(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 syndrome associated mutations of this site lower PIP2 affinity, resulting 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 modulation 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 termination of the cardiac action potential. Although exogenous expression 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 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 September 29, 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. Rundown oc- rundown (Fig. 1D). A lower molar ratio of KCNQ1∶KCNE1 curs when the PIP2 level falls below a threshold of saturation and mRNA results in a larger portion of the expressed channel channels become nonfunctional due to unbinding of PIP2. These population containing a higher stoichiometry of KCNE1∶ results suggest that KCNE1 coexpression increases the current KCNQ1, and these results show that channels with more KCNE1 amplitude by recruiting KCNQ1 channels that would be nonfunc- subunits have slower PIP2-dependent rundown (Fig. 1 D and E). tional due to a lack of PIP2 binding. Why does the association of KCNE1 affect the time course of PIP2-dependent rundown? To answer this question, we applied Key Structural Determinants of PIP2 Sensitivity in KCNE1 Proteins PIP2 to the intracellular face of membrane patches after com- associate with PIP2 through electrostatic interactions between basic residues and the negatively charged head group of PIP2 (4, 14). Application of diC4-PIP2 and diC8-PIP2, which have different fatty acid tail groups as the PIP2 purified from cell membrane (see Materials and Methods) has the same effect on KCNQ1 þ KCNE1 currents (Fig. 2B), supporting that the PIP2 head group is important in interactions with the channel protein. In order to identify the key structural determinants in KCNE1 contributing to enhanced PIP2 sensitivity, we made mutations that individually neutralized each of the 11 basic residues located in the cytosolic C terminus of KCNE1 and measured the time course of rundown after patch excision for each mutant coexpressed with KCNQ1. Of the 11 neutralizing mutations, 4 (R67Q, K69C, K70C, and H73N) abolished the delay and significantly reduced the time constant of rundown, indicating that neutraliza- tion of these basic residues leads to a decreased PIP2 sensitivity (Fig. 3 A and B). In the KCNE1 solution NMR structure in micelles (15), these residues are positioned on a helical stretch C α Fig. 2. KCNE1 enhances PIP2 sensitivity. (A) Normalized tail current ampli- at the bottom of the transmembrane domain (Fig. 3 ). The - tude at various times following patch excision and PIP2 application. PIP2 helicity of the juxtamembranous portion of the KCNE1 C termi- was applied at all times after the arrow. Voltage protocol to elicit the currents nus is also supported by mutagenic perturbation analysis (16). is the same as in Fig. 1. (B)PIP2 dose response of WT KCNQ1 and WT and The basic residues at positions 67, 69, 70, and 73 lie on one face 1 þ 1 mutant KCNQ KCNE channels. Irec is tail current amplitude at steady state of the α-helix, which provide a dense cluster of positive charges after recovery of channel activity following PIP2 application. I0 is the tail cur- that would be attractive for the negatively charged head group of rent amplitude immediately following patch excision. KCNQ1 (filled circles), PIP2. Our mutational study cannot definitively identify these KCNQ1 þ KCNE1 (open circles), R539W þ KCNE1 (open diamonds), and residues as a PIP2 binding site because the mutations could affect R555C þ KCNE1 (open squares). Solid curves are fits to the Hill equation. Hill coefficients are 2.0, 1.5, 1.6, and 1.0 for WT KCNQ1 þ KCNE1,R555C þ KCNE1, PIP2 binding through an allosteric mechanism. However, the R539W þ KCNE1, and KCNQ1, respectively. For KCNQ1 þ KCNE1, the re- clustering of these basic residues juxtaposed with the inner leaflet sponses of currents to diC8-PIP2 (up triangles) and diC4-PIP2 (down triangles) of the plasma membrane suggests that they could directly interact (5 and 500 μM) are also plotted. (C)EC50 calculated by the Hill fit (n ¼ 6–9). with PIP2. Consistent with this idea, a triple charge-reversal mu-

