KCNE1 and KCNE3 Modulate KCNQ1 Channels by Affecting

KCNE1 and KCNE3 modulate KCNQ1 channels by PNAS PLUS affecting different gating transitions

Rene Barro-Soriaa,1,2, Rosamary Ramentola, Sara I. Liina,3, Marta E. Pereza, Robert S. Kassb, and H. Peter Larssona,2

aDepartment of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, FL 33136; and bDepartment of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY 10032

Edited by Ramon Latorre, Universidad de Valparaíso, Valparaíso, Chile, and approved July 19, 2017 (received for review June 16, 2017) KCNE β-subunits assemble with and modulate the properties of on the activation of KCNQ1 channels is still debated. Here, we voltage-gated K+ channels. In the heart, KCNE1 associates with simultaneously track changes in voltage sensor movement and in the α-subunit KCNQ1 to generate the slowly activating, voltage- gate opening of KCNQ1/KCNE1 and KCNQ1/KCNE3 channels dependent potassium current (IKs) in the heart that controls the using voltage clamp fluorometry (VCF) to determine the differ- repolarization phase of cardiac action potentials. By contrast, in ences in the molecular mechanisms by which KCNE1 and KCNE3 epithelial cells from the colon, stomach, and kidney, KCNE3 coas- alter KCNQ1 channel gating. + sembles with KCNQ1 to form K channels that are voltage- Pore-forming KCNQ1 channel α-subunits form tetramers. Each + independent K channels in the physiological voltage range and α-subunit has six transmembrane (TM) segments (S1–S6) divided important for controlling water and salt secretion and absorption. into two functional domains: a voltage sensor domain comprising How KCNE1 and KCNE3 subunits modify KCNQ1 channel gating so four peripheral TM helices (S1–S4) and a centrally located pore differently is largely unknown. Here, we use voltage clamp fluor- domain (S5–S6) (14). The fourth TM segment, S4, has several pos- ometry to determine how KCNE1 and KCNE3 affect the voltage itively charged amino acids and has been shown to move and func- sensor and the gate of KCNQ1. By separating S4 movement and tion as the voltage sensor (15–19). In Kv channels, it is thought that gate opening by mutations or phosphatidylinositol 4,5-bisphos- the S4 movement upon membrane depolarization causes a lateral phate depletion, we show that KCNE1 affects both the S4 move- pull on the S4–S5 linker, which, in turn, is coupled to S6 and thereby ment and the gate, whereas KCNE3 affects the S4 movement and opens the S6 gate (14, 20–22). All five KCNE regulatory β-subunits only affects the gate in KCNQ1 if an intact S4-to-gate coupling is have only a single TM segment (23). Disulfide cross-linking studies present. Further, we show that a triple mutation in the middle of suggest that KCNE1 localizes between the voltage sensor domain the transmembrane (TM) segment of KCNE3 introduces KCNE1-like and the pore domain of KCNQ1 channels (24–26), such that KCNE1 effects on the second S4 movement and the gate. In addition, we could affect the voltage sensor movements, the pore, or the coupling show that differences in two residues at the external end of the between the voltage sensor and the pore. Because of the high ho- KCNE TM segments underlie differences in the effects of the dif- mology within the KCNEs family, it is likely that KCNE3 is posi- ferent KCNEs on the first S4 movement and the voltage sensor-to- tioned in the KCNQ1 channel structure in a similar location. gate coupling. However, how KCNE1 and KCNE3 differently affect the S4 and the pore in KCNQ1 channel to produce voltage-dependent KCNQ1 | KCNE1 | KCNE3 | Kv7.1 | voltage clamp fluorometry

