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Long polyamines act as cofactors in PIP2 activation of inward rectifier potassium (Kir2.1) channels.

Xie LH, John SA, Ribalet B, Weiss JN - J. Gen. Physiol. (2005)

Bottom Line: Using neomycin as a measure of PIP2 affinity, we found that long polyamines were capable of strengthening either the wild type or K188Q channels' interaction with PIP2.Sustained pore block by polyamines was neither sufficient nor necessary for this effect.We conclude that long polyamines serve a dual role as both blockers and coactivators (with PIP2) of Kir2.1 channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, CA 90095, USA.

ABSTRACT
Phosphatidylinosital-4,5-bisphosphate (PIP2) acts as an essential factor regulating the activity of all Kir channels. In most Kir members, the dependence on PIP2 is modulated by other factors, such as protein kinases (in Kir1), G(betagamma) (in Kir3), and the sulfonylurea receptor (in Kir6). So far, however, no regulator has been identified in Kir2 channels. Here we show that polyamines, which cause inward rectification by selectively blocking outward current, also regulate the interaction of PIP2 with Kir2.1 channels to maintain channel availability. Using spermine and diamines as polyamine analogs, we demonstrate that both spontaneous and PIP2 antibody-induced rundown of Kir2.1 channels in excised inside-out patches was markedly slowed by long polyamines; in contrast, polyamines with shorter chain length were ineffective. In K188Q mutant channels, which have a low PIP2 affinity, application PIP2 (10 microM) was unable to activate channel activity in the absence of polyamines, but markedly activated channels in the presence of long diamines. Using neomycin as a measure of PIP2 affinity, we found that long polyamines were capable of strengthening either the wild type or K188Q channels' interaction with PIP2. The negatively charged D172 residue inside the transmembrane pore region was critical for the shift of channel-PIP2 binding affinity by long polyamines. Sustained pore block by polyamines was neither sufficient nor necessary for this effect. We conclude that long polyamines serve a dual role as both blockers and coactivators (with PIP2) of Kir2.1 channels.

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Stabilization of Kir2.1 channel activity by PIP2. Representative current traces elicited by the ramp pulses between −100 mV and +100 mV from a holding potential of 0 mV are shown. (A) After patch excision into Mg- and polyamine-free bath solution, inward rectification disappeared and current transiently increased before running down. Application of phosphatidylinositol-4,5-bisphosphate (PIP2, 10 μM) after rundown recovered channel activity. (B) Current–voltage (I-V) relationships obtained at times a–d in A. (C) Application of PIP2 (10 μM) immediately after patch excision prevented rundown. (D) MgATP (2 mM), but not K2ATP (2 mM), reactivated current after rundown. (E) After rundown, the PIP2 synthesis inhibitor wortmanin (WMN, 100 μM) prevented reactivation by MgATP (2 mM). The triangle to the right of each trace indicates zero current level. The upward arrow under each trace indicates the patch excision from cell-attached to inside-out configuration. Applications of reagents are indicated by the horizontal bars. The same markings are applicable to subsequent figures.
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fig1: Stabilization of Kir2.1 channel activity by PIP2. Representative current traces elicited by the ramp pulses between −100 mV and +100 mV from a holding potential of 0 mV are shown. (A) After patch excision into Mg- and polyamine-free bath solution, inward rectification disappeared and current transiently increased before running down. Application of phosphatidylinositol-4,5-bisphosphate (PIP2, 10 μM) after rundown recovered channel activity. (B) Current–voltage (I-V) relationships obtained at times a–d in A. (C) Application of PIP2 (10 μM) immediately after patch excision prevented rundown. (D) MgATP (2 mM), but not K2ATP (2 mM), reactivated current after rundown. (E) After rundown, the PIP2 synthesis inhibitor wortmanin (WMN, 100 μM) prevented reactivation by MgATP (2 mM). The triangle to the right of each trace indicates zero current level. The upward arrow under each trace indicates the patch excision from cell-attached to inside-out configuration. Applications of reagents are indicated by the horizontal bars. The same markings are applicable to subsequent figures.

Mentions: Kir2.1 channels were expressed in Xenopus oocytes and macroscopic currents recorded in giant inside-out membrane patches. Upon excising and perfusing the patch in a Mg2+- and polyamine-free solution, the outward current increased gradually, as endogenous Mg2+ and polyamines were washed out (Fig. 1, A, D, and E). Since outward current through Kir2.1 channels is very sensitive to contamination by residual polyamines and other cations, we used the inward current at −100 mV to index channel activity. In most patches, channel activity started to rundown after the outward current reached a maximal level, until eventually all channels closed. The average half-time for spontaneous rundown was 4.8 ± 0.4 min (n = 27). Channel activity could be recovered by applying exogenous PIP2 (10 μM), without further rundown thereafter (Fig. 1, A and B). Early application of PIP2 prevented rundown completely (Fig. 1 C). (The inhibition of the outward current during PIP2 perfusion might be caused by ammonium ions present in the PIP2 formulation.) Fig. 1 D shows that after spontaneous rundown, channels could also be reactivated by 2 mM MgATP, but not with K2ATP or AMP-PNP (not depicted). This reactivating effect was blocked by wortmannin (100 μM), an inhibitor of phosphatidylinositol kinases (Fig. 1 E), which prevents resynthesis of PIP2 by MgATP-sensitive PI kinases. These results are reminiscent of Kir6.2 channels (Xie et al., 1999a,b) and indicate that membrane PIP2 levels are also the main determinant of the Kir2.1 channel activity.


