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KCNE1 Enhances Phosphatidylinositol 4,5-Bisphosphate (PIP2) Sensitivity of Iks to Modulate Channel Activity

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KCNE1 Enhances Phosphatidylinositol 4,5-Bisphosphate (PIP2) Sensitivity of Iks to Modulate Channel Activity KCNE1 enhances phosphatidylinositol 4,5-bisphosphate (PIP2) sensitivity of IKs to modulate channel activity Yang Li, Mark A. Zaydman, Dick Wu1, Jingyi Shi, Michael Guan, Brett Virgin-Downey, and Jianmin Cui2 Department of Biomedical Engineering, Center for the Investigation of Membrane Excitability Disorders, Cardiac Bioelectricity and Arrhythmia Center, Washington University, St. Louis, MO 63130 Edited by Richard W. Aldrich, University of Texas at Austin, Austin, TX, and approved April 18, 2011 (received for review January 17, 2011) Phosphatidylinositol 4,5-bisphosphate (PIP2) is necessary for the function of various ion channels. The potassium channel, IKs,is important for cardiac repolarization and requires PIP2 to activate. Here we show that the auxiliary subunit of IKs, KCNE1, increases PIP2 sensitivity 100-fold over channels formed by the pore-forming KCNQ1 subunits alone, which effectively amplifies current because native PIP2 levels in the membrane are insufficient to activate all KCNQ1 channels. A juxtamembranous site in the KCNE1 C terminus is a key structural determinant of PIP2 sensitivity. Long QT syn- drome associated mutations of this site lower PIP2 affinity, result- ing in reduced current. Application of exogenous PIP2 to these mutants restores wild-type channel activity. These results reveal a vital role of PIP2 for KCNE1 modulation of IKs channels that may represent a common mechanism of auxiliary subunit modula- tion of many ion channels. BIOPHYSICS AND CNQ1 α-subunits coassemble with KCNE1 β-subunits to COMPUTATIONAL BIOLOGY Kform the cardiac slow-delayed rectifier channel, IKs, which conducts a potassium current that is important for the termina- tion of the cardiac action potential. Although exogenous expres- sion of KCNQ1 alone is sufficient to produce a voltage-gated channel, coexpression of KCNE1 with KCNQ1 leads to changes in current properties to resemble the physiologically important A Fig. 1. KCNE1 slows the PIP2-dependent channel rundown. (A) KCNQ1 and cardiac IKs current (Fig. 1 ) (1, 2). These changes include in- KCNQ1 þ KCNE1 currents at various times after patch excision. Voltage was creased current amplitude, shift of the voltage dependence of ac- stepped from a holding potential of −80 to þ80 mV and then back to the tivation toward more positive potentials, slowed activation and holding potential. (B) Time dependence of current amplitude after patch deactivation kinetics, and suppressed inactivation, all of which excision. Normalized tail current amplitude at various time, It ∕I0, is plotted; are essential for the physiological role of IKs (3). After many years I0 is the tail current amplitude immediately following patch excision. KCNQ1 of investigation, it is still poorly understood how the KCNE1 (filled circles), n ¼ 9; KCNQ1 þ KCNE1 (open circles), n ¼ 12; KCNQ1þ KCNE1 þ 10 μMPIP2 (open squares), n ¼ 6; KCNQ1 þ KCNE1 coexpressed peptide exerts such a dramatic effect on the IKs current. Loss-of- with Ci-VSP (open diamonds), n ¼ 6. Solid lines are monoexponential fits function mutations in IKs lead to delayed cardiac cell repolariza- to data. Voltage was stepped from a holding potential of −80 mV to tion that manifests clinically as long QT (LQT) syndrome. þ80 mV for 5 s and then back to the holding potential for 5 s. (C) Time con- Patients with LQT syndrome are predisposed to ventricular stants of exponential fits and initial delay to current rundown. (D) Normal- arrhythmia and suffer from syncope and a high risk of sudden ized tail current amplitude at various times, It ∕I0, with KCNQ1: KCNE1 mRNA cardiac death. At present, the molecular mechanisms of disease ratio 1∶1 (open circles), n ¼ 12; 1∶0.05 (open diamonds), n ¼ 6; 1∶0.01 (open pathogenesis are still unknown for many LQT-associated muta- squares), n ¼ 6; 1∶0 (filled circles), n ¼ 9.(E) Time constants of exponential fits and initial delay to current rundown. In this and other figures, the data tions in IKs. are presented as mean Æ SEM. Phosphatidylinositol 4,5-bisphosphate (PIP2) is a minor acidic membrane lipid found primarily in the inner leaflet of the plasma currents using patch-clamp and two-electrode voltage-clamp membrane. PIP2 has been shown to be a necessary cofactor for a wide variety of ion channels, e.g., voltage-gated Kþ and Ca2þ techniques. Currents recorded from inside-out membrane channels, transient receptor potential channels, inward rectifying patches expressing KCNQ1 immediately and rapidly decayed fol- þ þ K channels, and epithelial Na channels (4). Recently, IKs has lowing patch excision such that channel activity was completely been shown to require PIP2 for channel activity, and several LQT- associated mutations located in KCNQ1 have been suggested to decrease the PIP2 affinity of IKs (5, 6). Here we provide evidence Author contributions: Y.L., M.A.Z., D.W., and J.C. designed research; Y.L., M.A.Z., J.S., M.G., and B.V.