KCNQ1 α-subunits coassemble with KCNE1 β-subunits to

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1 KCNE1 enhances phosphatidylinositol 4,5-bisphosphate (PIP 2 ) sensitivity of I Ks to modulate channel activity Yang Li, Mark A. Zaydman, Dick Wu 1, Jingyi Shi, Michael Guan, Brett Virgin-Downey, and Jianmin Cui 2 Department of Biomedical Engineering, Center for the Investigation of Membrane Excitability Disorders, Cardiac Bioelectricity and Arrhythmia Center, Washington University, St. Louis, MO 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 (PIP 2 ) is necessary for the function of various ion channels. The potassium channel, I Ks,is important for cardiac repolarization and requires PIP 2 to activate. Here we show that the auxiliary subunit of I Ks, KCNE1, increases PIP 2 sensitivity 100-fold over channels formed by the pore-forming KCNQ1 subunits alone, which effectively amplifies current because native PIP 2 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 PIP 2 sensitivity. Long QT syndrome associated mutations of this site lower PIP 2 affinity, resulting in reduced current. Application of exogenous PIP 2 to these mutants restores wild-type channel activity. These results reveal a vital role of PIP 2 for KCNE1 modulation of I Ks channels that may represent a common mechanism of auxiliary subunit modulation of many ion channels. KCNQ1 α-subunits coassemble with KCNE1 β-subunits to form the cardiac slow-delayed rectifier channel, I Ks, 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 cardiac I Ks current (Fig. 1A) (1, 2). These changes include increased current amplitude, shift of the voltage dependence of activation toward more positive potentials, slowed activation and deactivation kinetics, and suppressed inactivation, all of which are essential for the physiological role of I Ks (3). After many years of investigation, it is still poorly understood how the KCNE1 peptide exerts such a dramatic effect on the I Ks current. Loss-offunction mutations in I Ks lead to delayed cardiac cell repolarization that manifests clinically as long QT (LQT) syndrome. Patients with LQT syndrome are predisposed to ventricular arrhythmia and suffer from syncope and a high risk of sudden cardiac death. At present, the molecular mechanisms of disease pathogenesis are still unknown for many LQT-associated mutations in I Ks. Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is a minor acidic membrane lipid found primarily in the inner leaflet of the plasma membrane. PIP 2 has been shown to be a necessary cofactor for a wide variety of ion channels, e.g., voltage-gated K þ and Ca 2þ channels, transient receptor potential channels, inward rectifying K þ channels, and epithelial Na þ channels (4). Recently, I Ks has been shown to require PIP 2 for channel activity, and several LQTassociated mutations located in KCNQ1 have been suggested to decrease the PIP 2 affinity of I Ks (5, 6). Here we provide evidence that KCNE1, as a β-subunit, alters the function of I Ks by modulating the interaction between PIP 2 and the heteromeric ion channel complex. Knowledge of the molecular mechanisms of channel modulation by KCNE1 is of great importance as at least 36 LQT-associated mutations reside within KCNE1 (7 9). Results KCNE1 Slows the PIP 2 -Dependent Channel Rundown. To investigate the regulation of I Ks by PIP 2, we expressed KCNQ1 with and without KCNE1 in oocytes from Xenopus laevis and recorded Fig. 1. KCNE1 slows the PIP 2 -dependent channel rundown. (A) KCNQ1 and KCNQ1 þ KCNE1 currents at various times after patch excision. Voltage was stepped from a holding potential of 80 to þ80 mv and then back to the holding potential. (B) Time dependence of current amplitude after patch excision. Normalized tail current amplitude at various time, I t I 0, is plotted; I 0 is the tail current amplitude immediately following patch excision. KCNQ1 (filled circles), n ¼ 9; KCNQ1 þ KCNE1 (open circles), n ¼ 12; KCNQ1þ KCNE1 þ 10 μm PIP 2 (open squares), n ¼ 6; KCNQ1 þ KCNE1 coexpressed with Ci-VSP (open diamonds), n ¼ 6. Solid lines are monoexponential fits to data. Voltage was stepped from a holding