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1100872108 Li et al. Downloaded by guest on September 29, 2021 Fig. 3. Molecular determinants of PIP2 sensitivity in KCNE1. (A) Normalized tail current amplitude at various times following patch excision. WT KCNQ1 (filled circles), n ¼ 9; WT KCNQ1 þ KCNE1 (⋈), n ¼ 9; KCNQ1 þ mutant KCNE1: R67Q (up triangles), n ¼ 6; K69C (open diamonds), n ¼ 6; K70C (open circles), n ¼ 6; H73N (open squares), n ¼ 6; R67E/K69E/K70E triple mutation (TM), (down triangles), n ¼ 6.(B) Time constants of exponential fits and initial delay to current rundown. The axis is labeled with the KCNE1 construct Fig. 4. LQT mutations in KCNE1 reduce PIP2 sensitivity. (A) Whole-cell that was coexpressed with KCNQ1. (-) indicates KCNQ1 alone. (C) The trans- currents and (B) conductance-voltage (G-V) relation in oocytes. WT KCNQ1 þ membrane segment and juxtamembraneous C-terminal region of the KCNE1 KCNE1 (open squares), n ¼ 6; KCNQ1 þ mutant KCNE1: R67C (filled circles), NMR structure (PDB ID code 2K21). The residues important for PIP2 sensitivity n ¼ 6; R67H (filled diamonds), n ¼ 6; K70N (filled triangles), n ¼ 6; K70M BIOPHYSICS AND are shown in stick presentation. (filled squares), n ¼ 6; Voltages were stepped from a holding potential of COMPUTATIONAL BIOLOGY −80 mV to a test potential (−80 to þ80 mV) and then repolarized to −40 mV. (C) Whole-cell current amplitude measured þ80 mV normalized tation, R67E/K69E/K70E, renders the time course of rundown by that of WT channels (filled bars) and V1∕2 measured from G-V relation even faster than that of KCNQ1 alone (Fig. 3 A and B), as if (open bars). (D)PIP2 dose response of KCNQ1 þ mutant KCNE1 channels: PIP2 were repulsed by the negative charges. K70N (filled circles), R67C (open circles). Solid curves are fits to the Hill equation. Hill coefficients are 3 and 4, and EC50 are 114 μM and 99 μMfor KCNQ1 þ K70N and KCNQ1 þ R67C, respectively. (E) Tail current amplitude PIP2 Restores the Loss of Function Due to LQT Mutations in KCNE1 in response to the same voltage protocol as described in Fig. 1 for WT Mutations of the putative PIP2 interaction site in KCNE1 and mutant channels. (filled bars) Indicate the tail current amplitude imme- (R67C, R67H, K70M, and K70N) have been previously identified diately following patch excision. (open bars) Indicate the tail current ampli- in LQT patients as disease-associated mutations (7–9). We find tude after steady-state recovery of channel activity in response to 300 uM that coexpressing the KCNE1 mutation with KCNQ1 decreases exogenous PIP2.(F) G-V relations of KCNQ1 þ K70N (filled symbols) and 1 þ 1 current amplitude and shifts the voltage dependence of activation WT KCNQ KCNE (unfilled) before and after patch excision in PIP2- 1 þ containing solutions. KCNQ1 þ K70N: on cell (filled circles), V1∕2 ¼ 48.5 toward more depolarized potentials compared to WT KCNQ 2 2 μ ¼ 50 7 0 8 1 A–C . mV; excised in 50 MPIP2 (filled diamonds), V1∕2 . . mV; excised KCNE channels (Fig. 4 ). Each of these changes in channel in 150 μMPIP2, (filled triangles), V1∕2 ¼ 29.9 3.2 mV; excised in 300 μM properties would decrease the contribution of IKs to termination PIP2 (filled squares), V1∕2 ¼ 8.9 2.6 mV. KCNQ þ KCNE1: on cell (open ¼ 21 6 3 2 μ of cardiac action potentials, resulting in prolongation of action circles), V1∕2 . . mV; excised in 300 M PIP2 (open squares), ¼ 8 5 1 2 n ¼ 2 1 þ 70 μ potential duration and creating a substrate for potentially fatal V1∕2 . . mV. for G-V relation of KCNQ K Nin50 M PIP2, n ¼ 6–12 for all other experiments. arrhythmias. The PIP2 dose-response curves of KCNQ1 þ R67C and KCNQ1 þ K70N (Fig. 4D) are significantly right-shifted in 1 þ 1 Discussion comparison to that of WT KCNQ KCNE , indicating that Ion channel β-subunits modulate pore-forming α-subunits, these LQT-associated mutations decrease PIP2 sensitivity of the increasing the diversity of ion channel properties that can be gen- channel complex. Furthermore, the current amplitude measured erated from a limited repertoire of genes encoding α- and β- by applying saturating levels of PIP2 exceeded the current ampli- subunits. To understand the function of ion channels in vivo re- tude immediately following patch excision, suggesting that a quires structure-function knowledge of not only the pore-forming reduction of PIP2 affinity decreases the number of PIP2-bound subunits, but also their heteromeric complexes with β-subunits. channels in the native membrane resulting in less current. The interactions