Wu S et al. (2012), Inner activation gate in S6 contributes to the ...

XB-ART-46099

J Gen Physiol 2012 Jul 01;1401:29-39. doi: 10.1085/jgp.201110749.

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Inner activation gate in S6 contributes to the state-dependent binding of cAMP in full-length HCN2 channel.

Wu S , Gao W , Xie C , Xu X , Vorvis C , Marni F , Hackett AR , Liu Q , Zhou L .

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Recently, applications of the patch-clamp fluorometry (PCF) technique in studies of cyclic nucleotide-gated (CNG) and hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channels have provided direct evidence for the long-held notion that ligands preferably bind to and stabilize these channels in an open state. This state-dependent ligand-channel interaction involves contributions from not only the ligand-binding domain but also other discrete structural elements within the channel protein. This insight led us to investigate whether the pore of the HCN channel plays a role in the ligand-whole channel interaction. We used three well-characterized HCN channel blockers to probe the ion-conducting passage. The PCF technique was used to simultaneously monitor channel activity and cAMP binding. Two ionic blockers, Cs(+) and Mg(2+), effectively block channel conductance but have no obvious effect on cAMP binding. Surprisingly, ZD7288, an open channel blocker specific for HCN channels, significantly reduces the activity-dependent increase in cAMP binding. Independent biochemical assays exclude any nonspecific interaction between ZD7288 and isolated cAMP-binding domain. Because ZD7228 interacts with the inner pore region, where the activation gate is presumably located, we did an alanine scanning of the intracellular end of S6, from T426 to A435. Mutations of three residues, T426, M430, and H434, which are located at regular intervals on the S6 α-helix, enhance cAMP binding. In contrast, mutations of two residues in close proximity, F431A and I432A, dampen the response. Our results demonstrate that movements of the structural elements near the activation gate directly affect ligand binding affinity, which is a simple mechanistic explanation that could be applied to the interpretation of ligand gating in general.

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Species referenced: Xenopus laevis
Genes referenced: camp hcn2 kcna2 tbx2 ttl

