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J Gen Physiol
2019 Feb 04;1512:200-213. doi: 10.1085/jgp.201812019.
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cAMP binds to closed, inactivated, and open sea urchin HCN channels in a state-dependent manner.
Idikuda V
,
Gao W
,
Su Z
,
Liu Q
,
Zhou L
.
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Hyperpolarization-activated cyclic-nucleotide-modulated (HCN) channels are nonselective cation channels that regulate electrical activity in the heart and brain. Previous studies of mouse HCN2 (mHCN2) channels have shown that cAMP binds preferentially to and stabilizes these channels in the open state-a simple but elegant implementation of ligand-dependent gating. Distinct from mammalian isoforms, the sea urchin (spHCN) channel exhibits strong voltage-dependent inactivation in the absence of cAMP. Here, using fluorescently labeled cAMP molecules as a marker for cAMP binding, we report that the inactivated spHCN channel displays reduced cAMP binding compared with the closed channel. The reduction in cAMP binding is a voltage-dependent process but proceeds at a much slower rate than the movement of the voltage sensor. A single point mutation in the last transmembrane domain near the channel''s gate, F459L, abolishes inactivation and concurrently reverses the response to hyperpolarizing voltage steps from a decrease to an increase in cAMP binding. ZD7288, an open channel blocker that interacts with a region close to the activation/inactivation gate, dampens the reduction of cAMP binding to inactivated spHCN channels. In addition, compared with closed and "locked" closed channels, increased cAMP binding is observed in channels purposely locked in the open state upon hyperpolarization. Thus, the order of cAMP-binding affinity, measured by the fluorescence signal from labeled cAMP, ranges from high in the open state to intermediate in the closed state to low in the inactivated state. Our work on spHCN channels demonstrates intricate state-dependent communications between the gate and ligand-binding domain and provides new mechanistic insight into channel inactivation/desensitization.
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30541772
???displayArticle.pmcLink???PMC6363418 ???displayArticle.link???J Gen Physiol ???displayArticle.grants???[+]
Figure 1. Chemical structure of NBD-cAMP and the modulation of WT spHCN channels. (A) Chemical structure of the NBD-cAMP (Axxora). (B) Top: Voltage protocol. Bottom: Current traces of the WT spHCN channel recorded in the absence of cAMP (black), 5 μM NBD-cAMP (green), 10 μM NBD-cAMP (red), or 10 μM regular cAMP (blue). (C) Competitive binding assay for the specificity of NBD cAMP binding to spHCN channels. Raw fluorescent images of membrane patch showing a decrease in the 0.5-μM NBD-cAMP fluorescence signal upon addition of saturating concentration of 50 μM nonfluorescent cAMP. (D) Normalized fluorescent intensities from competitive binding experiments. The fluorescence intensity of NBD-cAMP (black, control, n = 5) decreased upon adding regular cAMP (red, 10 μM, n = 6; blue, 50 μM, n = 6). Error bars here and after represent SEM (see Statistics in Materials and methods).
Figure 5. Both ZD7288 and Cs+ can block the spHCN current, but only ZD7288 affects the binding of NBD-cAMP. (A) ZD7288 blocks the currents of the WT spHCN channel. Top: Voltage step, laser pulse, and image collection protocol. Bottom: Current traces before (black) and after (red) adding 60 µM ZD7288. (B) Normalized fluorescence intensity before (black) and after (red) ZD7288 application. Voltage step, +80 to −80 mV (n = 17). (C) Normalized fluorescence intensity before (black) and after (red) ZD7288 application. Voltage step, +80 to −100 mV. (D) 2 mM Cs+ was added to the pipette solution to block the WT spHCN current from the extracellular side. Top: Voltage step, laser pulse, and image collection protocol. Bottom: Current traces collected with Cs+ added to the pipette solution. (E) Normalized fluorescence intensity without (black) or with Cs+ (red). Voltage step, +80 to −80 mV. Because the pipette solution was not exchanged during the experiments, control results and Cs+ results were collected from different patches (n = 13). (F) Normalized fluorescence intensity without (black) or with Cs+ (red). Voltage step, +80 to −80 mV.
Figure 6. Strategy to specifically lock the spHCN channel in either the open or closed state. (A) Alignment of primary protein sequences of representative HCN and other potassium channels in the region encompassing the selectivity filter and the last transmembrane segment (S2 in KcsA or S6 in other channels). Relevant residues are shown in bold and marked with a different color. (B) Mutations introduced to the S6 of the spHCN channel to make the locked-open (H462C-L466C) or locked-closed (H462Y-Q468C) channel. (C) Current traces of the WT spHCN channel. Black, control in the absence of cAMP and Cd2+. Red, 10 μM cAMP. Blue, 10 μM cAMP and 1µM Cd2+. (D) Current traces of the locked-open spHCN channel. Black, control in the absence of cAMP and Cd2+. Red, 10 μM cAMP. Blue, 1 µM Cd2+ without cAMP. The locked-open effect was persistent after the washing off of cAMP (bath solution containing Cd2+). (E) Current traces of the locked-closed spHCN channel. Black, control in the absence of cAMP and Cd2+. Red, 10 μM cAMP. Blue, 2 μM Cd+2 and 10 µM cAMP.
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