between KCNQ1 and KCNE1 have been studied α β Remarkably, application of a saturating concentration of PIP2 intensely for many years as this pair of - and -subunits coassem- (300 μM) to the intracellular face of membrane patches expres- bles in the cardiac myocyte and generates the IKs current that is sing KCNQ1 þ mutant KCNE1 restored the wild-type channel important in the termination of cardiac action potentials. How- current characteristics. Specifically, the current amplitude in- ever, despite these efforts, the molecular mechanisms by which KCNE1 changes the KCNQ1 channel to generate the IKs current creased 2- to 5-fold (Fig. 4E), accounting for all of the reduction remain elusive. In this work we show that KCNE1 increases the in the whole cell current of the mutant channels (Fig. 4 A and C). PIP2 sensitivity of IKs by several orders of magnitude (Fig. 2). Additionally, the voltage dependence of channel activation Because of the low PIP2 sensitivity of KCNQ1, the PIP2 level shifted back toward less depolarized voltages to nearly superim- in biological membrane is too low to saturate binding of KCNQ1, pose with that of the WT KCNQ1 þ KCNE1 (Fig. 4F). These leaving a fraction of the channel population nonfunctional in the results show that a decrease in PIP2 sensitivity of the IKs channel absence of KCNE1. This unique mechanism of current amplifi- due to these KCNE1 mutations can lead to LQT syndrome. cation is independent of KCNE1 effects on single channel con-

Li et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 29, 2021 ductance for which previous reports have provided conflicting tical for the PKA-dependent modulation. A previous study sug- results (17–19). gested a cross-talk between PKA and PIP2 modulations of the IKs In this study we identify four residues (R67, K69, K70, and channel (31). To examine whether phosphorylation of IKs channel H73) in proximal C terminus of KCNE1 as key determinants affects PIP2-dependent modulation, we studied the properties of of PIP2 sensitivity (Fig. 3). Previous studies have highlighted the mutation S27D/S92D in KCNQ1 coexpressed with KCNE1, the KCNE1 C terminus as critical for the modulation of IKs cur- which has been shown to mimic the effects of PKA phosphoryla- rent characteristics. A C-terminal deleted KCNE1 mutant as- tion of IKs channels (31, 32). Consistent with previous results, we sembled with KCNQ1, but did not modify current properties found that S27D/S92D affects IKs function, including a shift of (20). Functional scan of KCNE1 point mutations revealed that voltage-dependent activation to more negative voltages and slow- the juxtamembranous C-terminal region of KCNE1 is especially ing of deactivation kinetics (Fig. 5 A–C). However, the phospho- intolerant to mutation, indicating that this region likely plays an mimetic mutation S27D/S92D does not alter the time course of important role in controlling channel activity (21). Docking of the PIP2-dependent rundown of the IKs current (Fig. 5D), suggesting NMR structure of KCNE1 to a KCNQ1 homology model indi- that PKA phosphorylation does not alter PIP2 modulation. Like- cates that the proximal C terminus of KCNE1 may participate in wise, the slowing of deactivation caused by the phosphomimetic intimate interaction with the S4–S5 linker suggesting that KCNE1 mutation is not dependent on the time after patch excision during could directly influence the channel gating machinery (15). The which PIP2 levels decrease (Fig. 5E), suggesting that PIP2 does colocalization of the KCNE1 proximal C terminus and the gating not affect the functional changes caused by PKA phosphoryla- machinery of KCNQ1 have also been shown experimentally by tion. These results suggest that, if a cross-talk between PKA and the formation of disulfide bonds between pairs of cysteine muta- PIP2 modulations of the IKs channel exists, it does not happen in tions engineered in the KCNE1 C terminus and the S4–S5 linker the channel protein. or the bottom of S6 (22). Interestingly, basic residues in the bot- The KCNE family of ion channel β-subunits contains five tom of S6 and in the S4–S5 linker have been proposed to interact family members that have been reported to modulate the activity with PIP2 (5). Purified fragments of the KCNQ1 proximal C ter- of a variety of channel α-subunits in ion channel complexes. Many minus demonstrated