Iaa

20 ms

Fig. 3. Experimental steps for establishing the direct activation of CNG channels by NO groups or SH-modifying reagents.

donor cysteine groups must be on the intracellular portion of the channel protein because, unlike NO, these reagents are only poorly membrane permeable,22 and so must have acted at the intracellular face of the membrane patch. In addition, application of a membrane-impermeant form of NEM, 1 mM dextran-NEM (kind gift of S. J. Kleene, University of Cincinnati; College of Medicine, Cincinnati, OH), can also activate the channel in inside-out patches.

From the known amino acid sequences, the olfactory CNG channel contains eight cysteine residues.9 Of these, four are both highly conserved and reside intra-cellularly; three of them are located in the cAMP-binding site of the channel.

Molecular Approach

Site-Directed Mutagenesis

Cysteine-to-serine CNG channel mutants can be generated by substituting the specific cysteine residues with serines by polymerase chain reaction (PCR)-based mutagenesis described by Nelson and Long.23 Pju polymerase (Stratagene, La Jolla, CA) should be used to reduce the rate of contaminating mutations. All constructs must be verified by sequencing.

Channel Protein Expression

Human embryonic kidney (HEK) 293 cells grown at 37° in minimal essential medium supplemented with 10% (v/v) horse serum and 1% (w/v) gentamicin can be used as an expression system for the wild-type and mutant CNG channels (Fig. 3). A pCIS expression vector (Genentech, South San Francisco, CA) containing either the wild-type rat olfactory CNG channel subunit (CNGA2)24 or one of the different types of mutant channel is used to perform transient transfections according to a standard calcium phosphate protocol.25 The cells are cotransfected with a vector carrying the gene for the green fluorescent protein (GFP) (Stratagene) at a 1:1 molar ratio. Patch-clamp recordings are made 2-3 days after transfec-tion. GFP is used as an indicator of transfection success,26'27 efficiency, and probable expression of wild-type or mutant CNG channels. GFP fluorescence can be visualized in living cells, without histological processing, by using a fluorescence

22 K. Donner, S. Hemila, G. Kalamkarov, A. Koskelainen, I. Pogozheva, and T. Rebrik, Exp. Eye Res. 51, 97 (1990).

23 R. M. Nelson and L. L. Long, Anal. Biochem. 180, 147 (1989).

24T.-Y. Chen, Y.-W. Peng, R. S. Dhallan, B. Ahamed, R. R. Reed, and K.-W. Yau, Nature (London) 362, 764 (1993).

25 C. M. Gorman, D. R. Gies, and G. McCray, DNA Protein Eng. Techn. 2, 3 (1990).

26 A. B. Cubitt, R. Heim, S. R. Adams, A. E. Boyd, L. A. Gross, and R. Y. Tsien, Trends Biochem. Sci. 20,448(1995).

27 M.-C. Broillet and S. Firestein, Neuron 18,951 (1997).

microscope equipped with fluorescein isothiocyanate (FITC) filter sets that span the excitation wavelengths of 450-500 nm. Observation of the degree of fluorescence after cotransfection with a vector carrying the gene for GFP allows us to reliably select for cells with a high probability of either single-channel or macroscopic currents (hundreds of channels) in the membrane patch. Macroscopic currents can also be recorded with an Axopatch ID patch-clamp amplifier (Axon Instruments) and stored on a Power Mac G4 using the software Pulse (HEKA Elektronik) and analyzed with the software Igor (WaveMetrics).

Northern Blot Analysis

It is necessary to verify by Northern blot analysis that the mutant CNG channels are still expressed in HEK 293 cells. The cells can be harvested 2 days after transfection with wild-type or mutant cDNAs of the CNG channel subunits. The total RNA can be extracted by TRIzol reagent (GIBCO-BRL, Gaithersburg, MD). Fifteen micrograms of total RNA is then size fractionated on a formaldehyde gel and blotted. The blot is hybridized at 42° with a digoxigenin (dig)-ll-dUTP-labeled 500-bp cDNA fragment located at the wild-type CNG channel (CNGA2) bp 1251-1751 coding region. The hybridized blot is detected with a digoxigenin nucleic acid detection kit (Boehringer Mannheim, Indianapolis, IN). As a control, nontransfected HEK 293 cells should be used.

Specificity of S-Nitrosylation

It is not uncommon for several cysteine residues on a given protein to be candidates for nitrosylation. In the ryanodine receptor, of a total of 364 cysteines, 84 provide free SH groups, but only 12 are thought to undergo nitrosylation.5 Although the precise parameters governing accessibility by NO are unknown, the existence of a consensus nitrosylation acid-base motif has been postulated on the basis of large database screenings.28 The proposed motif is XYCZ, where X can be any of G, S, T, C, Y, N, or Q; Y can be K, R, H, D, or E; and Z can be D or E. The most important element of the sequence is believed to be the aspartate/glutamate residues following the cysteine. In spite of this rather degenerate motif, in the CNG channel only Cys-460, identified by our biochemical and mutation experiments as the NO target site, possesses the required motif (i.e., Q, D, C, E) (Fig. 4).

Because a functional CNG channel is most probably made up of four subunits,29,30 there are four potential nitrosylation sites per channel. However, factors other than those noted above may also determine the likelihood of NO

29 D. T. Liu, G. R. Tibbs, and S. A. Siegelbaum, Neuron 16, 983 (1996).

30 D. T. Liu, G. R. Tibbs, P. Paoletti, and S. A. Siegelbaum, Neuron 21,235 (1998).

Wild type

50 hMCAMP

0 0

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