Mechanism of cGMP-gated channel block by intracellular polyamines

Abstract

Polyamines block the retinal cyclic nucleotide-gated channel from both the intracellular and extracellular sides. The voltage-dependent mechanism by which intracellular polyamines inhibit the channel current is complex: as membrane voltage is increased in the presence of polyamines, current inhibition is not monotonic, but exhibits a pronounced damped undulation. To understand the blocking mechanism of intracellular polyamines, we systematically studied the endogenous polyamines as well as a series of derivatives. The complex channel-blocking behavior of polyamines can be accounted for by a minimal model whereby a given polyamine species (e.g., spermine) causes multiple blocked channel states. Each blocked state represents a channel occupied by a polyamine molecule with characteristic affinity and probability of traversing the pore, and exhibits a characteristic dependence on membrane voltage and cGMP concentration.

Figure 1

Effects of mutation at E363 on CNG channel block by spermine. (A and B) Macroscopic I-V curves of wild-type and mutant E363G channels, respectively, in the absence and presence of various concentrations of intracellular spermine. (C and D) The fractions of unblocked currents of wild-type and mutant channels in the presence of various concentrations of spermine are plotted as a function of membrane voltage.

Figure 2

Comparison of CNG channel block by intracellular spermine and putrescine. (A) I-V curves of the CNG channel in the absence and presence of 10 μM spermine (SPM). (B) The fraction of current not blocked by 10 μM spermine is plotted against membrane voltage. (C and E) I-V curves of the channel in the absence and presence of 10 mM of putrescine (PUT), over different voltage ranges. (D and F) The fractions of current not blocked by 10 mM putrescine, obtained from C and E, are plotted against membrane voltage.

Figure 3

Effect of diamines on the current–voltage relationship of the CNG channel. I-V curves were obtained in the absence and presence of nine diamines (DM_C2 through DM_C10, labeled as C₂ through C₁₀) at the various concentrations indicated.

Figure 4

Voltage dependence of CNG channel block by various diamines. The fraction of current not blocked by nine diamines (DM_C2 through DM_C10, labeled as C₂ through C₁₀) at various concentrations is plotted against membrane voltage. The curves superimposed on the data are fits of or (see discussion).

Figure 5

Intracellular pH dependence of CNG channel block by intracellular spermine. (A) I-V curves of the CNG channel in the absence and presence of 10 μM spermine at intracellular pH 7.6 and 8.6. Extracellular pH was 7.6 in both cases. (B) Ratios of the I-V curves at the corresponding pH, shown in A, are plotted against membrane voltage. The smooth curves were obtained by simultaneous fitting of the two data curves using . The values of all parameters obtained from fitting are: K ₁ ^a = 0.51 ± 0.04 μM, Z ₁ ^a = 3.0 ± 0.1, k ₋₂ ^a/k ₋₁ ^a = 8.5 ± 0.3, “z ₋₂ ^a+ z ₋₁ ^a” = 5.5 ± 0.1, K ₁ ^b = 45.9 ± 0.9 μM; Z ₋₁ ^b = 1.8 ± 0.1, and pKa = 9.1 ± 0.1 (mean ± SEM, n = 3).

Figure 6

Block of the CNG channel by intracellular spermine and spermidine over a wider voltage range. (A and B) I-V curves of the CNG channel without or with spermine and spermidine, respectively, at the concentrations indicated. (C and D) The fractions of current not blocked by spermine and spermidine, respectively, are plotted against membrane voltage.

Figure 7

Cyclic GMP concentration dependence of CNG channel block by spermine. (A–F) I-V curves of the channel without and with spermine at various cGMP concentrations. (G–L) The fractions of current not blocked by spermine at various spermine and cGMP concentrations are plotted against membrane voltage.

Figure 8

Block of the CNG channel by a spermine derivative. (A) I-V curves without or with three concentrations of a spermine derivative, PhTx. (B) The fractions of current not blocked by PhTx are plotted against membrane voltage. The curves superimposed on the data are fits of the Woodhull equation, I/I _o = 1/(1 + [PhTx]/K _d e ⁻ ZF ^V/ RT. The K _d and Z determined from the fits are 2.8 ± 0.5 μM and 1.8 ± 0.3 (mean ± SEM, n = 4).

Figure 9

A kinetic model for CNG channel block by diamines. (A) Reaction scheme. Ch represents the CNG channel, DM_i and DM_o denote intra- and extracellular diamines, and Ch·DM^a and Ch·DM^b denote the CNG channels blocked by DM in two conformations, respectively. Rate constants at 0 mV (k _x or k _−x) and the corresponding valence number (z _x or z _−x) are indicated. (B) Noisy experimental trace plots the fraction of current not blocked by 10 mM putrescine against membrane voltage. Curve a superimposed on the data is a fit of . The fit gives K ₁ ^a = 2.0 mM, Z ₁ ^a = 1.1, k ₋₂ ^a/k ₋₁ ^a = 2.0, “z ₋₂ ^a+ z ₋₁ ^a” = 1.9, K ₁ ^b = 388.2 mM, and Z ₋₁ ^b = 0.5. The other three curves correspond to three hypothetical cases using the corresponding parameters from the fit, except that putrescine is assumed to act (b) as a pure high-affinity permeant blocker (K ₁ ^b = ∞), (c) as a pure high-affinity non-permeant blocker (K ₁ ^b = ∞ and k ₋₂ ^a = 0), or (d) as a low affinity nonpermeant blocker for curve d (K ₁ ^a = ∞ and k ₋₂ ^b = 0).

Figure 10

Summary of the fitting parameters obtained from analyzing the diamine-blocking curves. As illustrated in Fig. 4, all diamine-blocking curves were fitted with either or , depending on whether the fitted curve contains a second descending phase. The six adjustable parameters, plotted in A–F against number of methylene groups in the diamine chain, were K ₁ ^a, Z ₁ ^a, k ₋₂ ^a/k ₋₁ ^a, and “z ₋₂ ^a+ z ₋₁ ^a” needed in both equations, and K ₁ ^b and Z ₁ ^b needed in only. All parameters, obtained from the fits, are presented as mean ± SEM (n = 6–10).

Figure 11

A kinetic model for pH dependence of CNG channel block by polyamines. The model essentially is the same as that in Fig. 9 A, except that polyamine molecules can be in either a more or a less protonated form.