potential of 80 mv to þ80 mv for 5 s and then back to the holding potential for 5 s. (C) Time constants of exponential fits and initial delay to current rundown. (D) Normalized tail current amplitude at various times, I t I 0, with KCNQ1: KCNE1 mrna ratio 1 1 (open circles), n ¼ 12; (open diamonds), n ¼ 6; (open 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 are presented as mean SEM. currents using patch-clamp and two-electrode voltage-clamp techniques. Currents recorded from inside-out membrane patches expressing KCNQ1 immediately and rapidly decayed following patch excision such that channel activity was completely 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 Fig. 1 5; M.A.Z. and M.G. performed voltage clamp experiments in Fig. 4. J.S., M.G., and B.V.-D. performed molecular biology; Y.L. analyzed data; and Y.L., M.A.Z., D.W., and J.C. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1 Present address: Stanford Institute for Neuro-innovation and Translational Neurosciences, Stanford, CA To whom correspondence should be addressed. jcui@biomed.wustl.edu. This article contains supporting information online at doi: /pnas /-/dcsupplemental. BIOPHYSICS AND COMPUTATIONAL BIOLOGY PNAS Early Edition 1of6

2 Fig. 2. KCNE1 enhances PIP 2 sensitivity. (A) Normalized tail current amplitude at various times following patch excision and PIP 2 application. PIP 2 was applied at all times after the arrow. Voltage protocol to elicit the currents is the same as in Fig. 1. (B) PIP 2 dose response of WT KCNQ1 and WT and mutant KCNQ1 þ KCNE1 channels. I rec is tail current amplitude at steady state after recovery of channel activity following PIP 2 application. I 0 is the tail current amplitude immediately following patch excision. KCNQ1 (filled circles), KCNQ1 þ KCNE1 (open circles), R539W þ KCNE1 (open diamonds), and 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, R539W þ KCNE1, and KCNQ1, respectively. For KCNQ1 þ KCNE1, the responses of currents to dic8-pip 2 (up triangles) and dic4-pip 2 (down triangles) (5 and 500 μm) are also plotted. (C) EC 50 calculated by the Hill fit (n ¼ 6 9). lost within 5 min (Fig. 1A). This rundown of current was fit well with a single exponential (Fig. 1B). However, after excising inside-out membrane patches expressing KCNQ1 þ KCNE1, currents remained stable over 3 min prior to rundown, which was not observed with KCNQ1 alone (Fig. 1B). The subsequent rundown of KCNQ1 þ KCNE1 current was slower than that observed with KCNQ1 alone (Fig. 1 B and C). This rundown of I Ks activity is attributed to the loss of PIP 2 from the excised membrane patch (6). The PIP 2 dependence of current rundown after patch excision was supported by the following experiments. The voltage sensitive phosphatase from Ciona intestinalis (CiVSP) that dephosphorylates PIP 2 upon membrane depolarization (10) was coexpressed with KCNQ1 þ KCNE1. CiVSP hastens PIP 2 loss from the membrane patch, resulting in a faster rate of current rundown. Conversely, directly applying 10 μm of exogenous PIP 2 to the intracellular face of membrane patches expressing KCNQ1 þ KCNE1 prevented current rundown for longer than 10 min (Fig. 1 B and C). These results show that PIP 2 loss after patch excision leads to loss of channel activity. KCNQ1 activity is lost more quickly after patch excision than KCNQ1 þ KCNE1, indicating that KCNE1 slows PIP 2 -dependent rundown. To further demonstrate this effect of KCNE1, we injected oocytes with the same amount of KCNQ1 mrna and various amounts of KCNE1 mrnas in molar ratios of 1 0, , , and 1 1. The activation kinetics and the steadystate voltage dependence of activation of the expressed channels depend on the KCNQ1 KCNE1 ratio, which suggested that poreforming KCNQ1 subunits are associated with varying numbers of KCNE1 subunits (11 13) (Fig. S1). The time course of current rundown became progressively longer with higher concentrations of KCNE1 due to a longer delay and a larger time constant of rundown (Fig. 1D). A lower molar ratio of KCNQ1 KCNE1 mrna results in a larger portion of the expressed channel population containing a higher stoichiometry of KCNE1 KCNQ1, and