Lu M et al. (2002), Hydrolyzable ATP and PIP(2) modulate the small-...

XB-ART-6293

J Gen Physiol 2002 Nov 01;1205:603-15. doi: 10.1085/jgp.20028677.

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Hydrolyzable ATP and PIP(2) modulate the small-conductance K+ channel in apical membranes of rat cortical-collecting duct (CCD).

Lu M , Hebert SC , Giebisch G .

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The small-conductance K+ channel (SK) in the apical membrane of the cortical-collecting duct (CCD) is regulated by adenosine triphosphate (ATP) and phosphorylation-dephosphorylation processes. When expressed in Xenopus oocytes, ROMK, a cloned K+ channel similar to the native SK channel, can be stimulated by phosphatidylinositol bisphosphate (PIP2), which is produced by phosphoinositide kinases from phosphatidylinositol. However, the effects of PIP2 on SK channel activity are not known. In the present study, we investigated the mechanism by which hydrolyzable ATP prevented run-down of SK channel activity in excised apical patches of principal cells from rat CCD. Channel run-down was significantly delayed by pretreatment with hydrolyzable Mg-ATP, but ATP gamma S and AMP-PNP had no effect. Addition of alkaline phosphatase also resulted in loss of channel activity. After run-down, SK channel activity rapidly increased upon addition of PIP2. Exposure of inside-out patches to phosphoinositide kinase inhibitors (LY294002, quercetin or wortmannin) decreased channel activity by 74% in the presence of Mg-ATP. PIP2 added to excised patches reactivated SK channels in the presence of these phosphoinositide kinase inhibitors. The protein kinase A inhibitor, PKI, reduced channel activity by 36% in the presence of Mg-ATP. PIP2 was also shown to modulate the inhibitory effects of extracellular and cytosolic ATP. We conclude that both ATP-dependent formation of PIP2 through membrane-bound phosphoinositide kinases and phosphorylation of SK by PKA play important roles in modulating SK channel activity.

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Species referenced: Xenopus
Genes referenced: kcnj1 pnp runx2

