Sunkara MR , Schwabe T , Ehrlich G , Kusch J , Benndorf K .

	Figure 1. Series of representative currents generated by the wwww concatemer for recording steady-state activation. The pulse protocol is shown above the traces. The channels were activated in the sequence to more hyperpolarizing voltages. The amplitude of the tail current at −100 mV was evaluated.
	Figure 2. Function of concatenated homotetrameric channels. (A) Steady-state activation relationships in the absence and presence of 50 µM cAMP for w4 channels. The data points were fitted with Eq. 1, yielding for the parameters Vh and zδ −116.5 ± 1.8 mV (n = 10) and 6.57 ± 0.40 (n = 10); −94.8 ± 1.5 mV (n = 9) and 6.42 ± 0.48 (n = 9), respectively. (B) Same as A for wwww channels. The respective parameters for Vh and zδ are −123.9 ± 2.3 mV (n = 17) and 6.41 ± 0.26 (n = 17); −103.9 ± 3.0 mV (n = 12) and 6.43 ± 0.26 (n = 12). (C) Comparison of the Vh values for the three wild-type and three mutated channels; 0 cAMP (control), black squares, 50 µM cAMP, green squares. (D) Comparison of the cAMP-induced voltage shift of Vh, ΔVh. (E) Comparison of zδ. Symbols correspond to C. (F) Comparison of cAMP-evoked current increase, ΔI−150mV. 4–17 recordings per data point were included for analyses in C to F. In the cartoons here and below, green circles represent functional binding sites, whereas empty crossed circles represent mutated binding sites. Error bars indicate SEM.
	Figure 3. Effect of different combinations of liganded subunits on steady-state activation. Eight tetrameric and one dimeric concatemer are compared. cAMP is applied at a saturating concentration of 50 µM. (A) cAMP-induced voltage shift ΔVh (mV), determined as described for Fig. 2A by using Eq. 1. The amount of voltage shift by liganded subunits is additive. In channels with two functional CNBDs, the cis and the trans position are indistinguishable. (B) Effect of saturating cAMP on current increase at the saturating voltage of −150 mV, ΔI−150mV. Two liganded subunits suffice to generate the maximum effect. Data points contain 8–17 recordings. Error bars indicate SEM.
	Figure 4. Effects of subunit liganding on activation kinetics. (A) Superimposition of activation time courses in the presence and in the absence of cAMP at −140 mV for wwww and illustration of determining the activation time constant τa (monoexponential fits are shown in red). Late currents are normalized. (B) Voltage dependence of τa and effect of cAMP for wwww (n = 13–17 recordings per data point). Green and black symbols show values obtained at 50 µM and zero cAMP, respectively. (C) Comparison of τa for the eight tetrameric concatemers and the dimeric concatemer wm2 at hyperpolarization to −140 mV. Data points contain 9–17 recordings. (D) τa values in dependence on normalized voltage. Shown are individual data points. Green and black symbols show values obtained at 50 µM and zero cAMP, respectively. Normalization was performed by the respective Vh value of the individual recordings. Error bars indicate SEM.
	Figure 5. Effects of subunit liganding on deactivation kinetics. The currents were activated by pulses of 6-s duration to −140 mV, and deactivation was measured at a subsequent pulse to −30 mV. (A) Superimposition of two current time courses of the concatemer wwww in the absence and presence of cAMP and expanded deactivation time courses at −30 mV. Determination of the time of half-maximum deactivation thd is illustrated. (B) Comparison of τd,cAMP/τd,cont for the eight tetrameric concatemers and the dimeric concatemer wm2 (see text). At least two liganded subunits in trans position are required to decelerate deactivation. Data points contain 3–32 recordings. Error bars indicate SEM.
	Figure 6. Cartoon model for the energetics of activation by voltage and cAMP in a HCN2 channel. A channel is assumed to adopt only one open (O) and one closed (C) state. According to the Eyring rate theory (Glasstone et al., 1941), the transition between the two states requires an amount of free energy ΔG to reach the activation energy Ea of the transition state TS. The major effect on activation is that of voltage (thick red arrows): At −30 mV, ΔG is much higher in O than in C, whereas at −140 mV, ΔG is moderately higher in C than in O. The binding of cAMP to the four subunits (green circles) is assumed to increase and decrease ΔG in the closed and open state, respectively. In the closed state, the energy contributions for the four binding steps are additive, whereas in the open state, these energy contributions are only additive for the quadruple, triple, and trans-double ligated channel and the cis-double and the single ligated channel do not change ΔG with respect to the empty channel.

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