these results show that channels with more KCNE1 subunits have slower PIP 2 -dependent rundown (Fig. 1 D and E). Why does the association of KCNE1 affect the time course of PIP 2 -dependent rundown? To answer this question, we applied PIP 2 to the intracellular face of membrane patches after complete current rundown, which restored channel activity in a dosedependent manner (Fig. 2A). The effective PIP 2 concentrations of half maximal activation (EC 50 ) for KCNQ1 and KCNQ1þ KCNE1 are >600 μm and 4.6 μm, respectively (Fig. 2 B and C). These results show that KCNE1 greatly enhances the PIP 2 sensitivity of the channel resulting in a longer time course of rundown as the channels can still function at lower PIP 2 levels. To further confirm the correlation between time course of PIP 2 - dependent rundown and PIP 2 sensitivity, we studied two LQT mutations of KCNQ1, R539W, and R555C, coexpressed with KCNE1, both of which decrease PIP 2 sensitivity (Fig. 2 B and C) (5). Consistently, these mutants shortened the time course of rundown by eliminating the delay and decreasing the time constant of rundown relative to WT KCNQ1 þ KCNE1 (Figs. 1C and 2B). It is important to note that application of high levels of PIP 2 could increase the current of KCNQ1 beyond the level measured immediately following patch excision (Fig. 2 A and B). This result suggests that the native PIP 2 level in the patch membrane is not sufficient to saturate the PIP 2 binding and activation of all KCNQ1 channels; therefore, supernormal levels of exogenous PIP 2 can activate channels that are PIP 2 unbound in the native membrane patch. In contrast, high doses of PIP 2 could not increase the current of KCNQ1 þ KCNE1 beyond the amplitude immediately following patch excision (Fig. 2 A and B), indicating that KCNE1 association enhances PIP 2 affinity, such that channel activation by PIP 2 is saturated immediately following patch excision. This conclusion is supported by the delay before PIP 2 - dependent current rundown that is present only when KCNE1 is coexpressed with KCNQ1 (Fig. 1 B E). This delay likely represents a period when the PIP 2 level in the patch membrane is supersaturating for activating KCNQ1 þ KCNE1. Rundown occurs when the PIP 2 level falls below a threshold of saturation and channels become nonfunctional due to unbinding of PIP 2. These results suggest that KCNE1 coexpression increases the current amplitude by recruiting KCNQ1 channels that would be nonfunctional due to a lack of PIP 2 binding. Key Structural Determinants of PIP 2 Sensitivity in KCNE1 Proteins associate with PIP 2 through electrostatic interactions between basic residues and the negatively charged head group of PIP 2 (4, 14). Application of dic4-pip 2 and dic8-pip 2, which have different fatty acid tail groups as the PIP 2 purified from cell membrane (see Materials and Methods) has the same effect on KCNQ1 þ KCNE1 currents (Fig. 2B), supporting that the PIP 2 head group is important in interactions with the channel protein. In order to identify the key structural determinants in KCNE1 contributing to enhanced PIP 2 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 neutralization of these basic residues leads to a decreased PIP 2 sensitivity (Fig. 3 A and B). In the KCNE1 solution NMR structure in micelles (15), these residues are positioned on a helical stretch at the bottom of the transmembrane domain (Fig. 3C). The α- helicity of the juxtamembranous portion of the KCNE1 C terminus is also supported by mutagenic perturbation analysis (16). The basic residues at positions 67, 69, 70, and 73 lie on one face of the α-helix, which provide a dense cluster of positive charges that would be attractive for the negatively charged head group of PIP 2. Our mutational study cannot definitively identify these residues as a PIP 2 binding site because the mutations could affect PIP 2 binding through an allosteric mechanism. However, the clustering of these basic residues juxtaposed with the inner leaflet of the plasma membrane suggests that they could directly interact with PIP 2. Consistent with this idea, a triple charge-reversal mu- 2of6 Li et al.