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	Figure 1. . Small-conductance K+ channel run-down in a representative excised patch. In the cell-attached patch, inward K+ current was recorded showing five active SK channels. Since the pipette solution contained 140 mM KCl, the channel shows inward current displaying typical SK channel kinetics with high open probability. When the patch was excised into a 5 mM K+ bath solution, the current amplitude immediately decreased due to loss of the membrane potential. Then pipette holding potential (−Vpipette) was elevated from 0 to −40 mV, current amplitude was increased. SK channels completely closed in 1.7 min in the Ringer's solution after excision. The downward deflection of the traces shows inward current and the line “C” indicates the zero current level (channel completely closed state).
	Figure 2. . Depletion of intracellular ATP concentration decreased SK channel activity. In a representative cell-attached patch, the recording showed seven open channels before addition of 20 μM antimycin A and 20 mM 2-deoxy-D-glucose to the bath solution. In 7.4 min after application of the metabolic inhibitors, the SK channel activity was reversibly inhibited. NPo decreased from 6.4 ± 0.6 to 1.9 ± 0.3. Pipette holding potential (−Vpipette) = 0 mV. C = channel-closed state.
	Figure 3. . Hydrolyzable ATP prevented SK channel run-down in inside-out patches. (A) In the absence of Mg-ATP, channel activity rapidly decreased in the Ca2+/Mg2+-free Ringer's solution. 0.5 mM Mg-ATP reactivated the run-down channel. (B) SK channel activity recorded from an inside-out patch shows run-down in the presence of ATPγS (0.5 mM)-containing, Ca2+-free bath solution. The SK channel activity could be reactivated by 0.5 mM Mg-ATP in a Ca2+-free Ringer solution. The time to run-down was similar to those shown in Figs. 1 and 3 A. C = channel-closed state. −Vpipette = −40 mV.
	Figure 4. . A representative inside-out patch recording showing that alkaline phosphatase (cAP, 20 U/ml) reversibly inhibited SK channel activity in the presence of 0.5 mM Mg-ATP. After removal of cAP, SK channel activity was rapidly restored. C = channel-closed state. −Vpipette = −40 mV.
	Figure 5. . PIP2 reactivated run-down SK channels in a representative inside-out patch. (A) After run-down in the Ca2+-free, ATP-free bath solution, SK channel activity was restored by application of 50 μM PIP2. (B) PIP2 antibodies (anti-PIP2, 20 μM) caused SK channel run-down in the presence of Mg-ATP. (C) After removal of PIP2 antibodies, SK channel activity was not restored by Mg-ATP. However, SK channel activity was observed following addition of reducing agent, 100 μM DL-dithiothreitol (DTT). C = channel-closed state. −Vpipette = −40 mV.
	Figure 6. . (A) The PI kinase inhibitor, LY294002 (20 μM), reversibly reduced SK channel activity in the presence of 0.5 mM Mg-ATP/Ca2+-free bath solution in an inside-out patch. (B) PIP2 (50 μM) reactivated SK channel activity inhibited by the PI kinase inhibitor, LY294002 (20 μM). Channel current was recorded in an inside-out patch bathed in 0.5 mM Mg-ATP/Ca2+-free solution. C = channel-closed state. −Vpipette = −40 mV.
	Figure 7. . Summary of SK channel activity inhibited by the PI kinase inhibitors, LY294002 (20 μM), quercetin (60 μM), and wortmannin (20 μM). Channel activity (NPo) was normalized to the control value. Ca2+-free bath solution contained 0.5 mM Mg-ATP. PIP2 concentrations was 50 μM. ** indicates P < 0.01 versus control.
	Figure 8. . (A) PKI reduced SK channel activity in the presence of 0.5 mM Mg-ATP by 36% in 10 min. Data were from inside-out patch configuration. * indicates P < 0.05 compared with control (without PKI). (B) A representative trace showing that alkaline phosphatase (cAP) + PKI reduced SK channel activity in the presence of 0.5 mM Mg-ATP. After cAP wash-out, ∼65% of channel activity was restored in the presence of PKI + 0.5 mM Mg-ATP. C = channel-closed state. −Vpipette = −40 mV.
	Figure 9. . PIP2 and PKA are required for restoration of SK channel activity after purinergic receptors mediated inhibition by extracellular Mg-ATP. Cell-attached apical membrane patches were obtained, extracellular Mg-ATP (0.2 mM) was added to the bath, and then patches were excised in the inside-out configuration. Representative SK channel traces are shown demonstrating inhibition of SK activity by extracellular Mg-ATP in the cell-attached configuration. In the inside-out configuration, no channel activity was observed in the absence of Mg-ATP or PIP2 (A). However, with addition of 0.5 mM Mg-ATP followed by 50 U of the catalytic subunit of PKA (B) SK channels were nearly completely reactivated. C = channel-closed state. −Vpipette = pipette-holding potential.
	Figure 10. . PIP2 reduces Mg-ATP sensitivity of SK channels. (A) Representative SK channel activity recording of the concentration dependence of Mg-ATP inhibition in the presence of 50 μM PIP2 in the bath solution. 1 mM Mg-ATP had little effect on channel activity in the presence of PIP2 (B, open circle), whereas this Mg-ATP concentration abolished channel activity in the absence of PIP2 (B, solid circle). C = channel-closed state. −Vpipette = −40 mV. (B) Mg-ATP concentration-dependence of SK channel activity in the presence of 50 μM PIP2 (open circles). EC50 = 1.8 mM; Curve was fitted to the equation: Y = 1/{1 + 10^[(logEC50 − X)*Hill]}. Hill = 1.2 ± 0.1.

References [+] :

Anderson, Phosphatidylinositol phosphate kinases, a multifaceted family of signaling enzymes. 1999, Pubmed