Mazzolini M et al. (2009), The analysis of desensitizing CNGA1 channels re...

XB-ART-41140

J Gen Physiol 2009 Apr 01;1334:375-86. doi: 10.1085/jgp.200810157.

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The analysis of desensitizing CNGA1 channels reveals molecular interactions essential for normal gating.

Mazzolini M , Anselmi C , Torre V .

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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.

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Species referenced: Xenopus laevis
Genes referenced: cnga1 kcna2 kcnb1 tbk1 tbx2

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	Figure 1. Desensitized mutant channels. cGMP-activated currents in the presence of 1 mM cGMP at +60 mV in the WT (A) and mutant channels E363A (B), T355A (C), L356A (D), and F380A (E). cGMP was added as indicated by the solid horizontal black bars. (F) Sequence alignment between CNGA1, Shaker, Kv1.2, Kv2.1, KcsA, and NaK channels. The TVGYG signature of K+ channels is highlighted in yellow. Amino acids that become more accessible upon desensitization or C-type inactivation are highlighted in green. The sequence alignment was performed by using Clustal W (Thompson et al., 1994).
	Figure 2. State-dependent modification of the mutant channel E363A+T364C. cGMP-activated current traces at +60 mV in three different conformations of mutant channel: desensitized (A), closed (D), and open (E) states. (B) Effect of 5 mM DTT added to patch pipette solution in desensitized conformation of channel. (F and G) Effect of 2.5 mM MTSET added to the extracellular medium in closed (F) and open (G) states. 1 mM cGMP, 5 mM DTT, and 2.5 mM MTSET was added as indicated by the solid horizontal black or red bars. (C) Comparison of 5 mM DTT added to bath solution (red solid triangle), in patch pipette (red open square), and in DTT absence (black solid circle) in desensitized state; the values represent the amplitude of the peak current (I) measured every 30 s and normalized (I/Imax) to peak current of control (Imax) at time zero. (H) State-dependent modification in the closed (black solid circle) and open (black open circle) states in the absence of 2.5 mM MTSET, and in closed (red solid triangle) and open (red open triangle) states in the presence of 2.5 mM MTSET in the extracellular medium. Single points of the plots represent the amplitude of the peak current (I) measured every 5 s in open state and 30 s in closed state and, as in C, normalized (I/Imax) to peak current of control (Imax) at time zero.
	Figure 3. Desensitization in mutant channels E363A+P366C, T355A, T355C, and T355C+C314S. cGMP-activated current traces in the desensitized state of four mutant channels: E363A+P366C (A) at −60 mV, T355A (C), T355C (D), and T355C+C314S (F) at +60 mV. 1 mM cGMP was added as indicated by the solid horizontal black bars. (B) State-dependent modification in closed (solid triangle), open (open triangle), and desensitized (plus) states of mutant channel E363A+P366C in the presence of 2.5 mM MTSET. The plot shows the state-dependent modification calculated from the amplitude of the peak current (I) every 30 s in the closed state and 5 s in the open state, whereas in the desensitized state the values of current were measured from the peak (I) to the steady state (I) every 2 s. The values were normalized (I/Imax) to the peak current of control (Imax) at time zero. (E) State-dependent modification in closed (solid triangle), open (open triangle), and desensitized (solid circle) states for mutant channel T355C in the absence of 2.5 mM MTSET. The values of current were measured as in B. (G) Comparison of mutant channels T355C and T355C+C314S in desensitizing state in the absence (black open circle and square) and in the presence (black solid circle and square) of 5 mM DTT applied to bath solution. Symbols represent the amplitude of the peak current (I) measured every 30 s for both mutant channels and normalized (I/Imax) to peak current of control (Imax) at time zero.
	Figure 4. Recovery from desensitization in double-mutant channels. cGMP-activated currents in the presence of 1 mM cGMP at +60 mV in mutant channels L356C (A), F380C (B), L356C+F380C (C), L356F (D), F380L (E), L356F+F380L (F), L356D (G), F380K (H), and L356D+F380K (I). cGMP was added as indicated by the solid horizontal bars.
	Figure 5. The interaction between L356 and F380 is within the same subunit. Diagrams representing subunit composition of the tandem L356XtandemWT (A, top row), the tandem L356XtandemF380X (B, top row), and the tandem L356X+F380XtandemWT (C, top row) in which X = C or A. Each subunit is represented by a circle, whereas the linker of 10 amino acids between two subunits of the tandem is represented by an arc (see Materials and methods). In the middle row, cGMP-activated currents in the presence of 1 mM cGMP at +60 mV in the tandem L356CtandemWT (A, middle row), in the tandem L356CtandemF380C (B, middle row), and in the tandem L356C+F380CtandemWT (C, middle row). In the bottom row, cGMP-activated currents in the presence of 1 mM cGMP at +60 mV in the tandem L356AtandemWT (A, bottom row), in the tandem L356AtandemF380A (B, bottom row), and in the tandem L356A+F380AtandemWT (C, bottom row).
	Figure 6. Illustrated representations of hypothesized molecular interactions between S6 and P helix during gating. (A) In the open (and closed) states of the WT CNGA1 channel, the hydrophobic interaction between Phe380 and Leu356 couples S6 and the P helix, and a presumed H-bond between Thr355 and Glu363 couples the P helix and the pore wall. (B) In mutant channels E363A, T355A, L356A, and F380A, these interactions are lost and, after opening, mutant channels desensitize. In mutant channel E363A, residues in positions 364 and 366 become more accessible to the reagents added to the extracellular medium. (C) A simplified model of gating in CNGA1 channels: in the closed state (gray) the tip of P helices occludes the pore lumen; during gating, the S6 helix rotates by 30° anticlockwise, the pore is not occluded (red), and ions can permeate through it. The hydrophobic interaction between Phe380 and Leu356 couples the rotation of S6 to the motion of the P helix.

References [+] :

Becchetti, Cyclic nucleotide-gated channels: intra- and extracellular accessibility to Cd2+ of substituted cysteine residues within the P-loop. 2000, Pubmed, Xenbase