Guo D and Lu Z (2003), Interaction mechanisms between polyamines and I...

XB-ART-4500

J Gen Physiol 2003 Nov 01;1225:485-500. doi: 10.1085/jgp.200308890.

Show Gene links Show Anatomy links

Interaction mechanisms between polyamines and IRK1 inward rectifier K+ channels.

Guo D , Lu Z .

???displayArticle.abstract???
Rectification of macroscopic current through inward-rectifier K+ (Kir) channels reflects strong voltage dependence of channel block by intracellular cations such as polyamines. The voltage dependence results primarily from the movement of K+ ions across the transmembrane electric field, which accompanies the binding-unbinding of a blocker. Residues D172, E224, and E299 in IRK1 are critical for high-affinity binding of blockers. D172 appears to be located somewhat internal to the narrow K+ selectivity filter, whereas E224 and E299 form a ring at a more intracellular site. Using a series of alkyl-bis-amines of varying length as calibration, we investigated how the acidic residues in IRK1 interact with amine groups in the natural polyamines (putrescine, spermidine, and spermine) that cause rectification in cells. To block the pore, the leading amine of bis-amines of increasing length penetrates ever deeper into the pore toward D172, while the trailing amine in every bis-amine binds near a more intracellular site and interacts with E224 and E299. The leading amine in nonamethylene-bis-amine (bis-C9) makes the closest approach to D172, displacing the maximal number of K+ ions and exhibiting the strongest voltage dependence. Cells do not synthesize bis-amines longer than putrescine (bis-C4) but generate the polyamines spermidine and spermine by attaching an amino-propyl group to one or both ends of putrescine. Voltage dependence of channel block by the tetra-amine spermine is comparable to that of block by the bis-amines bis-C9 (shorter) or bis-C12 (equally long), but spermine binds to IRK1 with much higher affinity than either bis-amine does. Thus, counterintuitively, the multiple amines in spermine primarily confer the high affinity but not the strong voltage dependence of channel block. Tetravalent spermine achieves a stronger interaction with the pore by effectively behaving like a pair of tethered divalent cations, two amine groups in its leading half interacting primarily with D172, whereas the other two in the trailing half interact primarily with E224 and E299. Thus, nature has optimized not only the blocker but also, in a complementary manner, the channel for producing rapid, high-affinity, and strongly voltage-dependent channel block, giving rise to exceedingly sharp rectification.

???displayArticle.pubmedLink??? 14581581
???displayArticle.pmcLink??? PMC2229578
???displayArticle.link??? J Gen Physiol
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: bag3 kcnj12 kcnj2

???attribute.lit??? ???displayArticles.show???