3 Fig. 3. Molecular determinants of PIP 2 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 that was coexpressed with KCNQ1. (-) indicates KCNQ1 alone. (C) The transmembrane segment and juxtamembraneous C-terminal region of the KCNE1 NMR structure (PDB ID code 2K21). The residues important for PIP 2 sensitivity are shown in stick presentation. tation, R67E/K69E/K70E, renders the time course of rundown even faster than that of KCNQ1 alone (Fig. 3 A and B), as if PIP 2 were repulsed by the negative charges. PIP 2 Restores the Loss of Function Due to LQT Mutations in KCNE1 Mutations of the putative PIP 2 interaction site in KCNE1 (R67C, R67H, K70M, and K70N) have been previously identified in LQT patients as disease-associated mutations (7 9). We find that coexpressing the KCNE1 mutation with KCNQ1 decreases current amplitude and shifts the voltage dependence of activation toward more depolarized potentials compared to WT KCNQ1 þ KCNE1 channels (Fig. 4 A C). Each of these changes in channel properties would decrease the contribution of I Ks to termination of cardiac action potentials, resulting in prolongation of action potential duration and creating a substrate for potentially fatal arrhythmias. The PIP 2 dose-response curves of KCNQ1 þ R67C and KCNQ1 þ K70N (Fig. 4D) are significantly right-shifted in comparison to that of WT KCNQ1 þ KCNE1, indicating that these LQT-associated mutations decrease PIP 2 sensitivity of the channel complex. Furthermore, the current amplitude measured by applying saturating levels of PIP 2 exceeded the current amplitude immediately following patch excision, suggesting that a reduction of PIP 2 affinity decreases the number of PIP 2 -bound channels in the native membrane resulting in less current. Remarkably, application of a saturating concentration of PIP 2 (300 μm) to the intracellular face of membrane patches expressing KCNQ1 þ mutant KCNE1 restored the wild-type channel current characteristics. Specifically, the current amplitude increased 2- to 5-fold (Fig. 4E), accounting for all of the reduction in the whole cell current of the mutant channels (Fig. 4 A and C). Additionally, the voltage dependence of channel activation shifted back toward less depolarized voltages to nearly superimpose with that of the WT KCNQ1 þ KCNE1 (Fig. 4F). These results show that a decrease in PIP 2 sensitivity of the I Ks channel due to these KCNE1 mutations can lead to LQT syndrome. Fig. 4. LQT mutations in KCNE1 reduce PIP 2 sensitivity. (A) Whole-cell currents and (B) conductance-voltage (G-V) relation in oocytes. WT KCNQ1 þ KCNE1 (open squares), n ¼ 6; KCNQ1 þ mutant KCNE1: R67C (filled circles), n ¼ 6; R67H (filled diamonds), n ¼ 6; K70N (filled triangles), n ¼ 6; K70M (filled squares), n ¼ 6; Voltages were stepped from a holding potential of 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 by that of WT channels (filled bars) and V 1 2 measured from G-V relation (open bars). (D) PIP 2 dose response of KCNQ1 þ mutant KCNE1 channels: 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 μm for KCNQ1 þ K70N and KCNQ1 þ R67C, respectively. (E) Tail current amplitude in response to the same voltage protocol as described in Fig. 1 for WT and mutant channels. (filled bars) Indicate the tail current amplitude immediately following patch excision. (open bars) Indicate the tail current amplitude after steady-state recovery of channel activity in response to 300 um exogenous PIP 2. (F) G-V relations of KCNQ1 þ K70N (filled symbols) and WT KCNQ1 þ KCNE1 (unfilled) before and after patch excision in PIP 2 - containing solutions. KCNQ1 þ K70N: on cell (filled circles), V 1 2 ¼ mv; excised in 50 μmpip 2 (filled diamonds), V 1 2 ¼ mv; excised in 150 μm PIP 2, (filled triangles), V 1 2 ¼ mv; excised in 300 μm PIP 2 (filled squares), V 1 2 ¼ mv. KCNQ þ KCNE1: on cell (open circles), V 1 2 ¼ mv; excised in 300 μm PIP 2 (open squares), V 1 2 ¼ mv. n ¼ 2 for G-V relation of KCNQ1 þ K70N in 50 μm PIP 2, n ¼ 6 12 for all other experiments. Discussion Ion channel β-subunits modulate pore-forming α-subunits, increasing the diversity of ion channel properties