Hua L and Gordon SE (2005), Functional interactions between A' helices in t...

XB-ART-2253

J Gen Physiol 2005 Mar 01;1253:335-44. doi: 10.1085/jgp.200409187.

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Functional interactions between A' helices in the C-linker of open CNG channels.

Hua L , Gordon SE .

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Cyclic nucleotide-gated (CNG) channels are nonselective cation channels that are activated by the direct binding of the cyclic nucleotides cAMP and cGMP. The region linking the last membrane-spanning region (S6) to the cyclic nucleotide binding domain in the COOH terminus, termed the C-linker, has been shown to play an important role in coupling cyclic nucleotide binding to opening of the pore. In this study, we explored the intersubunit proximity between the A' helices of the C-linker regions of CNGA1 in functional channels using site-specific cysteine substitution. We found that intersubunit disulfide bonds can be formed between the A' helices in open channels, and that inducing disulfide bonds in most of the studied constructs resulted in potentiation of channel activation. This suggests that the A' helices of the C-linker regions are in close proximity when the channel is in the open state. Our finding is not compatible with a homology model of the CNGA1 C-linker made from the recently published X-ray crystallographic structure of the hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channel COOH terminus, and leads us to suggest that the C-linker region depicted in the crystal structure may represent the structure of the closed state. The opening conformational change would then involve a movement of the A' helices from a position parallel to the axis of the membrane to one perpendicular to the axis of the membrane.

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Species referenced: Xenopus
Genes referenced: camp cnga1 hcn2

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	Figure 1. . Sequence alignment of the C-linker regions between CNGA1 and HCN2 and structural model of the C-linker regions of CNGA1. (A) Sequence alignment of the C-linker regions between CNGA1 and HCN2. Green indicates conserved sequence and yellow indicates identical sequence. The square indicates the cysteine mutation region. (B) Structural model of CNGA1 C-linker based on the crystal structure of HCN2 channel COOH termini, with red and blue used to show alternate subunits. The model was built using the software Swiss-PDB viewer in conjunction with the Swiss-Model protein modeling server (Guex and Peitsch, 1997). The yellow residues indicate 420H in each subunit. The distance between 420H is 35.12 Å for adjacent subunits, and 50.18 Å for opposite subunits.
	Figure 2. . Cu/P induced potentiation in three of the seven cysteine mutants. Dose–response curves of cysteine mutants and CNGA1cysless activated by cGMP initially (open circles) and after (filled circles) treatment with Cu/P plus 2 mM cGMP. Smooth curves represent fits with the Hill equation (see materials and methods), with mean K1/2 values listed in Table I.
	Figure 3. . Cu/P effect on the current activated by saturating cAMP. Fraction of the current activated by 20 mM cAMP versus 2 mM cGMP (IcAMP/Imax) initially (open circles) and after Cu/P treatment (filled circles). Points represent the mean, with error bars representing the SEM. Patch numbers range from 4 to 8. Red * denotes a statistical difference of P < 0.01 (Student's t test) between the initial data and that after Cu/P treatment.
	Figure 4. . DTT reversed the effect of Cu/P treatment. (A) DTT reversed Cu/P potentiation of 417C. cGMP dose–response curves initially (open circles), after a 5-min treatment with Cu/P plus 128 μM cGMP (black circles), and after treatment with 5 mM DTT + 64 μM cGMP for 10 min (red circles). The smooth curves are fits with the Hill equation, with the following parameters: initially K1/2 = 7.04 μM, Hill slope = 2.0 (black curve, right), after Cu/P K1/2 = 1.61 μM, Hill slope = 2.8 (black curve, left), after DTT K1/2 = 5.03 μM, Hill slope = 2.2 (red curve). (B) Time course of DTT reversing the potentiation effect of Cu/P on 417C. Normalized currents activated by 2 μM cGMP (black circles) and 2 mM cGMP (open circles) at +100 mV after a 5-min treatment with Cu/P plus 128 μM cGMP, and then various cumulative time in 5 mM DTT plus 64 μM cGMP. Smooth curve represents single exponential fit of the data, with time constant = 37.0 s.
	Figure 5. . Cu/P produced two populations of channels in 418C. Single channel currents measured at +50 mV were recorded before and after a 10-min treatment with Cu/P plus 2 mM cGMP for 418C (A and B) and CNGA1cysless channels (C). For 418C, 4 μM cGMP was used to activate the channels, and for CNGA1cysless, 2 μM cGMP was used to activate the channels. All-points histograms were made from the data collected under each condition (right, in all cases >5 s of data was used).
	Figure 6. . Mechanisms of the dual effects of Cu/P on 418C. (A) Disulfide bonds formed between opposite and adjacent subunits could produce different effects. (B) The number of disulfide bonds could determine whether potentiation or inhibition would be observed. (C and D) Kinetics of the Cu/P effect on 418C with currents activated by either (C) 2 μM cGMP or (D) 2 mM cGMP. The smooth curves represent Gaussian fits (C) and exponential fits (D).
	Figure 7. . Cartoon model of closed and open channels. Side (top) and top (bottom) views of the CNGA1 C-linker made from the HCN2 structure (A) and from a model based on our data (B). The A' helices are shown in green and the B' helices are shown in blue. A and B differ only by the conformation of the B'-C' loop.

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Falke, Structure of a bacterial sensory receptor. A site-directed sulfhydryl study. 1988, Pubmed