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	Figure 1. Extracellular Cs+ blocks the ionic conductance but has no effect on cAMP binding. (A) A schematic model showing the pore region and the CNBD in HCN channels. (B) Voltage protocol (top), timing of image collection (middle), and ionic current traces (bottom). The black line indicates control condition without Cs+; the red indicates 2 mM Cs+ added to the pipette (extracellular) solution. (C) Brightfield (BF) images of the membrane patch (indicated by arrows) and the corresponding fluorescence images collected before (C) or near the end of the hyperpolarizing voltage step (V). (D) Comparison of current amplitude (top) and fluorescence intensity (bottom) between control (black) and with Cs+ (red) in the pipette solution. The difference in the current amplitude between control and Cs+ was significant (independent samples t test: t = 3.57, P < 0.01). No significant difference in the fluorescence intensity was found (independent samples t test: t = 0.05, P = 0.96). Error bars indicate standard error.
	Figure 2. Intracellular Mg2+ blocks the ionic conductance but has no effect on cAMP binding. (A) A hyperpolarizing voltage step was used to activate the channel. Tail current was measured at 50 mV. (B) The corresponding current trace with no Mg2+ or 6 mM Mg2+. The inset shows an expanded view of the tail currents. (C) TTL signals from the charge-coupled device camera exposure port showing the timing of image collections. A series of images were collected before the voltage step (C, cAMP binding to the channels in the closed/resting state), immediately after the voltage step (V, maximal cAMP binding to channels in the open state), and during channel deactivation. (D) Raw fluorescence images collected before (C) and immediately after the voltage step (V) without Mg2+ in the bath solution. (E) Fluorescence images collected with 6 mM Mg2+ in the bath. (F) Mg2+ significantly inhibited both the inward macroscopic current (left, recorded at â150 mV) and outward tail current (right, recorded at 50 mV; independent samples t test: t = 6.74, P < 0.01). (G) Time course of fluorescence intensity changes without Mg2+ or with 6 mM Mg2+ in the bath. No significant difference was found (independent samples t test results at 3.65 s: t = 0.11, P = 0.99). (F and G) Error bars indicate standard error.
	Figure 3. Intracellular Mg2+ reduces the efficacy of ZD7288 in blocking HCN channel conductance. (A) Macroscopic currents blocked by 100 ÂµM ZD7288 (application indicated by the blue bar). The black line indicates 6 mM Mg2+ added in the bath solution; the red line indicates 0 Mg2+. The difference between 0 Mg2+ and 6 Mg2+ at time 255 s is statistically significant (independent samples t test: t = 13.85, P < 0.01). (B, top) Voltage protocol used. (middle) Current traces recorded with 6 mM Mg2+ in the bath solution. (bottom) 0 Mg2+. Current traces recorded with ZD7288 are shown in blue. Control traces without ZD7288 are shown in either black (6 mM Mg2+) or red (0 Mg2+). (C) Washing off the ZD7288 block (blue bar) by exposing the membrane patch to different concentrations of Mg2+ (open bar). The red line indicates without Mg2+; the black line indicates 6 mM Mg2+; the green line indicates 20 mM Mg2+. Recovery of HCN current was tested in the absence of Mg2+. (A and C) Error bars indicate standard error. (D) Corresponding voltage protocol and representative current traces.
	Figure 4. ZD7288 blocks ionic conductance and reduces activity-dependent cAMP binding. (A, top) Voltage protocol used for channel activation (â150 mV) and tail current recording (50 mV). (middle) Time course of image collection. (bottom) Current traces of control (black) and with 100 ÂµM ZD7288 (red). (B) The same experimental protocol used as in A except fluorescence images were collected during channel deactivation (50 mV). (C) Brightfield (BF) image of the patch pipette and fluorescence images collected before (0), at the beginning (1), and near the end (16) of channel deactivation. Control without ZD7288. (D) Brightfield and fluorescence images collected during channel deactivation with ZD7288 added in the bath solution. (E) Normalized fluorescence intensity change during channel activation. The fluorescence intensity measured from image 0 was used as a reference. The black line indicates control; the red line indicates 100 ÂµM ZD7288. At 5.45 s, the difference between control and ZD7288 was significant (independent samples t test: t = 2.46, P < 0.05). (F) Normalized fluorescence intensity change during channel deactivation. The black line indicates control; the red line indicates 100 ÂµM ZD7288. At 3.65 s, the difference between control and ZD7288 was significant (independent samples t test: t = 2.96, P < 0.05). Because of photobleaching, the fluorescence intensity measured near the end of the time course was slightly lower than that at the beginning. (E and F) Error bars indicate standard error.
	Figure 5. Biochemical assays exclude the possibility of direct contact between ZD7288 and the CL-CNBD fragment and the interference with cAMP binding. (A) ZD7288 has no effect on the excitation (top) and emission (bottom) spectra of 8-NBD-cAMP in complex with purified HCN channel C-terminal protein. The effect of Mg2+ was tested separately, and no difference was found between 0 and 6 mM Mg2+. (B) ITC results showing that ZD7288 does not interfere with normal cAMP binding. (top) Raw trace showing the rate of heat exchange versus the time of cAMP injection (with 300 ÂµM ZD7288). (bottom) Binding curves of cAMP to mHCN2 protein in the absence (black) and presence (red) of ZD7288.
	Figure 6. Alanine scanning of residues near the intracellular end of S6. (A) Alignment of primary sequences from mHCN2, MthK, KcsA, and Kv1.2 channels. Selectivity filter and S6 segment are indicated by red squares. Numbers on the top refer to the residue positions in mHCN2. Asterisks indicate the sequence number for the mHCN2 channel. Underlining indicates the region scanned by alanine replacement. Green and magenta letters indicate that the corresponding alanine replacements either increase (green) or decrease (magenta) the dynamic cAMP binding. (B) Normalized macroscopic current traces for WT (black), mHCN2/I432A (magenta), and mHCN2/H434A (green) channels. Voltage step and timing of fluorescent image collection are shown on top of the current traces. (C) Representative fluorescence images for I432A and H434A mutant channels. Timing of fluorescence image collection is shown in B. (D) Profiles of cAMP binding in response to a hyperpolarizing voltage step for WT mHCN2 (black), mHCN2/I432A (magenta), and mHCN2/H434A (green) channels. Fluorescence intensities are normalized to the value collected at â40 mV. (E) Summary of the PCF results. The mHCN2 channel contains two endogenous alanine residues in this region, A429 and A435. A435 was tested by a glycine replacement. A429 was not tested so the result represents the WT channel value. Independent samples t test shows that each of the following alanine mutants, T426A, M430A, F431A, I432A, and H434A, are significantly different from WT (A429; P < 0.01). (D and E) Error bars indicate standard error. (F) Structure model of the S5-pore-S6 region from the mHCN2 channel. The crystal structure of Kv1.2/2.1 chimera (Protein Data Bank accession no. 2R9R; Long et al., 2007) was used as the template for modeling. Side chains of T426, M430, and H434, of which the corresponding alanine replacement mutations increased cAMP binding, are shown in green. Side chains of F431 and I432, of which the alanine replacement mutations reduced cAMP binding, are shown in magenta.

References [+] :

Altomare, Integrated allosteric model of voltage gating of HCN channels. 2001, Pubmed