KCNE1 enhances phosphatidylinositol 4,5-bisphosphate (PIP₂) sensitivity of I_Ks to modulate channel activity

Abstract

Phosphatidylinositol 4,5-bisphosphate (PIP₂) is necessary for the function of various ion channels. The potassium channel, I_Ks, is important for cardiac repolarization and requires PIP₂ to activate. Here we show that the auxiliary subunit of I_Ks, 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 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-of-function 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.

Fig. 1.

KCNE1 slows the PIP₂-dependent channel rundown. (A) KCNQ1 and KCNQ1 + KCNE1 currents at various times after patch excision. Voltage was stepped from a holding potential of -80 to +80 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/I₀, is plotted; I₀ is the tail current amplitude immediately following patch excision. KCNQ1 (filled circles), n = 9; KCNQ1 + KCNE1 (open circles), n = 12; KCNQ1 + KCNE1 + 10 μM PIP₂ (open squares), n = 6; KCNQ1 + KCNE1 coexpressed with Ci-VSP (open diamonds), n = 6. Solid lines are monoexponential fits to data. Voltage was stepped from a holding potential of -80 mV to +80 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/I₀, with KCNQ1: KCNE1 mRNA ratio 1∶1 (open circles), n = 12; 1∶0.05 (open diamonds), n = 6; 1∶0.01 (open squares), n = 6; 1∶0 (filled circles), n = 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.

Phosphatidylinositol 4,5-bisphosphate (PIP₂) is a minor acidic membrane lipid found primarily in the inner leaflet of the plasma membrane. PIP₂ has been shown to be a necessary cofactor for a wide variety of ion channels, e.g., voltage-gated K⁺ and Ca²⁺ channels, transient receptor potential channels, inward rectifying K⁺ channels, and epithelial Na⁺ channels (4). Recently, I_Ks has 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 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 PIP₂ 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 PIP₂-Dependent Channel Rundown.

To investigate the regulation of I_Ks by PIP₂, we expressed KCNQ1 with and without KCNE1 in oocytes from Xenopus laevis and recorded 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 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 + KCNE1, currents remained stable over 3 min prior to rundown, which was not observed with KCNQ1 alone (Fig. 1B). The subsequent rundown of KCNQ1 + 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 PIP₂ from the excised membrane patch (6). The PIP₂ dependence of current rundown after patch excision was supported by the following experiments. The voltage sensitive phosphatase from Ciona intestinalis (CiVSP) that dephosphorylates PIP₂ upon membrane depolarization (10) was coexpressed with KCNQ1 + KCNE1. CiVSP hastens PIP₂ loss from the membrane patch, resulting in a faster rate of current rundown. Conversely, directly applying 10 μM of exogenous PIP₂ to the intracellular face of membrane patches expressing KCNQ1 + KCNE1 prevented current rundown for longer than 10 min (Fig. 1 B and C). These results show that PIP₂ loss after patch excision leads to loss of channel activity.

KCNQ1 activity is lost more quickly after patch excision than KCNQ1 + KCNE1, indicating that KCNE1 slows PIP₂-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, 1∶0.01, 1∶0.05, and 1∶1. The activation kinetics and the steady-state voltage dependence of activation of the expressed channels depend on the KCNQ1∶KCNE1 ratio, which suggested that pore-forming 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 PIP₂-dependent rundown (Fig. 1 D and E).

Why does the association of KCNE1 affect the time course of PIP₂-dependent rundown? To answer this question, we applied PIP₂ to the intracellular face of membrane patches after complete current rundown, which restored channel activity in a dose-dependent manner (Fig. 2A). The effective PIP₂ concentrations of half maximal activation (EC₅₀) for KCNQ1 and KCNQ1 + KCNE1 are > 600 μM and 4.6 μM, respectively (Fig. 2 B and C). These results show that KCNE1 greatly enhances the PIP₂ sensitivity of the channel resulting in a longer time course of rundown as the channels can still function at lower PIP₂ levels. To further confirm the correlation between time course of PIP₂-dependent rundown and PIP₂ sensitivity, we studied two LQT mutations of KCNQ1, R539W, and R555C, coexpressed with KCNE1, both of which decrease PIP₂ 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 + KCNE1 (Figs. 1C and 2B).

Fig. 2.