that can be generated from a limited repertoire of genes encoding α- and β- subunits. To understand the function of ion channels in vivo requires structure-function knowledge of not only the pore-forming subunits, but also their heteromeric complexes with β-subunits. The interactions between KCNQ1 and KCNE1 have been studied intensely for many years as this pair of α- and β-subunits coassembles in the cardiac myocyte and generates the I Ks current that is important in the termination of cardiac action potentials. However, despite these efforts, the molecular mechanisms by which KCNE1 changes the KCNQ1 channel to generate the I Ks current remain elusive. In this work we show that KCNE1 increases the PIP 2 sensitivity of I Ks by several orders of magnitude (Fig. 2). Because of the low PIP 2 sensitivity of KCNQ1, the PIP 2 level in biological membrane is too low to saturate binding of KCNQ1, leaving a fraction of the channel population nonfunctional in the absence of KCNE1. This unique mechanism of current amplification is independent of KCNE1 effects on single channel con- BIOPHYSICS AND COMPUTATIONAL BIOLOGY Li et al. PNAS Early Edition 3of6

4 ductance for which previous reports have provided conflicting results (17 19). In this study we identify four residues (R67, K69, K70, and H73) in proximal C terminus of KCNE1 as key determinants of PIP 2 sensitivity (Fig. 3). Previous studies have highlighted the KCNE1 C terminus as critical for the modulation of I Ks current characteristics. A C-terminal deleted KCNE1 mutant assembled with KCNQ1, but did not modify current properties (20). Functional scan of KCNE1 point mutations revealed that the juxtamembranous C-terminal region of KCNE1 is especially intolerant to mutation, indicating that this region likely plays an important role in controlling channel activity (21). Docking of the NMR structure of KCNE1 to a KCNQ1 homology model indicates that the proximal C terminus of KCNE1 may participate in intimate interaction with the S4 S5 linker suggesting that KCNE1 could directly influence the channel gating machinery (15). The colocalization of the KCNE1 proximal C terminus and the gating machinery of KCNQ1 have also been shown experimentally by the formation of disulfide bonds between pairs of cysteine mutations engineered in the KCNE1 C terminus and the S4 S5 linker or the bottom of S6 (22). Interestingly, basic residues in the bottom of S6 and in the S4 S5 linker have been proposed to interact with PIP 2 (5). Purified fragments of the KCNQ1 proximal C terminus demonstrated broad binding of anionic lipids in biochemical assays that depended on several juxtamembranous basic residues in KCNQ1 (23). These results indicate that the key determinants of PIP 2 sensitivity in KCNE1 (residues R67, K69, K70, and H73) reside in the same region that is critical for KCNE1 modulation of I Ks and interaction with the gating machinery. Thus, PIP 2 may be an integral component in the I Ks channel complex, and the interactions among PIP 2, KCNE1, and KCNQ1 may hold the key to resolve the long-standing absence of a mechanistic understanding of how KCNE1 shapes the I Ks channel function. Mutations of the key residues in KCNE1 that are determinants of PIP 2 sensitivity, R67C, R67H, K70M, and K70N, are associated with long QT syndrome (7 9). These mutations reduce I Ks current and PIP 2 sensitivity (Fig. 4). Remarkably, we are able to rescue wild-type channel characteristics by applying supernormal levels of exogenous PIP 2, suggesting that the disease pathogenesis of these mutations can be explained by a decrease in PIP 2 sensitivity of the channel complex. PIP 2 levels can be changed in the cardiac myocyte by activation of phospholipase C (PLC) via G-protein coupled receptors, such as α 1A -adrenergic receptors and M 1 -muscarinic receptors, that are coupled to Gqα. PLC hydrolyzes PIP 2 to generate diacylglycerol and inositol 1,4,5-trisphosphate, 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 PIP 2 levels makes I Ks regulation by PLC activation complex as I Ks is known to be sensitive to PKC phosphorylation (24), intracellular