	Figure 1. . Effects of single mutations within M2 or the COOH terminus on current inhibition by polyamines. Shown are currents of wild-type and mutant channels containing a single mutation, D172N, E224G, or E299S, recorded from various patches in the absence (control) or presence of PUT, SPD, or SPM at concentrations indicated, and with the voltage protocol shown at the top. Dashed lines identify zero current levels.
	Figure 2. . Polyamine inhibition of currents of IRK1 with a double mutation in the COOH terminus. Shown are currents of mutant channels containing the double mutation E224G+E299S recorded from various patches in the absence (control) or presence of 0.3 mM PUT, SPD, or SPM. Dashed lines identify zero current levels.
	Figure 3. . Effects of single mutations within M2 or the COOH terminus on the voltage-dependent block of IRK1 current by polyamines. The fraction of wild-type and mutant channel currents (mean Â± SEM; n = 4â6) not blocked by PUT, SPD, or SPM (at the concentrations indicated) is plotted against membrane voltage. The curves fitted to the data are described in results.
	Figure 4. . Effects of a double mutation (E224G+E299S) in the COOH terminus on the voltage-dependent block of IRK1 current by polyamines. The fraction of mutant channel currents (mean Â± SEM; n = 4â6) not blocked by PUT, SPD, or SPM (at the concentrations indicated) is plotted against membrane voltage. The curves fitted to the data are described in results.
	Figure 5. . Summary of dissociation constants and valences for channel block by polyamines. Shown are values of Kd(0 mV) (A) and Z (B) (mean Â± SEM; n = 4â6; determined from the fits as shown in Figs. 3 and 4) for block of wild-type and various mutant channels by PUT, SPD, or SPM.
	Figure 6. . Kinetics of voltage jumpâinduced current relaxations in the presence of spermine. Current traces of wild-type and three mutant channels, each at three appropriate concentrations of SPM, elicited by stepping membrane voltage from 0 to 70 mV. The curves superimposed on the data are single-exponential fits. Dashed lines identify zero current levels.
	Figure 7. . Polyamine concentration dependence of the time constant of current transients induced by voltage steps. Reciprocals of the time constants of current transients (mean Â± SEM; n = 4â6; obtained from fits such as those shown in Fig. 6) are plotted against the concentration of SPD or SPM for wild-type and several mutant channels. The lines through the data are linear fits. The individual slopes reflect, in ascending order, the kinetics of current transients elicited by stepping membrane voltage from 0 mV to 70, 80, 90, or 100 mV.
	Figure 8. . Dependence of the rate constant of channel block on membrane voltage. Natural logarithm of the rate constant for formation of the (first) blocked state (kon; mean Â± SEM; n = 4â6; determined from the slopes of fits in Fig. 7) is plotted against membrane voltage for wild-type and several mutant channels. The lines through the data represent Boltzmann functions.
	Figure 9. . Summary of rate constants and valences of channel block by spermidine and spermine. A and B show, respectively, values of kon(0 mV) and zon (mean Â± SEM; n = 4â6; determined from fits such as those shown in Fig. 8) for block of wild-type and several mutant channels.
	Figure 10. . Effects of mutations on current inhibition by octamethylene-bis-amine. Shown are currents of wild-type and mutant channels containing a single mutation D172N, E224G, or E299S recorded in the absence (control) or presence of bis-C8 at the concentrations indicated. Dashed lines identify zero current levels.
	Figure 11. . Effects of mutations on voltage-dependent block of IRK1 current by octomethylene-bis-amine. The fraction of wild-type and mutant channel currents (mean Â± SEM; n = 4â€“6) not blocked by bis-C8 (at the concentrations indicated) is plotted against membrane voltage. The curves superimposed on the data are fits as described for the case of PUT in results.
	Figure 12. . Summary of dissociation constants and valences for steady-state channel block by alkyl-bis-amines. Shown in A and B are values of Kd(0 mV) and Z, respectively (mean Â± SEM; n = 4â€“6; determined from fits as shown in Fig. 11), for block of wild-type and three single mutant channels by a series of bis-amines of varying chain length. Bis-C11 is not available commercially.
	Figure 13. . Kinetics of voltage jumpâ€“induced current relaxations in the presence of octamethylene-bis-amine. Current traces of wild-type and three mutant channels at three concentrations of bis-C8 elicited by stepping membrane voltage from 0 to 70 mV. The curves superimposed on the data are single-exponential fits. Dashed lines identify zero current levels.
	Figure 14. . Octamethylene-bis-amine concentration dependence of the time constant of current transients induced by voltage steps. Reciprocals of the time constants of current transients (mean Â± SEM; n = 4â€“6; obtained from fits such as those shown in Fig. 13) are plotted against the concentration of bis-C8 for wild-type and three mutant channels. The lines through the data are linear fits. The individual slopes reflect, in ascending order, the kinetics of current transients elicited by stepping membrane voltage from 0 to 70, 80, 90, or 100 mV.
	Figure 15. . Dependence of the rate constant of channel block by octamethylene-bis-amine on membrane voltage. Natural logarithm of the rate constant for formation of the (first) blocked state (kon; mean Â± SEM; n = 4â€“6; determined from the slopes of fits in Fig. 14) is plotted against membrane voltage for wild-type and three mutant channels. The lines through the data represent Boltzmann functions.
	Figure 16. . Summary of rate constants and valences of channel block by alkyl-bis-amines. Values of kon(0 mV) and zon (mean Â± SEM; n = 4â€“6; determined from fits such as those shown for bis-C8 in Fig. 15) for block of wild-type and three mutant channels are plotted in A and B, respectively.
	Figure 17. . Thermodynamic cycle analyses. The four corners are Kds (0 mV) of the wild-type and a mutant channel for a polyamine (PM) and for a bis-amine (bis-Cn). Î© = (wtKdPM Ã— mtKdbis-Cn)/(mtKdPM Ã— wtKdbis-Cn), where Kds (all at 0 mV) are taken from Figs. 5 A and 12 A. The results are plotted as â€œRT ln Î©â€. Shown in A, B, or C are â€œRT ln Î©â€ values computed for PUT, SPD, or SPM, with respect to a series of bis-amines of varying length. Each panel shows values computed for each of the three mutant channels (D172N, E224G, and E229S).
	Figure 18. . Blocker structures, models for blocker-bound channels, and blocking reaction scheme. (A) Chemical structures of bis-Cn, PUT (bis-C4), SPD, and SPM. (B) Models for channel block by bis- and polyamines. K+ ions present in the blocked inner pores are not drawn. (C) A kinetic model of channel block with one open (O) and n blocked states (B1â€¦Bn).

References [+] :

ARMSTRONG, ANOMALOUS RECTIFICATION IN THE SQUID GIANT AXON INJECTED WITH TETRAETHYLAMMONIUM CHLORIDE. 1965, Pubmed