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Figure 1. Location of spHCN1 mutations involved in reversed voltage dependence. (A) Topology diagram of spHCN1, showing the location of the S4–S5 linker and post-S6 C-linker regions. Also shown are the S1–S6 transmembrane segments including the S4 voltage sensor, and the cyclic nucleotide-binding domain (CNBD). Locations of the residues F359 and K482 discussed in the text are indicated by circles. Also shown are alignments of sequences in these regions for spHCN1 and hHCN2, with F359 and K482 of spHCN1 shown in red. (B) Clues to the positions of the mutations from related crystal structures of the Kv1.2 pore-forming domain (top) and the HCN2 cyclic nucleotide binding domain (bottom). The S4–S6 regions of one subunit of Kv1.2 (Long et al., 2005) are shown (aa288–416); the S5 and S6 are roughly in the plane of the paper with the N-terminal end of S4–S5 running ∼35° into the paper. The alignment of HCN with Kv channels is difficult in the linker and S5 regions, making it hard to speculate on the location of individual residues. Three subunits of the HCN2 CNBD (Zagotta et al. 2003) are shown, with the position corresponding to spHCN1-482 indicated for the green subunit in the foreground. The relative position of the pore-forming and CNB domains is not known. The Kv1.2 structure is apparently an open state; the CNBD structure has four molecules of cAMP bound, but the C-linker conformation may correspond more to a closed-preferring form (Craven and Zagotta, 2004).
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Figure 2. Cross-linking S4–S5 linker and post-S6 regions reverses voltage dependence of spHCN1. Effect of cAMP (100 μM), Cd2+ (130 nM), or DTNB treatment, on 359C-482C channel currents from independent patches. Fold increases in peak time-dependent current at −120 mV in response to application were as follows: cAMP (8.0 ± 1.2, n = 9), Cd2+ (3.5 ± 1.5, n = 9), and DTNB (4.7 ± 1.7, n = 9), i.e., cAMP induced a greater increase of peak current than either Cd2+ or DTNB. DTNB results were obtained after washout of the reagent (see Materials and methods).
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Figure 3. Quantitative assessment of reversed voltage dependence. Properties of 359C-482C currents after modification with DTNB. (A) Depolarization-activated currents elicited by steps from −120 mV (500-ms pulse) to test potentials ranging from −110 mV to +90 mV (1 s duration), in +20-mV steps. Pulse voltage is indicated alongside the corresponding current. (B) Normalized conductance–voltage relationship for modified channels (filled circles, from tail current relaxation amplitudes at −120 mV in A after 500-ms depolarizing prepulses), or unmodified channels (open triangles, from tails at +50 mV after hyperpolarizing prepulses; measured in the presence of cAMP [100 μM] for experimental ease, as parameters of G-V fits are not markedly different ± cAMP; e.g. Shin et al., 2004). Values for modified and unmodified channels were independently normalized. Mean parameters from individual fits to four patches were as follows: modified channels, V1/2 = −4.1 ± 8 mV and slope = 25.8 ± 1.3 mV; unmodified channels, V1/2 = −69 ± 2 mV and slope = 5.7 ± 0.2 mV (similar to wild-type spHCN1 under these conditions; e.g., Shin et al., 2004). Note that normalized tail current relaxation amplitudes are shown, and hence residual steady-state current at −120 mV is not represented; we did not routinely measure ZD7288-blockable currents in these patches and therefore cannot reliably estimate the fraction of channel current contributing to leak. (C) Current declines elicited by steps from +10 mV to test potentials ranging from −20 to −120 mV, in −10 mV steps (along with a 400-ms decrement in duration, to give shorter durations at hyperpolarized potentials, for increased patch stability). (D) Time constants for time-dependent relaxations: rising depolarization-induced relaxations in A (τrising), and declining hyperpolarization-induced relaxations in C (τdeclining). Mean parameters from individual fits were as follows: rising, slope = +0.0080 ± 6.4 × 10−4 mV−1 (n = 4; i.e., e-fold in ∼54 mV); declining, slope = −0.0091 ± 2.4 × 10−4 mV−1 (n = 3; i.e., e-fold in ∼48 mV).
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Figure 4. Reversed gating is sensitive to cAMP and to a mutation in the pore. (A) Effect of cAMP (100 μM) on 359C-482C currents modified with Cd2+ (130 nM, left) or DTNB (right). (B) Effect of DTNB on 359C-482C-459L currents, in the absence of cAMP.
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Figure 5. State-dependent block by ZD7288 during reversed gating. (A) 359C-482C currents after modification with DTNB. ZD7288 (100 μM) was applied during the periods indicated. The sequential first and second current traces during which ZD7288 was applied are shown. Block at +50 mV occurred with τ = 300 ± 12 ms (n = 3 patches). The graph shows cumulative block of currents by ZD7288 (100 μM) applied for 1 s during each pulse at the voltage indicated. Pulses were applied every 5 s. Block was virtually irreversible over the time course studied, shown by the absence of recovery upon washout. Cumulative block occurred slowly when ZD7288 was applied at −120 mV (τ ∼ 13 s), but much more rapidly when applied at +50 mV (τ ∼ 460 ms). Note ZD7288-sensitive residual steady-state current was evident at −120 mV, comprising ∼8% of maximum ZD7288-blockable inward current in this patch. (B) 359C-482C current traces after modification with DTNB (left) and after subsequent application of cAMP (100 μM). ZD7288 was then applied for 1 s during pulses to −120 mV in the presence of cAMP (right). Sequential first and second traces during which ZD7288 was applied are shown. Block occurred with τ ∼ 460 ms.
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Figure 6. Cross-linking S4–S5 and post-S6 produces tonic inactivation that can no longer be completely removed by cAMP. (A) Effects of Cd2+ (130 nM, left) or DTNB (right) on 359C-482C currents in the constant presence of cAMP. (B) Effects of Cd2+ (130 nM, left) or DTNB (right) on 359C-482C-459L currents in the constant presence of cAMP.
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