calcium (25), and PIP 2 (6). This complex regulation in response to activation of Gqα may be reflected in the biphasic response of heterologously expressed I Ks channels (26) and the conflicting reports of effects on endogenous I Ks in myocytes (27 29). Our current results show that the PIP 2 sensitivity of the I Ks channel is dependent on the KCNE1 expression level relative to that of KCNQ1 (Fig. 1), suggesting a possibility that KCNE1 may modulate the response to PLC activation. Further studies with the consideration of this possibility may provide previously undescribed insights on the effects of the Gqα signaling pathway on I Ks channels. I Ks channels are also modulated by β-adrenergic receptor stimulation due to the phosphorylation of residues S27 and S92 in KCNQ1 by protein kinase A (PKA) (30, 31), which increases I Ks currents and shortens ventricular action potentials. Importantly, this phosphorylation of KCNQ1 alters channel function only with KCNE1 coexpression (32), indicating that, similar to PIP 2 modulation, the KCNE1 interaction with KCNQ1 is critical for the PKA-dependent modulation. A previous study suggested a cross-talk between PKA and PIP 2 modulations of the I Ks channel (31). To examine whether phosphorylation of I Ks channel affects PIP 2 -dependent modulation, we studied the properties of the mutation S27D/S92D in KCNQ1 coexpressed with KCNE1, which has been shown to mimic the effects of PKA phosphorylation of I Ks channels (31, 32). Consistent with previous results, we found that S27D/S92D affects I Ks function, including a shift of voltage-dependent activation to more negative voltages and slowing of deactivation kinetics (Fig. 5 A C). However, the phosphomimetic mutation S27D/S92D does not alter the time course of PIP 2 -dependent rundown of the I Ks current (Fig. 5D), suggesting that PKA phosphorylation does not alter PIP 2 modulation. Likewise, the slowing of deactivation caused by the phosphomimetic mutation is not dependent on the time after patch excision during which PIP 2 levels decrease (Fig. 5E), suggesting that PIP 2 does not affect the functional changes caused by PKA phosphorylation. These results suggest that, if a cross-talk between PKA and PIP 2 modulations of the I Ks channel exists, it does not happen in the channel protein. The KCNE family of ion channel β-subunits contains five family members that have been reported to modulate the activity of a variety of channel α-subunits in ion channel complexes. Many of these channel α-subunits or channel complexes are also modulated by PIP 2 (Table 1). Sequence alignment shows that the basic residues that are essential for KCNE1 modulation of I Ks PIP 2 sensitivity are highly conserved across all members of the KCNE family of peptides (Fig. 6), suggesting that modulation of PIP 2 sensitivity may be a common mechanism of current modulation by the KCNE β-subunits. Interestingly, some members of the KCNE family may modulate more than one channel α-subunit. Likewise, multiple KCNE family members may modulate the same channel α-subunit (Table 1). Because of structural differ- Fig. 5. PIP 2 modulation is independent of phosphomimetic mutations of I Ks channels. (A) Whole-cell currents of S27D/S92D channels. To measure deactivation time constants, voltages were stepped from a holding potential of 80 mv to a depolarization potential (þ40 mv) and then repolarized to test potentials ( 70 to 0 mv) (Left). To measure the G-V relation, voltages were stepped from a holding potential of 80 mv to a test potential ( 80 to þ80 mv) and then repolarized to 40 mv (Right). (B) Time constant of deactivation vs. voltage and (C) G-V relation. WT KCNQ1 þ KCNE1 (open circles), n ¼ 6; S27D S92D þ KCNE1 (filled circles), n ¼ 7. (D) Time dependence of normalized tail current amplitude after patch excision. WT KCNQ1þ KCNE1 (open circles), n ¼ 12; S27D S92D þ KCNE1 (filled circles), n ¼ 6. Solid lines are monoexponential fits to data. (E) Time constant of tail currents in D. The deactivation time constant of the mutant channel is significantly larger than that of the WT (P < 0.05). 4of6 Li et al.