-D. performed research; Y.L. performed patch–clamp experiments in β Ks that KCNE1, as a -subunit, alters the function of I by modu- Fig. 1–5; M.A.Z. and M.G. performed voltage–clamp experiments in Fig. 4. J.S., M.G., lating the interaction between PIP2 and the heteromeric ion and B.V.-D. performed molecular biology; Y.L. analyzed data; and Y.L., M.A.Z., D.W., channel complex. Knowledge of the molecular mechanisms of and J.C. wrote the paper. channel modulation by KCNE1 is of great importance as at least The authors declare no conflict of interest. 36 LQT-associated mutations reside within KCNE1 (7–9). This article is a PNAS Direct Submission. 1Present address: Stanford Institute for Neuro-innovation and Translational Neurosciences, Results Stanford, CA 94305. KCNE1 Slows the PIP2-Dependent Channel Rundown. To investigate 2To whom correspondence should be addressed. E-mail: [email protected]. the regulation of IKs by PIP2, we expressed KCNQ1 with and This article contains supporting information online at www.pnas.org/lookup/suppl/ without KCNE1 in oocytes from Xenopus laevis and recorded doi:10.1073/pnas.1100872108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1100872108 PNAS Early Edition ∣ 1of6 Downloaded by guest on October 1, 2021 lost within 5 min (Fig. 1A). This rundown of current was fit well plete current rundown, which restored channel activity in a dose- with a single exponential (Fig. 1B). However, after excising dependent manner (Fig. 2A). The effective PIP2 concentrations inside-out membrane patches expressing KCNQ1 þ KCNE1, of half maximal activation (EC50) for KCNQ1 and KCNQ1þ currents remained stable over 3 min prior to rundown, which KCNE1 are >600 μM and 4.6 μM, respectively (Fig. 2 B and C). was not observed with KCNQ1 alone (Fig. 1B). The subsequent These results show that KCNE1 greatly enhances the PIP2 sen- rundown of KCNQ1 þ KCNE1 current was slower than that sitivity of the channel resulting in a longer time course of run- observed with KCNQ1 alone (Fig. 1 B and C). This rundown down as the channels can still function at lower PIP2 levels. To of IKs activity is attributed to the loss of PIP2 from the excised further confirm the correlation between time course of PIP2- membrane patch (6). The PIP2 dependence of current rundown dependent rundown and PIP2 sensitivity, we studied two LQT after patch excision was supported by the following experi- mutations of KCNQ1, R539W, and R555C, coexpressed with ments. The voltage sensitive phosphatase from Ciona intestinalis KCNE1, both of which decrease PIP2 sensitivity (Fig. 2 B and C) (CiVSP) that dephosphorylates PIP2 upon membrane depolari- (5). Consistently, these mutants shortened the time course of run- zation (10) was coexpressed with KCNQ1 þ KCNE1. CiVSP down by eliminating the delay and decreasing the time constant hastens PIP2 loss from the membrane patch, resulting in a faster of rundown relative to WT KCNQ1 þ KCNE1 (Figs. 1C and 2B). rate of current rundown. Conversely, directly applying 10 μMof It is important to note that application of high levels of PIP2 exogenous PIP2 to the intracellular face of membrane patches could increase the current of KCNQ1 beyond the level measured expressing KCNQ1 þ KCNE1 prevented current rundown for immediately following patch excision (Fig. 2 A and B). This result longer than 10 min (Fig. 1 B and C). These results show that suggests that the native PIP2 level in the patch membrane is not PIP2 loss after patch excision leads to loss of channel activity. sufficient to saturate the PIP2 binding and activation of all KCNQ1 activity is lost more quickly after patch excision than KCNQ1 channels; therefore, supernormal levels of exogenous KCNQ1 þ KCNE1, indicating that KCNE1 slows PIP2-depen- PIP2 can activate channels that are PIP2 unbound in the native dent rundown. To further demonstrate this effect of KCNE1, membrane patch. In contrast, high doses of PIP2 could not in- we injected oocytes with the same amount of KCNQ1 mRNA crease the current of KCNQ1 þ KCNE1 beyond the amplitude and various amounts of KCNE1 mRNAs in molar ratios of 1∶0, immediately following patch excision (Fig. 2 A and B), indicating 1∶0.01, 1∶0.05, and 1∶1. The activation kinetics and the steady- that KCNE1 association enhances PIP2 affinity, such that channel state voltage dependence of activation of the expressed channels activation by PIP2 is saturated immediately following patch exci- depend on the KCNQ1∶KCNE1 ratio, which suggested that pore- sion. This conclusion is supported by the delay before PIP2- forming KCNQ1 subunits are associated with varying numbers of dependent current rundown that is present only when KCNE1 KCNE1 subunits (11–13) (Fig. S1). The time course of current is coexpressed with KCNQ1 (Fig. 1 B–E). This delay likely repre- rundown became progressively longer with higher concentrations sents a period when the PIP2 level in the patch membrane is of KCNE1 due to a longer delay and a larger time constant of supersaturating for activating KCNQ1 þ KCNE1.
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