The analysis of desensitizing CNGA1 channels reveals molecular interactions essential for normal gating.
The pore region of cyclic nucleotide-gated (CNG) channels acts as the channel gate. Therefore, events occurring in the cyclic nucleotide-binding (CNB) domain must be coupled to the movements of the pore walls. When Glu363 in the pore region, Leu356 and Thr355 in the P helix, and Phe380 in the upper portion of the S6 helix are mutated into an alanine, gating is impaired: mutant channels E363A, L356A, T355A, and F380A desensitize in the presence of a constant cGMP concentration, contrary to what can be observed in wild-type (WT) CNGA1 channels. Similarly to C-type inactivation of K(+) channels, desensitization in these mutant channels is associated with rearrangements of residues in the outer vestibule. In the desensitized state, Thr364 residues in different subunits become closer and Pro366 becomes more accessible to extracellular reagents. Desensitization is also observed in the mutant channel L356C, but not in the double-mutant channel L356C+F380C. Mutant channels L356F and F380K did not express, but cGMP-gated currents with a normal gating were observed in the double-mutant channels L356F+F380L and L356D+F380K. Experiments with tandem constructs with L356C, F380C, and L356C+F380C and WT channels indicate that the interaction between Leu356 and Phe380 is within the same subunit. These results show that Leu356 forms a hydrophobic interaction with Phe380, coupling the P helix with S6, whereas Glu363 could interact with Thr355, coupling the pore wall to the P helix. These interactions are essential for normal gating and underlie the transduction between the CNB domain and the pore.
Mazzolini
Monica
M
International School for Advanced Studies, I-34014 Trieste, Italy.
Anselmi
Claudio
C
Torre
Vincent
V
D000595:Amino Acid Sequence; D000818:Animals; D006152:Cyclic GMP / Q000235:genetics / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D005260:Female; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009712:Nucleotides, Cyclic / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D017354:Point Mutation; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D014982:Xenopus laevis
C475741:CNGA1 protein, bovine; D054815:Cyclic Nucleotide-Gated Cation Channels; D009712:Nucleotides, Cyclic; D006152:Cyclic GMP