Long polyamines act as cofactors in PIP2 activation of inward rectifier potassium (Kir2.1) channels.

Xie LH, John SA, Ribalet B, Weiss JN - J. Gen. Physiol. (2005)

Stabilization of Kir2.1 channel activity by PIP2. Representative current traces elicited by the ramp pulses between −100 mV and +100 mV from a holding potential of 0 mV are shown. (A) After patch excision into Mg- and polyamine-free bath solution, inward rectification disappeared and current transiently increased before running down. Application of phosphatidylinositol-4,5-bisphosphate (PIP2, 10 μM) after rundown recovered channel activity. (B) Current–voltage (I-V) relationships obtained at times a–d in A. (C) Application of PIP2 (10 μM) immediately after patch excision prevented rundown. (D) MgATP (2 mM), but not K2ATP (2 mM), reactivated current after rundown. (E) After rundown, the PIP2 synthesis inhibitor wortmanin (WMN, 100 μM) prevented reactivation by MgATP (2 mM). The triangle to the right of each trace indicates zero current level. The upward arrow under each trace indicates the patch excision from cell-attached to inside-out configuration. Applications of reagents are indicated by the horizontal bars. The same markings are applicable to subsequent figures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266595&req=5

fig1: Stabilization of Kir2.1 channel activity by PIP2. Representative current traces elicited by the ramp pulses between −100 mV and +100 mV from a holding potential of 0 mV are shown. (A) After patch excision into Mg- and polyamine-free bath solution, inward rectification disappeared and current transiently increased before running down. Application of phosphatidylinositol-4,5-bisphosphate (PIP2, 10 μM) after rundown recovered channel activity. (B) Current–voltage (I-V) relationships obtained at times a–d in A. (C) Application of PIP2 (10 μM) immediately after patch excision prevented rundown. (D) MgATP (2 mM), but not K2ATP (2 mM), reactivated current after rundown. (E) After rundown, the PIP2 synthesis inhibitor wortmanin (WMN, 100 μM) prevented reactivation by MgATP (2 mM). The triangle to the right of each trace indicates zero current level. The upward arrow under each trace indicates the patch excision from cell-attached to inside-out configuration. Applications of reagents are indicated by the horizontal bars. The same markings are applicable to subsequent figures.
Mentions: Kir2.1 channels were expressed in Xenopus oocytes and macroscopic currents recorded in giant inside-out membrane patches. Upon excising and perfusing the patch in a Mg2+- and polyamine-free solution, the outward current increased gradually, as endogenous Mg2+ and polyamines were washed out (Fig. 1, A, D, and E). Since outward current through Kir2.1 channels is very sensitive to contamination by residual polyamines and other cations, we used the inward current at −100 mV to index channel activity. In most patches, channel activity started to rundown after the outward current reached a maximal level, until eventually all channels closed. The average half-time for spontaneous rundown was 4.8 ± 0.4 min (n = 27). Channel activity could be recovered by applying exogenous PIP2 (10 μM), without further rundown thereafter (Fig. 1, A and B). Early application of PIP2 prevented rundown completely (Fig. 1 C). (The inhibition of the outward current during PIP2 perfusion might be caused by ammonium ions present in the PIP2 formulation.) Fig. 1 D shows that after spontaneous rundown, channels could also be reactivated by 2 mM MgATP, but not with K2ATP or AMP-PNP (not depicted). This reactivating effect was blocked by wortmannin (100 μM), an inhibitor of phosphatidylinositol kinases (Fig. 1 E), which prevents resynthesis of PIP2 by MgATP-sensitive PI kinases. These results are reminiscent of Kir6.2 channels (Xie et al., 1999a,b) and indicate that membrane PIP2 levels are also the main determinant of the Kir2.1 channel activity.

Bottom Line: Using neomycin as a measure of PIP2 affinity, we found that long polyamines were capable of strengthening either the wild type or K188Q channels' interaction with PIP2.Sustained pore block by polyamines was neither sufficient nor necessary for this effect.We conclude that long polyamines serve a dual role as both blockers and coactivators (with PIP2) of Kir2.1 channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, CA 90095, USA.

ABSTRACT
Phosphatidylinosital-4,5-bisphosphate (PIP2) acts as an essential factor regulating the activity of all Kir channels. In most Kir members, the dependence on PIP2 is modulated by other factors, such as protein kinases (in Kir1), G(betagamma) (in Kir3), and the sulfonylurea receptor (in Kir6). So far, however, no regulator has been identified in Kir2 channels. Here we show that polyamines, which cause inward rectification by selectively blocking outward current, also regulate the interaction of PIP2 with Kir2.1 channels to maintain channel availability. Using spermine and diamines as polyamine analogs, we demonstrate that both spontaneous and PIP2 antibody-induced rundown of Kir2.1 channels in excised inside-out patches was markedly slowed by long polyamines; in contrast, polyamines with shorter chain length were ineffective. In K188Q mutant channels, which have a low PIP2 affinity, application PIP2 (10 microM) was unable to activate channel activity in the absence of polyamines, but markedly activated channels in the presence of long diamines. Using neomycin as a measure of PIP2 affinity, we found that long polyamines were capable of strengthening either the wild type or K188Q channels' interaction with PIP2. The negatively charged D172 residue inside the transmembrane pore region was critical for the shift of channel-PIP2 binding affinity by long polyamines. Sustained pore block by polyamines was neither sufficient nor necessary for this effect. We conclude that long polyamines serve a dual role as both blockers and coactivators (with PIP2) of Kir2.1 channels.

Show MeSH
Related in: MedlinePlus