KCNE1 enhances PIP₂ sensitivity. (A) Normalized tail current amplitude at various times following patch excision and PIP₂ application. PIP₂ was applied at all times after the arrow. Voltage protocol to elicit the currents is the same as in Fig. 1. (B) PIP₂ dose response of WT KCNQ1 and WT and mutant KCNQ1 + KCNE1 channels. I_rec is tail current amplitude at steady state after recovery of channel activity following PIP₂ application. I₀ is the tail current amplitude immediately following patch excision. KCNQ1 (filled circles), KCNQ1 + KCNE1 (open circles), R539W + KCNE1 (open diamonds), and R555C + 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 + KCNE1, R555C + KCNE1, R539W + KCNE1, and KCNQ1, respectively. For KCNQ1 + KCNE1, the responses of currents to diC8-PIP₂ (up triangles) and diC4-PIP₂ (down triangles) (5 and 500 μM) are also plotted. (C) EC₅₀ calculated by the Hill fit (n = 6–9).

It is important to note that application of high levels of PIP₂ 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 PIP₂ level in the patch membrane is not sufficient to saturate the PIP₂ binding and activation of all KCNQ1 channels; therefore, supernormal levels of exogenous PIP₂ can activate channels that are PIP₂ unbound in the native membrane patch. In contrast, high doses of PIP₂ could not increase the current of KCNQ1 + KCNE1 beyond the amplitude immediately following patch excision (Fig. 2 A and B), indicating that KCNE1 association enhances PIP₂ affinity, such that channel activation by PIP₂ is saturated immediately following patch excision. This conclusion is supported by the delay before PIP₂-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 PIP₂ level in the patch membrane is supersaturating for activating KCNQ1 + KCNE1. Rundown occurs when the PIP₂ level falls below a threshold of saturation and channels become nonfunctional due to unbinding of PIP₂. These results suggest that KCNE1 coexpression increases the current amplitude by recruiting KCNQ1 channels that would be nonfunctional due to a lack of PIP₂ binding.

Key Structural Determinants of PIP₂ Sensitivity in KCNE1

Proteins 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 + 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 neutralization 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 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 PIP₂. Our mutational study cannot definitively identify these residues as a PIP₂ binding site because the mutations could affect PIP₂ 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 PIP₂. Consistent with this idea, a triple charge-reversal mutation, R67E/K69E/K70E, renders the time course of rundown even faster than that of KCNQ1 alone (Fig. 3 A and B), as if PIP₂ were repulsed by the negative charges.

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 = 9; WT KCNQ1 + KCNE1 (⋈), n = 9; KCNQ1 + mutant KCNE1: R67Q (up triangles), n = 6; K69C (open diamonds), n = 6; K70C (open circles), n = 6; H73N (open squares), n = 6; R67E/K69E/K70E triple mutation (TM), (down triangles), n = 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 PIP₂ sensitivity are shown in stick presentation.

PIP₂ Restores the Loss of Function Due to LQT Mutations in KCNE1

Mutations of the putative PIP₂ 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 + 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 PIP₂ dose-response curves of KCNQ1 + R67C and KCNQ1 + K70N (Fig. 4D) are significantly right-shifted in comparison to that of WT KCNQ1 + KCNE1, indicating that these LQT-associated mutations decrease PIP₂ sensitivity of the channel complex. Furthermore, the current amplitude measured by applying saturating levels of PIP₂ exceeded the current amplitude immediately following patch excision, suggesting that a reduction of PIP₂ affinity decreases the number of PIP₂-bound channels in the native membrane resulting in less current. Remarkably, application of a saturating concentration of PIP₂ (300 μM) to the intracellular face of membrane patches expressing KCNQ1 + 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 + KCNE1 (Fig. 4F). These results show that a decrease in PIP₂ sensitivity of the I_Ks channel due to these KCNE1 mutations can lead to LQT syndrome.

Fig. 4.

