during the activation of rod cyclic nucleotidegated channels. Biophys. J. 75:825-833
Chen, T.Y., Yau, K.W. 1994. Direct modulation by Ca(2+)-calmodulin of cyclic nucleotideactivated channel of rat olfactory receptor neurons. Nature. 368:545-548[Medline].
Doyle, D.A., Morais Cabral, J., Pfuetzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S.L., Chait, B.T., MacKinnon, R. 1998. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 280:69-77
Eismann, E., Muller, F., Heinemann, S.H., Kaupp, U.B. 1994. A single negative charge within the pore region of a cGMP-gated channel controls rectification, Ca2+ blockage, and ionic selectivity. Proc. Natl. Acad. Sci. USA. 91:1109-1113[Abstract].
Fodor, A.A., Gordon, S.E., Zagotta, W.N. 1997. Mechanism of tetracaine block of cyclic nucleotidegated channels. J. Gen. Physiol. 109:3-14
Gavazzo, P., Picco, C., Menini, A. 1997. Mechanisms of modulation by internal protons of cyclic nucleotidegated channels cloned from sensory receptor cells. Proc. R. Soc. Lond. B Biol. Sci 264:1157-1165[Medline].
Gordon, S.E., Oakley, J.C., Varnum, M.D., Zagotta, W.N. 1996. Altered ligand specificity by protonation in the ligand binding domain of cyclic nucleotidegated channels. Biochemistry. 35:3994-4001[Medline].
Gordon, S.E., Varnum, M.D., Zagotta, W.N. 1997. Direct interaction between amino- and carboxyl-terminal domains of cyclic nucleotidegated channels. Neuron. 19:431-441[Medline].
Gordon, S.E., Zagotta, W.N. 1995a. A histidine residue associated with the gate of the cyclic nucleotideactivated channels in rod photoreceptors. Neuron. 14:177-183[Medline].
Gordon, S.E., Zagotta, W.N. 1995b. Localization of regions affecting an allosteric transition in cyclic nucleotideactivated channels. Neuron. 14:857-864[Medline].
Gordon, S.E., Zagotta, W.N. 1995c. Subunit interactions in coordination of Ni2+ in cyclic nucleotidegated channels. Proc. Natl. Acad. Sci. USA. 92:10222-10226[Abstract].
Goulding, E.H., Tibbs, G.R., Siegelbaum, S.A. 1994. Molecular mechanism of cyclic-nucleotidegated channel activation. Nature. 372:369-374[Medline].
Grossweiner, L.I. 1976. Photochemical inactivation of enzymes. Curr. Top. Radiat. Res. Q. 11:141-199[Medline].
Hodgkin, A.L., Huxley, A.F. 1952. A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. 117:500-544[Medline].
Hsu, S.F., Ahern, G.P., Jackson, M.B. 1999. Ultra-violet light-induced changes in membrane properties in secretory cells. J. Neurosci. Methods. 90:67-79[Medline].
Liu, D.T., Tibbs, G.R., Paoletti, P., Siegelbaum, S.A. 1998. Constraining ligand-binding site stoichiometry suggests that a cyclic nucleotidegated channel is composed of two functional dimers. Neuron. 21:235-248[Medline].
Matulef, K., Flynn, G.E., Zagotta, W.N. 1999. Cysteine-scanning mutagenesis of the cyclic nucleotidegated channel ligand binding domain. Biophys. J. 76:A337. (Abstr.).
Means, G.E., Feeney, R.E. 1971. Chemical modification of proteins. Monograph. San Francisco, CA, Holden-Day, Inc, pp. 254.
Mendez, F., Penner, R. 1998. Near-visible ultraviolet light induces a novel ubiquitous calcium-permeable cation current in mammalian cell lines. J. Physiol 507:365-377
Middendorf, T.R., Aldrich, R.W. 2000. Effects of ultraviolet modification on the gating energetics of cyclic nucleotidegated channels. J. Gen. Physiol. 116:253-282
Middendorf, T.R., Aldrich, R.W., Baylor, D.A. 2000. Modification of cyclic nucleotidegated ion channels by ultraviolet light. J. Gen. Physiol. 116:227-252
Monod, J., Wyman, J., Changeux, J.P. 1965. On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12:88-118[Medline].
Paoletti, P., Young, E.C., Siegelbaum, S.A. 1999. C-Linker of cyclic nucleotidegated channels controls coupling of ligand binding to channel gating. J. Gen. Physiol. 113:17-34
Root, M.J., MacKinnon, R. 1993. Identification of an external divalent cation-binding site in the pore of a cGMP-activated channel. Neuron. 11:459-466[Medline].
Shammat, I.M., Gordon, S.E. 1999. Stoichiometry and arrangement of subunits in rod cyclic nucleotidegated channels. Neuron. 23:809-819[Medline].
Su, Y., Dostmann, W.R.G., Herberg, F.W., Durick, K., Xuong, N.-h., Ten Eyck, L., Taylor, S.S., Varughese, K.I. 1995. Regulatory subunit of protein kinase A: structure of deletion mutant with cAMP binding domains. Science. 269:807-813[Medline].
Sun, Z.P., Akabas, M.H., Goulding, E.H., Karlin, A., Siegelbaum, S.A. 1996. Exposure of residues in the cyclic nucleotidegated channel pore: P region structure and function in gating. Neuron. 16:141-149[Medline].
Varnum, M.D., Black, K.D., Zagotta, W.N. 1995. Molecular mechanism for ligand discrimination of cyclic nucleotidegated channels. Neuron. 15:619-625[Medline].
Varnum, M.D., Zagotta, W.N. 1997. Interdomain interactions underlying activation of cyclic nucleotidegated channels. Science. 278:110-113
Vladimirov, Y.A., Roshchupkin, D.I., Fesenko, E.E. 1970. Photochemical reactions in amino acid residues and inactivation of enzymes during U.V.-irradiation. A review. Photochem. Photobiol. 11:227-246[Medline].
Wang, L., Xu, D., Dai, W., Lu, L. 1999. An ultraviolet-activated K+ channel mediates apoptosis of myeloblastic leukemia cells. J. Biol. Chem. 274:3678-3685
Weber, I.T., Steitz, T.A. 1987. Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 A resolution. J. Mol. Biol. 198:311-326[Medline].
Zong, X., Zucker, H., Hofmann, F., Biel, M. 1998. Three amino acids in the C-linker are major determinants of gating in cyclic nucleotidegated channels. EMBO (Eur. Mol. Biol. Organ.) J. 17:353-362