broad binding of anionic lipids in biochem- of these channel α-subunits or channel complexes are also modu- ical assays that depended on several juxtamembranous basic lated by PIP2 (Table 1). Sequence alignment shows that the basic residues in KCNQ1 (23). These results indicate that the key de- residues that are essential for KCNE1 modulation of IKs PIP2 terminants of PIP2 sensitivity in KCNE1 (residues R67, K69, K70, sensitivity are highly conserved across all members of the KCNE and H73) reside in the same region that is critical for KCNE1 family of peptides (Fig. 6), suggesting that modulation of PIP2 modulation of IKs and interaction with the gating machinery. sensitivity may be a common mechanism of current modulation Thus, PIP2 may be an integral component in the IKs channel com- by the KCNE β-subunits. Interestingly, some members of the plex, and the interactions among PIP2, KCNE1, and KCNQ1 may KCNE family may modulate more than one channel α-subunit. hold the key to resolve the long-standing absence of a mechanistic Likewise, multiple KCNE family members may modulate the understanding of how KCNE1 shapes the IKs channel function. same channel α-subunit (Table 1). Because of structural differ- Mutations of the key residues in KCNE1 that are determinants of PIP2 sensitivity, R67C, R67H, K70M, and K70N, are associated with long QT syndrome (7–9). These mutations reduce IKs current and PIP2 sensitivity (Fig. 4). Remarkably, we are able to rescue wild-type channel characteristics by applying supernormal levels of exogenous PIP2, suggesting that the disease pathogenesis of these mutations can be explained by a decrease in PIP2 sensitivity of the channel complex. PIP2 levels can be changed in the cardiac myocyte by activation of phospholipase C (PLC) via G-protein coupled receptors, such as α1A-adrenergic receptors and M1-muscarinic receptors, that are coupled to Gqα. PLC hydrolyzes PIP2 to generate diacylglycerol and inositol 1,4,5-tri- sphosphate, which lead to protein kinase C (PKC) activation and increased intracellular calcium, respectively (4). The generation of second messengers in addition to the effective decrease of PIP2 levels makes IKs regulation by PLC activation complex as IKs is known to be sensitive to PKC phosphorylation (24), intracellular calcium (25), and PIP2 (6). This complex regulation in response to activation of Gqα may be reflected in the biphasic response of heterologously expressed IKs channels (26) and the conflicting reports of effects on endogenous IKs in myocytes (27–29). Our current results show that the PIP2 sensitivity of the IKs channel is dependent on the KCNE1 expression level relative to that of Fig. 5. PIP2 modulation is independent of phosphomimetic mutations of KCNQ1 (Fig. 1), suggesting a possibility that KCNE1 may mod- IKs channels. (A) Whole-cell currents of S27D/S92D channels. To measure ulate the response to PLC activation. Further studies with the deactivation time constants, voltages were stepped from a holding potential consideration of this possibility may provide previously unde- of −80 mV to a depolarization potential (þ40 mV) and then repolarized scribed insights on the effects of the Gqα signaling pathway on to test potentials (−70 to 0 mV) (Left). To measure the G-V relation, voltages −80 −80 IKs channels. were stepped from a holding potential of mV to a test potential ( þ80 −40 IKs channels are also modulated by β-adrenergic receptor to mV) and then repolarized to mV (Right). (B) Time constant of deactivation vs. voltage and (C) G-V relation. WT KCNQ1 þ KCNE1 (open cir- stimulation due to the phosphorylation of residues S27 and n ¼ 6 27 ∕ 92 þ 1 n ¼ 7 S92 in KCNQ1 by protein kinase A (PKA) (30, 31), which in- cles), ;S D S D KCNE (filled circles), .(D) Time dependence of normalized tail current amplitude after patch excision. WT KCNQ1þ creases IKs currents and shortens ventricular action potentials. KCNE1 (open circles), n ¼ 12;S27D∕S92D þ KCNE1 (filled circles), n ¼ 6. Solid Importantly, this phosphorylation of KCNQ1 alters channel func- lines are monoexponential fits to data. (E) Time constant of tail currents in tion only with KCNE1 coexpression (32), indicating that, similar D. The deactivation time constant of the mutant channel is significantly to PIP2 modulation, the KCNE1 interaction with KCNQ1 is cri- larger than that of the WT (P < 0.05).