LQT mutations in KCNE1 reduce PIP₂ sensitivity. (A) Whole-cell currents and (B) conductance-voltage (G-V) relation in oocytes. WT KCNQ1 + KCNE1 (open squares), n = 6; KCNQ1 + mutant KCNE1: R67C (filled circles), n = 6; R67H (filled diamonds), n = 6; K70N (filled triangles), n = 6; K70M (filled squares), n = 6; Voltages were stepped from a holding potential of -80 mV to a test potential (-80 to +80 mV) and then repolarized to -40 mV. (C) Whole-cell current amplitude measured +80 mV normalized by that of WT channels (filled bars) and V_1/2 measured from G-V relation (open bars). (D) PIP₂ dose response of KCNQ1 + 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 + K70N and KCNQ1 + 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 PIP₂. (F) G-V relations of KCNQ1 + K70N (filled symbols) and WT KCNQ1 + KCNE1 (unfilled) before and after patch excision in PIP₂-containing solutions. KCNQ1 + K70N: on cell (filled circles), V_1/2 = 48.5 ± 2.2 mV; excised in 50 μM PIP₂ (filled diamonds), V_1/2 = 50.7 ± 0.8 mV; excised in 150 μM PIP₂, (filled triangles), V_1/2 = 29.9 ± 3.2 mV; excised in 300 μM PIP₂ (filled squares), V_1/2 = 8.9 ± 2.6 mV. KCNQ + KCNE1: on cell (open circles), V_1/2 = 21.6 ± 3.2 mV; excised in 300 μM PIP₂ (open squares), V_1/2 = 8.5 ± 1.2 mV. n = 2 for G-V relation of KCNQ1 + K70N in 50 μM PIP₂, n = 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 PIP₂ sensitivity of I_Ks by several orders of magnitude (Fig. 2). Because of the low PIP₂ sensitivity of KCNQ1, the PIP₂ 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 conductance 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 PIP₂ 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 PIP₂ (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 PIP₂ 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, PIP₂ may be an integral component in the I_Ks channel complex, and the interactions among PIP₂, 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 PIP₂ sensitivity, R67C, R67H, K70M, and K70N, are associated with long QT syndrome (7–9). These mutations reduce I_Ks 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 α_1A-adrenergic receptors and M₁-muscarinic receptors, that are coupled to Gqα. PLC hydrolyzes PIP₂ 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 PIP₂ 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 PIP₂ (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 PIP₂ 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 PIP₂ modulation, the KCNE1 interaction with KCNQ1 is critical for the PKA-dependent modulation. A previous study suggested a cross-talk between PKA and PIP₂ modulations of the I_Ks channel (31). To examine whether phosphorylation of I_Ks channel affects PIP₂-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 PIP₂-dependent rundown of the I_Ks current (Fig. 5D), suggesting that PKA phosphorylation does not alter PIP₂ modulation. Likewise, the slowing of deactivation caused by the phosphomimetic mutation is not dependent on the time after patch excision during which PIP₂ levels decrease (Fig. 5E), suggesting that PIP₂ does not affect the functional changes caused by PKA phosphorylation. These results suggest that, if a cross-talk between PKA and PIP₂ modulations of the I_Ks channel exists, it does not happen in the channel protein.

Fig. 5.

PIP₂ 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 (+40 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 +80 mV) and then repolarized to -40 mV (Right). (B) Time constant of deactivation vs. voltage and (C) G-V relation. WT KCNQ1 + KCNE1 (open circles), n = 6; S27D/S92D + KCNE1 (filled circles), n = 7. (D) Time dependence of normalized tail current amplitude after patch excision. WT KCNQ1 + KCNE1 (open circles), n = 12; S27D/S92D + KCNE1 (filled circles), n = 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).

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 PIP₂ (Table 1). Sequence alignment shows that the basic residues that are essential for KCNE1 modulation of I_Ks PIP₂ sensitivity are highly conserved across all members of the KCNE family of peptides (Fig. 6), suggesting that modulation of PIP₂ 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 differences in these α- and β-subunits, the putative interactions among KCNE β-subunits, channel α-subunits and PIP₂ may vary within different ion channel complexes, resulting in diverse effects of KCNE peptides.

Table 1.

K⁺ channel α-subunits that interact with KCNE families and show PIP₂ sensitivity

KCNE	α-subunit*	PIP2 sensitivity†
KCNE1	KCNQ1 (1, 2)	α + β (6)
	HERG (33)	α (34)
	Kv 4.3 (35)
KCNE2	KCNQ1 (36)	α + 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.

^†PIP₂ sensitivity of the channel either with the α-subunits alone (α) or with the α-subunits and a KCNE coexpression (α + β) has been reported.

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 PIP₂ sensitivity.

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 CaCl₂, 1 MgCl₂, 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 0.5–1.0 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 KMeSO₃, 20 Hepes, 2 KCl, and 2 MgCl₂, pH 7.2. The internal solution contained (in mM) 140 KMeSO₃, 20 Hepes, 2 KCl, 5 EGTA, 1.5 MgATP, and PIP₂ as indicated in the text, pH 7.2. PIP₂ (bovine brain, Sigma-Aldrich), diC8-, and diC4-PIP₂ (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:

[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).