5 Table 1. K þ channel α-subunits that interact with KCNE families and show PIP 2 sensitivity KCNE α-subunit* PIP 2 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) Kv4.2 (43) and Kv4.3 (35) KCNE3 KCNQ1 (44) Kv2.1 and Kv3.1 (45) α (46) Kv3.4 (47) α (48) HERG (44) α (34) KCNQ4 (44) α (49) KCNE4 KCNQ1 (50) KV1.1 and Kv1.3 (51) α (48) KCNE5 KCNQ1 (52) *Coexpression of a KCNE with the α-subunit changes the properties of the channel. PIP 2 sensitivity of the channel either with the α-subunits alone (α) or with the α-subunits and a KCNE coexpression (α þ β) has been reported. ences in these α- and β-subunits, the putative interactions among KCNE β-subunits, channel α-subunits and PIP 2 may vary within different ion channel complexes, resulting in diverse effects of KCNE peptides. Materials and Methods Mutagenesis and Oocyte Preparation. KCNQ1 and KCNE1 were subcloned into pcdna3.1(+) (Invitrogen). All mutations were generated by using overlap extension PCR and verified by sequencing. mrna was transcribed in vitro by using the mmessage mmachine T7 polymerase kit (Applied Biosystems). The follicle cells were removed by using type 1A collagenase (Sigma-Aldrich). Stage IV V Xenopus oocytes were selected and injected with 4.6 ng mrna per oocyte. Injected oocytes were incubated in ND96 solution (in mm: 96 NaCl, 2 KCl, 1.8 CaCl 2, 1 MgCl 2, and 5 Hepes, ph 7.60) at 18 C for 3 5 d before recording. Electrophysiology Macroscopic currents were recorded from inside-out patches formed with patch pipettes of MΩ resistance. The data were acquired using an Axopatch 200-B patch clamp amplifier (Axon Instruments) and pulse acquisition software (HEKA). Experiments were conducted at room temperature (20 22 C). The pipette solution contained (in mm) 140 KMeSO 3, 20 Hepes, 2 KCl, and 2 MgCl 2, ph 7.2. The internal solution contained (in mm) 140 KMeSO 3, 20 Hepes, 2 KCl, 5 EGTA, 1.5 MgATP, and PIP 2 as Fig. 6. Partial sequence alignment of all members of the KCNE family. Numbering indicates the position on KCNE1. The filled box highlights the predicted transmembrane domain (TMD) (53). Open boxes indicate conserved residues that are key determinants of PIP 2 sensitivity. indicated in the text, ph 7.2. PIP 2 (bovine brain, Sigma-Aldrich), dic8-, and dic4-pip 2 (Echelon) were sonicated in ice bath for 30 min to dissolve in the internal solution at 2 mm as stock and kept in aliquots in 80 C before use. Whole cell current recordings were obtained with the two-microelectrode voltage clamp technique. Microelectrodes were pulled from glass capillary tubes and filled with 3 M KCl. Oocytes were constantly superfused with ND96. The membrane potential was clamped using a voltage clamp amplifier (Dagan CA-1B; Dagan Corporation). Data acquisition was controlled using PULSE/PULSEFIT software (HEKA). Data Analysis The relative conductance was determined by measuring tail current amplitudes at indicated voltages. The conductance-voltage (G-V) relationships were fitted with the Boltzmann equation: G G max ¼ 1 1 þ expð zeðv V 1 2Þ kt Þ ; [1] where G G max is the ratio of conductance to maximum conductance, z is the number of the equivalent charges, V 1 2 is the voltage at which the channel is 50% activated, e is the elementary charge, k is Boltzmann s constant, and T is the absolute temperature. Data analysis and curve fitting were done using the Igor Pro software (WaveMetrics). 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 KCNQ1 and KCNE1 clones. The cdna of CiVSP was kindly provided by Y. Okamura (Osaka University, Osaka, Japan). 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7 Supporting Information Li et al /pnas A B 1.0 G/G max µ A 1 s Test Potential (mv) Fig. S1. Channel properties with different ratios of KCNQ1 to KCNE1. (A) Superimposed current traces recorded from 4 oocytes with KCNQ1: KCNE1 mrna ratios of 1 1 (open circle); (open triangle); (open square); 1 0 (filled circle). The currents were elicited by a depolarizing voltage pulse to 40 mv from a holding potential of 80 mv. (B) Normalized G-V curves for different ratios of KCNQ1: KCNE1, 1 1 (open circles), V 1 2 ¼ mv; (open triangles), V 1 2 ¼ mv; (open squares), V 1 2 ¼ mv; 1 0 (filled circles), V 1 2 ¼ mv. n ¼ 6 for all experiments. Li et al. 1of1