Rosenbaum T , Islas LD , Carlson AE , Gordon SE .

EY01730 NEI NIH HHS , R01 EY13007 NEI NIH HHS , P30 EY001730 NEI NIH HHS , R01 EY013007 NEI NIH HHS

	Figure 1. . Dequalinium blocks CNGA1 channels at a saturating cGMP concentration. (A) Chemical structure of dequalinium (quinolinium, 1,1′-(1, 10-decanediyl)bis(4-amino-2-methyl dichloride)). (B, left) Initial current obtained in the presence of 2 mM cGMP. (B, right) Block of current obtained from the same patch in the presence of 1 μM dequalinium. (C) Dose-response relation for dequalinium block. The smooth curve is a fit with the Hill equation (see materials and methods) with K1/2 = 189 nM and the Hill coefficient = 1.4. All currents were measured at 20 mV. Error bars represent SEM; n = 5 patches.
	Figure 2. . Dequalinium blocks heteromeric CNGA1+CNGB1 channels. Dose-response relation for block of heteromeric channels by dequalinium. A 1:4 ratio of CNGA1:CNGB1 RNA was injected in oocytes. The dotted curve was taken from the CNGA1 homomeric data shown in Fig. 1 C. Data from heteromeric channels (diamonds) were fit with the Hill equation with the following parameters: K1/2 = 385 nM, Hill coefficient = 1.5.
	Figure 3. . Block of cGMP-activated currents by dequalinium is more effective from the intracellular side of the channels. (A and B) Current families were recorded in response to voltage steps from 0 mV to between −100 and 100 mV in 20-mV steps. (A) Currents recorded in the inside-out configuration in the absence (left) and presence of 1 μM dequalinium (right). (B) Currents recorded in the outside-out configuration in the absence (left) and presence of 1 μM dequalinium (right).
	Figure 4. . Dequalinium applied to the extracellular surface appears to block from the inside. (A) Time-course for block by 1 μM dequalinium applied to the intracellular surface of an inside-out patch (open symbols) and 1 μM dequalinium applied to the extracellular surface of an outside-out patch (filled circles). Each time course was fit with a single exponential, with a τ = 53 ms for the inside-out patch and τ = 1,429 ms for the outside-out patch. The inset represents the voltage protocol used for these experiments. (B) Current response to a voltage protocol, as indicated above the data. Here, 1 μM dequalinium was applied to the extracellular surface of an outside out patch. The current decay at 100 mV indicates block and the relaxation at −100 mV represents unblock.
	Figure 5. . Block by dequalinium is state independent. (A) Dose-response relations for dequalinium block in the presence of saturating cGMP (2 mM cGMP, filled squares) and in the presence of subsaturating cGMP (32 μM cGMP, open circles). Data are presented as fraction of block for comparison purposes. The smooth line is a fit with Eq. 1, K1/2 = 189 nM. (B) Dose-response for cGMP in the absence (filled diamonds) and presence of 400 nM dequalinium (open diamonds). Error bars represent SEM, n = 4 patches. The smooth lines represent fits to the Eq.1 with K1/2 = 80 μM in the presence of dequalinium K1/2 = 90 μM in the absence of dequalinium. Dotted line represents normalization of the data obtained in the presence of 400 nM dequalinium to the data obtained in the absence of dequalinium, K1/2 = 80 μM. (C) Predicted fits with the models shown in Schemes 1–4. Filled and hollow diamonds are from the same data in B. The dashed line represents a fit with Scheme III (pure open channel block), the dotted line represents block only in the closed state (Scheme II, pure closed channel block) and the thin line represents block occurring equally during the closed and open states of the channel with a KD = 50 nM for both of the conformational states (Scheme IV). The parameter values for these fits were L = 19 and K = 3800 M−1 (in all cases). (D) Block of currents by 250 nM dequalinium in the presence of 2 mM cGMP (left) and in the presence of 20 mM cIMP, a partial agonist of these channels (right). In both cases block was of ∼48%.
	SCHEME I.
	SCHEME II.
	SCHEME III.
	SCHEME IV.
	Figure 6. . Voltage dependence of dequalinium block. (A) Current traces obtained at −60 and 60 mV (as indicated by the arrow) in the absence of dequalinium (left) and in the presence of 400 nM dequalinium (right). Dashed line marks the zero current level. (B) Current-voltage relation obtained from holding at the different voltages indicated until steady-state was reached. The smooth curve represents a fit with Eq. 2 with a value of zδ = 1.
	Figure 7. . Interaction of dequalinium with the permeant ion. (A) Dose-response relation for block by dequalinium in 130 mM NaCl (circles) with K1/2 = 189 nM and (B) triangles represent data obtained with 13 mM NaCl with K1/2 = 39 nM (knock-off experiment) and the dotted-line represents data shown in A (see materials and methods). Error bars represent SEM, n = 5.
	Figure 8. . Single-channel recordings. (A) Single-channel current traces in response to depolarizing voltage pulses to 60 mV in the presence of 2 mM cGMP. Channel open probability = 0.92. Open (O) and closed (C) levels are indicated in the figure. (B) All-points amplitude histogram that corresponds to a current amplitude of 1.9 pA. (C) Current traces from the same channel in A recorded in the presence of 5 nM dequalinium at the same potential. (D) The amplitude histogram shows that the single-channel conductance remains unchanged. (E) The closed distribution for cGMP alone can be fit with sum of two exponentials of time constants 0.34 and 8.4 ms. (F) Open time constant for cGMP alone. The distribution can be fitted with sums of two exponentials with time constants 3.4 and 16 ms. (G) Closed time distribution in the presence of 5 nm dequalinium. In the presence of dequalinium the same time constants are present (0.35 and 7.2 ms), but a third (43.7 ms) appears that corresponds to the blocked state. (H) Open time distribution in the presence of 5 nM dequalinium. The distribution can be fitted with sums to two exponentials (3.4 and 15.6 ms). Similar results were obtained from three other single-channel patches.

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