Subunit interactions in coordination of Ni2+ in cyclic nucleotide-gated channels.

Abstract

Cyclic nucleotide-gated (CNG) channels present a unique model for studying the molecular mechanisms of channel gating. We have studied the mechanism of potentiation of expressed rod CNG channels by Ni2+ as a first step toward understanding the channel gating process. Here we report that coordination of Ni2+ between histidine residues (H420) on adjacent channel subunits occurs when the channels are open. Mutation of H420 to lysine comptetely eliminated the potentiation by Ni2+ but did not markedly alter the apparent cGMP affinity of the channel, indicating that the introduction of positive charge at the Ni2+-binding site was not sufficient to produce potentiation. Deletion or mutation of most of the other histidines present in the channel did not diminish potentiation by Ni2+. We studied the role of subunit interactions in Ni2+ potentiation by generating heteromultimeric channels using tandem dimers of the rod CNG channel sequence. Injection of single heterodimers in which one subunit contained H420 and the other did not (wt/H420Q or H420Q/wt) resulted in channels that were not potentiated by Ni2+. However, coinjection of both heterodimers into Xenopus oocytes resulted in channels that exhibited potentiation. The H420 residues probably occurred predominantly in nonadjacent subunits when each heterodimer was injected individually, but, when the two heterodimers were coinjected, the H420 residues could occur in adjacent subunits as well. These results suggest that the mechanism of Ni2+ potentiation involves intersubunit coordination of Ni2+ by H420. Based on the preferential binding of Ni2+ to open channels, we suggest that alignment of H420 residues of neighboring subunits into the Ni2+-coordinating position may be associated with channel opening.Cyclic nucleotide-gated (CNG) channels transduce sensory information into electrical signals in rod and cone photoreceptors, avian pinealocyte photoreceptors, and olfactory receptors (1-3). These channels are also expressed in other tissues, such as kidney (4), aorta (5), and sperm (6), where their function is unknown. Although CNG channels are opened by the direct binding of cyclic nucleotides, they bear little functional or sequence similarity to other ligand-gated channels. Sequence similarity, especially in the pore region and transmembrane segments, places the CNG channels in the family of voltage-gated ion channels, along with voltage-gated K+, Na+, and Ca2+ channels (7). In addition, each CNG channel subunit has a region in its C-terminal domain that is similar in sequence to cAMP-and cGMP-dependent protein kinases (8, 9) and has been shown to bind a single cyclic nucleotide (10).CNG channels can be expressed as homomultimers of their primary subunit (a or subunit 1) (8,11 (16)(17)(18). When coexpressed with the a subunits, these 13 subunits (or subunit 2) appear to confer properties of the native channels previously absent from the expressed channels (16-18). However, even these expressed heteromultimeric channels do not match the native channels in all as...

C Termini of the Escherichia coli Mechanosensitive Ion Channel (MscS) Move Apart upon the Channel Opening

“…This affinity is similar to that estimated for a single imidazole group in solution (24). Coordination of Ni 2ϩ between multiple histidines on adjacent subunits of the cyclic nucleotide gating channel was reported to occur in the range of micromolar concentrations of Ni 2ϩ (33); however, histidines from the center of ␣-hemolysin polypeptide from Staphylococcus aureus were coordinated by Zn 2ϩ at a concentration of 0.1-0.6 mM (23). Even higher concentrations of Ni 2ϩ (0.1-50 mM) were reported to inhibit epithelial sodium channel by coordination of histidines from two extracellular subunits ␣ and ␥ (34).…”

Regulation of CNGA1 Channel Gating by Interactions with the Membrane

“…It has been suggested previously that His-420 residues on adjacent subunits coordinate metal ions (31,32), but the homology model of CNGA1 from the tetrameric HCN2 C-terminal region crystal structure shows that the residues are not close enough to support metal ion coordination (9). Instead we found that adding a metal-chelating lipid to the membrane (C18-NTA) increased the affinity of Co 2ϩ for potentiation.…”

“…It has been previously suggested that multiple His-420 residues on adjacent subunits coordinate transition metal ions (31,32). However, the tetrameric structure of the related HCN2 C-terminal fragment reveals that the His-420 equivalent positions in the AЈ helices of neighboring subunits are too far to support metal ion coordination in CNGA1 channels (9).…”

The carboxyl-terminal region of cyclic nucleotide-modulated channels is a gating ring, not a permeation path

“…Many studies have shown that mutagenic or chemical modification of this region have effects on channel gating (31,(44)(45)(46)(47)(48)(49)(50)(51). The central tunnel seen in the crystal structure of the carboxyl-terminal region of HCN2 suggests that it might also be a part of the channel ion-permeation pathway.…”