State-dependent block of CNG channels by dequalinium

Abstract

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels with a high permeability for Ca(2+). Not surprisingly, they are blocked by a number of Ca(2+) channel blockers including tetracaine, pimozide, and diltiazem. We studied the effects of dequalinium, an extracellular blocker of the small conductance Ca(2+)-activated K(+) channel. We previously noted that dequalinium is a high-affinity blocker of CNGA1 channels from the intracellular side, with little or no state dependence at 0 mV. Here we examined block by dequalinium at a broad range of voltages in both CNGA1 and CNGA2 channels. We found that dequalinium block was mildly state dependent for both channels, with the affinity for closed channels 3-5 times higher than that for open channels. Mutations in the S4-S5 linker did not alter the affinity of open channels for dequalinium, but increased the affinity of closed channels by 10-20-fold. The state-specific effect of these mutations raises the question of whether/how the S4-S5 linker alters the binding of a blocker within the ion permeation pathway.

Figure 1.

State-independent block of CNGA1 and CNGA2 channels observed at 0 mV. (A and B) Current traces obtained when holding the patch at 0 mV with 20-ms steps to +20 mV at 5-s intervals. (A) CNGA1 channels in the absence of dequalinium and in the presence of 250 nM dequalinium with 2 mM (left) and 32 μM cGMP (right). Dequalinium blocked the current ∼50% under both conditions. (B) CNGA2 channels in the absence of dequalinium and in the presence of 2 μM dequalinium with 2 mM (left) and 1.8 μM cGMP (right). Dequalinium blocked the current ∼50% under both conditions. (C) Dose–response for block of CNGA1 channels (squares) and CNGA2 channels (circles) by dequalinium. Filled squares represent block in the presence of 2 mM cGMP and open squares represent block in the presence of 32 μM cGMP for CNGA1 channels. For CNGA2 channels, filled circles represent block in the presence of 2 mM cGMP and open circles represent block in the presence of 1.8 μM cGMP. The smooth curves are fits with Eq. 1 (see materials and methods) with IC₅₀ = 189 nM and 192 nM for CNGA1 (high and low cGMP, respectively) and 2.4 μM (both for high and low cGMP) and the Hill coefficient = 1.4 in all cases. Error bars represent SEM, n = 5–7 patches.

SCHEME I

Figure 2.

Time course of block of CNGA1 channels during depolarizing and hyperpolarizing voltage steps. (A) Block by 50 nM dequalinium during a voltage step to +60 mV. The gray curve is a fit with a single exponential, with a time constant τ = 2.9 s. (B) Block by 50 nM dequalinium during a voltage step to −60 mV. The gray curve is a fit with a single exponential, with a time constant τ = 17 s. (C) Time course of block obtained by using a protocol in which the holding potential was held at 0 mV and pulsed for 20 ms to +20 mV every 5 s. The gray line is a fit with a single exponential, yielding τ = 59 min.

Figure 3.

Voltage dependence of block in CNGA1 and CNGA2 channels. (A) Block of CNGA1 channels by 50 nM dequalinium in the presence of 2 mM cGMP (filled circles) or 32 μM cGMP (open circles). The curves represent fits with Scheme I, with K_Do = 4 μM and K_Dc = 1.3 μM. The value of zδ was 1 in both cases. (B) Block of CNGA2 channels by 500 nM dequalinium in the presence of 2 mM cGMP (filled circles) or 1.8 μM cGMP (open circles). The curves represent fits with Scheme I, with K_Do = 20 μM, and K_Dc = 3.6 μM The value of zδ was 1 in both cases. Dequalinium concentrations below the IC₅₀ of block were used since otherwise block at depolarized potentials becomes rapidly saturated and the voltage-dependence of block cannot be correctly determined.

Figure 4.

State-dependent block of the S4/S5-linker chimeras by dequalinium at 0 mV. Block by different dequalinium concentrations of (A) S4/S5-CNGA1 and (B) S4/S5-CNGA2 channels. Filled symbols represent block in the presence of 2 mM cGMP and open symbols represent block in the presence of subsaturating cGMP concentrations (32 μM for S4/S5-CNGA1 and 1.8 μM for S4/S5-CNGA2). Fits to the Hill equation yielded mean IC₅₀ values for S4/S5-CNGA1 channels of 360 nM at 2 mM cGMP and 156 nM with 32 μM cGMP, and for S4/S5-CNGA2 channels of 2.7 μM at 2 mM cGMP and 650 nM at 1.8 μM cGMP. (C) Block of wild-type and mutant channels. A CNGA1 channel background (depicted in black) was used to construct chimeras where specific regions where replaced by the corresponding regions of CNGA2 (depicted in gray). The IC₅₀ values for each of the constructs obtained in the presence of saturating cGMP (2 mM, white boxes) and subsaturating cGMP (32 μM or 1.8 μM, gray boxes) are shown for each construct. For the box and whisker plot the line represent the median of the data, the box surrounds the 25th through 75th percentile, and the whiskers extend to the 5th and 95th percentiles.

Figure 5.

cGMP activation curves for S4/S5-chimeras. (A and C) Activation of the S4/S5-CNGA1 and S4/S5-CNGA2 chimeras by cGMP. Data were fit with Eq. 1 (see materials and methods). The dashed lines represents the activation curve for wild-type CNGA1 (A) and CNGA2 channels (C). The K_1/2 for activation by cGMP was 66 μM for S4/S5-CNGA1 and 2.6 μM for S4/S5-CNGA2. (B and D) Fractional activation by cAMP (thin traces) was of ∼6% of the maximal current activated by cGMP (thick traces) for both wild-type CNGA1 (B, left) and S4/S5-CNGA1 channels (B, right), and 86% for wild-type CNGA2 (D, left) and S4/S5-CNGA1 (D, right)

Figure 6.

Voltage dependence of block in S4/S5-CNGA1 and S4/S5-CNGA2 channels. (A) Block of S4/S5-CNGA1 channels by 50 nM dequalinium in the presence of 2 mM cGMP (filled circles) and 32 μM cGMP (open circles). The curves represent fits with Scheme I, with K_Do = 4 μM and K_Dc = 250 nM. (B) Block of S4/S5-CNGA2 channels by 500 nM dequalinium in the presence of 2 mM cGMP (filled circles) and 1.8 μM cGMP (open circles). The curves represent fits with Scheme I, with K_Do = 14 μM and K_Dc = 300 nM.

Figure 7.

Summary of the effects of mutations in the S4/S5 region on affinity of open and closed channels for dequalinium. Regions of sequence from CNGA1 are shown in black and regions of sequence from CNGA2 are shown in gray.

Figure 8.

Localization of amino acids of the S4/S5 chimeras in the channel structure. (A) Sequence alignment of four types of ion channels, CNGA1, CNGA2, KcsA, and KirBac 1.1. Asterisks denote amino acids mutated in the S4-S5 linker chimeras. (B) Structure of the KcsA K⁺ channel (Doyle et al., 1998). The circle with the “I” represents the position of one of the amino acids we mutated in the S4/S5 chimeras of CNG channels. The dotted line and the question mark indicated that the region of sequence containing the other S4-S5 linker mutations is not resolved. (C) Structure of the KirBac 1.1. K⁺ channel (Kuo et al., 2003). The positions analogous to the four mutations of the S4/S5 chimeras we produced are shown as circles.