Conformational rearrangements in the S6 domain and C-linker during gating in CNGA1 channels.

This work completes previous findings and, using cysteine scanning mutagenesis (CSM) and biochemical methods, provides detailed analysis of conformational changes of the S6 domain and C-linker during gating of CNGA1 channels. Specific residues between Phe375 and Val424 were mutated to a cysteine in the CNGA1 and CNGA1(cys-free) background and the effect of intracellular Cd(2+) or cross-linkers of different length in the open and closed state was studied. In the closed state, Cd(2+) ions inhibited mutant channels A406C and Q409C and the longer cross-linker reagent M-4-M inhibited mutant channels A406C(cys-free) and Q409C(cys-free). Cd(2+) ions inhibited mutant channels D413C and Y418C in the open state, both constructed in a CNGA1 and CNGA1(cys-free) background. Our results suggest that, in the closed state, residues from Phe375 to approximately Ala406 form a helical bundle with a three-dimensional (3D) structure similar to those of the KcsA; furthermore, in the open state, residues from Ser399 to Gln409 in homologous subunits move far apart, as expected from the gating in K(+) channels; in contrast, residues from Asp413 to Tyr418 in homologous subunits become closer in the open state.

A comparison of electrophysiological properties of the CNGA1, CNGA1tandem and CNGA1cys-free channels.

Three constructs are used for the analysis of biophysical properties of CNGA1 channels: the WT CNGA1 channel, a CNGA1 channel where all endogenous cysteines were removed (CNGA1cys-free) and a construct composed of two CNGA1 subunits connected by a small linker (CNGA1tandem). So far, it has been assumed, but not proven, that the molecular structure of these ionic channels is almost identical. The I/V relations, ionic selectivity to alkali monovalent cations, blockage by tetracaine and TMA+ were not significantly different. The cGMP dose response and blockage by TEA+ and Cd2+ were instead significantly different in CNGA1 and CNGA1cys-free channels, but not in CNGA1 and CNGA1tandem channels. Cd2+ blocked irreversibly the mutant channel A406C in the absence of cGMP. By contrast, Cd2+ did not block the mutant channel A406C in the CNGA1cys-free background (A406Ccys-free), but an irreversible and almost complete blockage was observed in the presence of the cross-linker M-4-M. Results obtained with different MTS cross-linkers and reagents suggest that the 3D structure of the CNGA1cys-free differs from that of the CNGA1 channel and that the distance between homologous residues at position 406 in CNGA1cys-free is longer than in the WT CNGA1 by several Angstroms.

Locking CNGA1 channels in the open and closed state.

With the aim of understanding the relation between structure and gating of CNGA1 channels from bovine rod, an extensive cysteine scanning mutagenesis was performed. Each residue from Phe-375 to Val-424 was mutated into a cysteine one at a time and the modification caused by various sulfhydryl reagents was analyzed. The addition of the mild oxidizing agent copper phenanthroline (CuP) in the open (presence of 1 mM cGMP) or closed state locked the channel in the respective states. A subsequent treatment with the reducing agent DTT restored normal gating fully in the open state and partially in the closed state. This action of CuP was not observed when F380 was mutated into a cysteine in the cysteine-free CNGA1 channel and in the double mutant C314S&F380C. These observations suggest that these effects are mediated by the formation of a disulfide bond (S-S) between F380C and the endogenous Cys-314 in the S5 segment. It can be rationalized by supposing that during gating the S6 segment rotates anticlockwise-when viewed from the extracellular side-by approximately 30 degrees .

Nickel differentially affects NMDA receptor channels in developing cultured rat neurons.

Nickel (Ni(2+)) is a transition metal that exerts multiple and complex effects on N-methyl-d-aspartate (NMDA) channels. In both HEK293 cells and Xenopus laevis oocytes expressing recombinant NMDA receptors, Ni(2+) (<100 microM) caused a potentiation of NR2B-containing channels but a voltage-independent inhibition in those containing NR2A. We took advantage of this different response to investigate the developmental switch between NR2B and NR2A subunits in neonatal rat cerebellar granule cells up to 16 days in vitro (DIV) and in rat embryo cortical neurons up to 35 DIV. In both cultures, the effect of Ni(2+) on the NMDA current gradually changed from potentiating to inhibitory with progressing DIV, and the decline of potentiation correlated well with the decrease in sensitivity for the NR2B specific antagonist ifenprodil. Dose-dependent experiments confirmed that Ni(2+) has a different effect in younger cultures with respect to older ones, in agreement with an increase of the percentage of NR2A-containing receptors. The developmental switch occurred within the first 5 DIV in cerebellar granule cells and after 20 DIV in cortical neurons. All these data indicate that Ni(2+) is a suitable marker for the identification of NR2A and NR2B native channel subunits and can be used to trace the development of NMDA receptor composition.

Movement of the C-helix during the gating of cyclic nucleotide-gated channels.

Movements within the cyclic nucleotide-binding domain of cyclic nucleotide-gated channels are thought to underlie the initial phase of channel gating (Tibbs, G. R., D. T. Liu, B. G. Leypold, and S. A. Siegelbaum. 1998. J. Biol. Chem. 273:4497-4505; Zong, X., H. Zucker, F. Hofmann, and M. Biel. 1998. EMBO J. 17:353-362; Matulef, K., G. E. Flynn, and W. N. Zagotta. 1999. Neuron. 24:443-452; Paoletti, P., E. C. Young, and S. A. Siegelbaum. 1999. J. Gen. Physiol. 113:17-33; Johnson, J. P., and W. N. Zagotta. 2001. Nature. 412:917-921). To investigate these movements, cysteine mutation was performed on each of the 28 residues (Leu-583 to Asn-610), which span the agonist-binding domain of the alpha-subunit of the bovine rod cyclic nucleotide-gated channel. The effects of Cd(2+) ions, 2-trimethylammonioethylmethane thiosulfonate (MTSET) and copper phenanthroline (CuP) on channel activity were examined, in excised inside-out patches in the presence and in the absence of a saturating concentration of cGMP. The application of 100 microM Cd(2+) in the presence of saturating concentration of cGMP caused an irreversible and almost complete reduction of the current in mutant channels E594C, I600C, and L601C. In the absence of cGMP, the presence of 100 microM Cd(2+) caused a strong current reduction in all cysteine mutants from Asp-588 to Leu-607, with the exception of mutant channels A589C, M592C, M602C, K603C, and L606C. The selective effect of Cd(2+) ions was very similar to that observed when adding the oxidizing agent CuP to the bath medium, except for mutant channel G597C, where CuP caused a stronger current decrease (67 +/- 7%) than Cd(2+) (23 +/- 4%). In the absence of cGMP, MTSET caused a reduction of the current by >40% in mutant channels L607C, L601C, I600C, G597C, and E594C, whereas in the presence of cGMP only mutant channel L601C was affected. The application of MTSET protected many mutant channels from the effects of Cd(2+) and CuP. These results suggest that, when CNG channels are in the open state, residues from Asp-588 to Leu-607 are in an alpha-helical structure, homologous to the C-helix of the catabolite gene activator protein (Weber, I. T., and T. A. Steitz. 1987. J. Mol. Biol. 198:311-326). Furthermore, residues Glu-594, Gly-597, Ile-600, and Leu-601 of these helices belonging to two different subunits must be in close proximity. In the closed state the C-helices are in a different configuration and undergo significant fluctuations.