Blockade of a retinal cGMP-gated channel by polyamines - PubMed (original) (raw)

Blockade of a retinal cGMP-gated channel by polyamines

Z Lu et al. J Gen Physiol. 1999 Jan.

Abstract

The cyclic nucleotide-gated (CNG) channel in retinal rods converts the light-regulated intracellular cGMP concentration to various levels of membrane potential. Blockade of the channel by cations such as Ca2+ and Mg2+ lowers its effective conductance. Consequently, the membrane potential has very low noise, which enables rods to detect light with extremely high sensitivity. Here, we report that three polyamines (putrescine, spermidine, and spermine), which exist in both the intracellular and extracellular media, also effectively block the CNG channel from both sides of the membrane. Among them, spermine has the greatest potency. Extracellular spermine blocks the channel as a permeant blocker, whereas intracellular spermine appears to block the channel in two conformations-one permeant, and the other non- (or much less) permeant. The membrane potential in rods is typically depolarized to approximately -40 mV in the dark. At this voltage, K1/2 of the CNG channel for extracellular spermine is 3 microM, which is 100-1,000-fold higher affinity than that of the NMDA receptor-channel for extracellular spermine. Blockade of the CNG channel by polyamines may play an important role in suppressing noise in the signal transduction system in rods.

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Figures

Figure 1

Figure 1

Blockade of the CNG channel by extracellular spermine. (A) Macroscopic current–voltage relations (I–V curves) of the CNG channel in the absence and presence of 1 μM extracellular spermine. (B) The fraction of unblocked current (I/Io), taken from A, is plotted against membrane voltage.

Figure 2

Figure 2

Comparison of CNG channel blockade by three extracellular polyamines. (A–C) Macroscopic I–V curves of the CNG channel in the absence and presence of various concentrations of extracellular putrescine (PUT), spermidine (SPD), and spermine (SPM). (D–F) The corresponding fraction of unblocked currents is plotted against membrane voltage.

Figure 3

Figure 3

Comparative potency of the three extracellular polyamines. The fraction of unblocked currents at −40 mV is plotted against the concentrations of putrescine, spermidine, and spermine. The solid curves are least-squares fits of equations of the form I/Io = PA K 1/2/(PA K 1/2 + [PA]), where PA K 1/2 is the concentration at which a given polyamine blocks half the current through the CNG channel, and [PA] is the concentration of the polyamine. The K 1/2 values for extracellular putrescine, spermidine, and spermine are 1.4 mM, 50 μM, and 3.1 μM, respectively.

Figure 4

Figure 4

Blockade of the CNG channel by 10 μM intracellular spermine. (A) Macroscopic I–V curves of the CNG channel in the absence and presence of spermine. (B) The fraction of unblocked current, taken from A, is plotted against membrane voltage.

Figure 5

Figure 5

CNG channel blockade by three intracellular polyamines. (A–C) Macroscopic I–V curves of the CNG channel in the absence and the presence of various concentrations of intracellular putrescine, spermidine, and spermine. (D–F) The fraction of unblocked currents, taken from A–C, is plotted against membrane voltage.

Figure 6

Figure 6

Comparative potency of the three intracellular polyamines. The fraction of unblocked currents at +40 mV is plotted against the concentrations of putrescine, spermidine, and spermine. The solid curves are least-squares fits of equations of the forms I/Io = PA K 1/2/(PA K 1/2 + [PA]); see Fig. 3. The K 1/2 values for putrescine, spermidine, and spermine are 3.0 mM, 80 μM, and 6.7 μM, respectively.

Figure 7

Figure 7

A kinetic model for the action of spermine. (A) Reaction scheme. Ch represents the CNG channel, SPMi and SPMo denote intra- and extracellular spermine, and Ch.SPMP and Ch.SPMNP denote the CNG channels blocked by spermine in the permeant and nonpermeant conformations, respectively. Kx and Qx are, respectively, the equilibrium dissociation constant at 0 mV and the total number of equivalent charges moving across the electrical field (in either direction) for nonpermeant spermine binding. Rate constants at 0 mV (kx or k−x) and the number of equivalent charges traversing the electrical field (qx or q−x) for movements of the permeant conformation of spermine are also shown. (The number of equivalent charges is defined as the actual number of charges of the blocker multiplied by the fraction of the electrical field transversed by the blocker.) (B) Noisy trace a is the same as that in Fig. 4 B, obtained in the presence of 10 μM of intracellular spermine. Curve b was drawn according to the scheme in A, with k1 = 1 * 106 M−1 s−1, k−1 = 8 s−1, k2 = 1.3 * 106 M−1 s−1, k−2 = 10.4 s−1, K3 = 333 μM, q1 = 2, q−1 = 0, q2 = 2, q−2 = 0, Q 3 = 1.5. (Any set of four rate constants in the given proportion will yield the same result.) Intracellular and extracellular spermine concentrations were set at 10 μM and zero, respectively. Curves c and d were drawn using the same parameters, except that both k1 and k−1 were zero for curve c and that K3 was infinite for curve d.

Figure 8

Figure 8

Modeling of channel blockade by various concentrations of intracellular and extracellular spermine. The experimental data (noisy traces) are the same as those in Figs. 2 F and 5 F. All smooth traces were computed according to the scheme in Fig. 7 A, using the same parameters as in Fig. 7 B and appropriate concentrations of intracellular and extracellular spermine. For example, in Fig. 8 A the model's intracellular spermine concentration was set at 1 μM and extracellular spermine concentration at zero.

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