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1100872108 Li et al. Downloaded by guest on September 29, 2021 Table 1. Kþ channel α-subunits that interact with KCNE families and show PIP2 sensitivity

† KCNE α-subunit* PIP2 sensitivity KCNE1 KCNQ1 (1, 2) α þ β (6) HERG (33) α (34) Kv 4.3 (35) KCNE2 KCNQ1 (36) α þ β (37) KCNQ2 and 2∕3 (38) α (39) HERG (40) α (34) HCN1&2 (41) α (42) Fig. 6. Partial sequence alignment of all members of the KCNE family. Kv4.2 (43) and Kv4.3 (35) Numbering indicates the position on KCNE1. The filled box highlights the KCNE3 KCNQ1 (44) predicted transmembrane domain (TMD) (53). Open boxes indicate Kv2.1 and Kv3.1 (45) α (46) conserved residues that are key determinants of PIP2 sensitivity. Kv3.4 (47) α (48) HERG (44) α (34) indicated in the text, pH 7.2. PIP2 (bovine brain, Sigma-Aldrich), diC8-, and KCNQ4 (44) α (49) diC4-PIP2 (Echelon) were sonicated in ice bath for 30 min to dissolve in KCNE4 KCNQ1 (50) the internal solution at 2 mM as stock and kept in aliquots in −80 °C before KV1.1 and Kv1.3 (51) α (48) use. Whole cell current recordings were obtained with the two-microelec- KCNE5 KCNQ1 (52) trode voltage–clamp technique. Microelectrodes were pulled from glass ca- *Coexpression of a KCNE with the α-subunit changes the properties of the pillary tubes and filled with 3 M KCl. Oocytes were constantly superfused channel. with ND96. The membrane potential was clamped using a voltage–clamp am- † PIP2 sensitivity of the channel either with the α-subunits alone (α) or with the plifier (Dagan CA-1B; Dagan Corporation). Data acquisition was controlled α-subunits and a KCNE coexpression (α þ β) has been reported. using PULSE/PULSEFIT software (HEKA).

ences in these α- and β-subunits, the putative interactions among Data Analysis The relative conductance was determined by measuring tail current amplitudes at indicated voltages. The conductance-voltage (G-V) KCNE β-subunits, channel α-subunits and PIP2 may vary within different ion channel complexes, resulting in diverse effects of relationships were fitted with the Boltzmann equation: KCNE peptides. 1 G ¼ ; [1] BIOPHYSICS AND −zeðV−V1∕2Þ G COMPUTATIONAL BIOLOGY Materials and Methods max 1 þ expð kT Þ Mutagenesis and Oocyte Preparation. KCNQ1 and KCNE1 were subcloned into pcDNA3.1(+) (Invitrogen). All mutations were generated by using over- ∕ z where G Gmax is the ratio of conductance to maximum conductance, is the lap extension PCR and verified by sequencing. mRNA was transcribed in vitro number of the equivalent charges, V1∕2 is the voltage at which the channel is by using the mMessage mMachine T7 polymerase kit (Applied Biosystems). 50% activated, e is the elementary charge, k is Boltzmann’s constant, and T is The follicle cells were removed by using type 1A collagenase (Sigma-Aldrich). the absolute temperature. Data analysis and curve fitting were done using Stage IV–V Xenopus oocytes were selected and injected with 4.6 ng mRNA the Igor Pro software (WaveMetrics). per oocyte. Injected oocytes were incubated in ND96 solution (in mM: 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 Hepes, pH 7.60) at 18 °C for 3–5 d before recording. ACKNOWLEDGMENTS. We thank Junqiu Yang for technical help in the first patch–clamp recording. We thank S. Goldstein (University of Chicago, Chicago, IL) and S. Nakanishi (Kyoto University, Kyoto, Japan) for human Electrophysiology Macroscopic currents were recorded from inside-out 0 5–1 0 Ω KCNQ1 and KCNE1 clones. The cDNA of CiVSP was kindly provided by patches formed with patch pipettes of . . M resistance. The data were Y. Okamura (Osaka University, Osaka, Japan). This study was funded by – acquired using an Axopatch 200-B patch clamp amplifier (Axon Instruments) Established Investigator Award 0440066N from the American Heart and pulse acquisition software (HEKA). Experiments were conducted at Association (AHA) and National Science Foundation of China Grant room temperature (20–22 °C). The pipette solution contained (in mM) 30528011 (to J.C.), AHA predoctoral fellowship 0910020G (to D.W.), and 140 KMeSO3, 20 Hepes, 2 KCl, and 2 MgCl2, pH 7.2. The internal solution con- 11PRE5720009 (to M.A.Z). J.C. is the Spencer T. Olin Professor of Biomedical tained (in mM) 140 KMeSO3, 20 Hepes, 2 KCl, 5 EGTA, 1.5 MgATP, and PIP2 as Engineering.

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