+ Significance oltage-gated K (Kv) channels are mainly expressed in ex- + Vcitable cells, where changes in the voltage across the mem- Regulatory β-subunits associate with voltage-gated K channels brane, such as action potentials, demand rapid channel activation to modulate their biophysical properties and physiological roles. and deactivation. Among Kv channels, the KCNQ1 channel (also KCNE1 and KCNE3 β-subunits turn voltage-dependent KCNQ1 called Kv7.1 or KvLQT1) differs from most other Kv channels in channels into delayed activating KCNQ1/KCNE1 channels and PHYSIOLOGY that KCNQ1 plays key physiological roles in nonexcitable cells, apparent voltage-independent KCNQ1/KCNE3 channels, respec- such as in epithelia, in addition to its roles in excitable cells, such tively, which are important for cardiac action potentials and as cardiomyocytes. The KCNQ1 channels display dramatically transport of water and salts across epithelial cells. Mutations in different biophysical properties in various cell types, differences ’ KCNQ1/KCNE1 and KCNQ1/KCNE3 channels are associated with that are thought to be mainly due to the KCNQ1 channel s ability diseases, such as cardiac arrhythmias, congenital deafness, se- to associate with five tissue-specific KCNE β-subunits to form + – cretory diarrhea, and tinnitus. Therefore, KCNQ1, KCNE1, and different K channel complexes (1 7). KCNE3 are potential drug targets. We here propose a model for KCNQ1 subunits expressed by themselves form voltage-dependent + how KCNE1 and KCNE3 differentially modulate KCNQ1 that will K channels that open at negative voltages (1, 2) (Fig. 1 A and D, allow for a better understanding of how mutations in KCNQ1, black squares). However, coassembly of KCNQ1 with KCNE1 (also KCNE1, and KCNE3 cause diseases and how to design drugs to called MinK) produces a much slower activating potassium current treat these diseases. (IKs) in the heart that activates at positive voltages (Fig. 1 B and D, black triangles) and shapes the repolarization phase of cardiac Author contributions: R.B.-S., S.I.L., R.S.K., and H.P.L. designed research; R.B.-S., R.R., and action potentials (1, 2). Mutations in the KCNQ1/KCNE1 complex M.E.P. performed research; R.B.-S., R.R., and H.P.L. analyzed data; and R.B.-S. and H.P.L. are linked to life-threatening cardiac arrhythmias, such as torsade wrote the paper. de pointes (8, 9). Association of KCNQ1 with KCNE3 (also called The authors declare no conflict of interest. MiRP2) produces channels that are voltage-independent in the This article is a PNAS Direct Submission. physiological voltage range (Fig. 1 C and D, black circles) and that 1Present address: Division of Endocrinology, Diabetes and Metabolism, Department of Med- are crucial in regulating the transport of water and salt in several icine, Miller School of Medicine, University of Miami, Miami, FL 33136. 2To whom correspondence may be addressed. Email: [email protected] or plarsson@med. epithelial tissues, including the colon, small intestine, and airways miami.edu. (3, 10, 11). Therefore, the KCNQ1/KCNE3 channel could be a 3Present address: Department of Clinical and Experimental Medicine, Linköping University, potential drug target to treat life-threatening diseases, such as hu- SE-581 85 Linköping, Sweden. man colonic secretory diarrhea (12, 13). How the different acces- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sory β-subunits KCNE1 and KCNE3 mediate such different effects 1073/pnas.1710335114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1710335114 PNAS | Published online August 14, 2017 | E7367–E7376 Downloaded by guest on September 29, 2021 Fig. 1. KCNQ1 channel with and without KCNE β-subunit. Representative current (black) and fluorescence (red) traces from KCNQ1 (A), KCNQ1/KCNE1 (B), and KCNQ1/KCNE3 (C) channels for voltage steps between −200 mV (KCNE1: −180 mV) and +80 mV (KCNE3: +20 mV) in steps of 20 mV, from a holding potential of −80 mV followed by a −40-mV tail potential. In B, a prepulse to −140 mV was used to deactivate the S4s more completely. The dashed-dotted line represents zero current. (D) Normalized G(V) (black) and F(V) (red) from KCNQ1 (squares), KCNQ1/KCNE1 (triangles), and KCNQ1/KCNE3 (circles) channels (mean ± SEM; n = 5). Black and red lines are single Boltzmann fits from G(V) and F(V) of KCNQ1 and KCNQ1/KCNE3 and G(V) of KCNQ1/KCNE1; the F(V) of KCNQ1/KCNE1 was fit with a double Boltzmann equation. Black arrows indicate left (KCNQ1/KCNE3) and right (KCNQ1/KCNE1) shifts of the G(V) relative to the G(V) of KCNQ1 alone. (E) Topology of KCNQ1, KCNE3, and KCNE1. Residues mutated in this study are indicated.

KCNQ1/KCNE1 channels and voltage-insensitive KCNQ1/KCNE3 in contrast to KCNE3, which primarily affects the S4 segment and channels is still unclear. Using VCF, we recently showed that only affects the gate indirectly through the S4-to-gate coupling. KCNE3 shifts the equilibrium of the S4 movement of KCNQ1 to very negative voltages (Fig. 1D, red circles), thereby making KCNQ1/ Results KCNE3 channels voltage-independent in the physiological voltage KCNE1 Shifts both the Equilibrium of S4 Movement and Gate Opening, range (27) (Fig. 1D, black circles). We also showed that two nega- Whereas KCNE3 only Shifts the S4 Movement. Using VCF, we si- tively charged residues at the extracellular end of the KCNE3 TM multaneously monitor gate opening (by ionic current) and S4 segment are necessary for this voltage shift (27, 28). The effect of movement (by fluorescence) in KCNQ1, KCNQ1/KCNE1, and KCNE3 on the KCNQ1 gate is only present with an intact voltage KCNQ1/KCNE3 channels by labeling a cysteine introduced at po- sensor-to-gate coupling (27). We refer to effects by KCNE β-subunits sition 219 close to the S4 of KCNQ1 with Alexa Fluor 488 5-mal- that require an intact voltage sensor-to-gate coupling as indirect ef- eimide (32) (Fig. 1). We will refer to the labeled KCNQ1 subunit fects and effects that remain even in the absence of an intact voltage simply as KCNQ1. We use the mutation F351A (Fig. 1E)in sensor-to-gate coupling as direct effects. We therefore concluded KCNQ1 to elucidate the differences in the effects of KCNE1 and that the effect of KCNE3 on the KCNQ1 gate is only indirect and KCNE3 on the S4 movement and the gate. As we have shown that the effect of KCNE3 is transmitted from the voltage sensor to earlier (27), the F351A mutation separates the fluorescence versus thegatebythevoltagesensor-to-gate coupling (27). We showed in voltage [F(V)] curve and the conductance versus voltage [G(V)] other studies that KCNE1 shifts a component of the S4 movement of curve by 47 mV (Fig. 2B, compare red and black triangles). Because KCNQ1 to more negative voltages relative to that of KCNQ1 alone the mutation F351A separates the fluorescence change (due to (although to a lesser extent than KCNE3) (Fig. 1D, red triangles), S4 movement) from the ionic current change (due to movement of but shifts the gate opening to more positive voltages (29, 30) (Fig. the gate) in both time and voltage (30, 31, 33), F351A makes it 1D, black triangles). In KCNQ1/KCNE1 channels, the S4 movement β occurs in two steps (Fig. 1D, red triangles; also see Fig. 3E, Top): The easier to distinguish effects of the different KCNE -subunits on the first step occurs at negative voltages and correlates with the main S4 movement and/or the gate. We have previously shown (27) that gating charge movement, whereas the second step occurs at positive KCNE3 does not modify the G(V) relation of KCNQ1-F351A (Fig. voltages and correlates with channel opening (29). The molecular 2B, compare black circles and triangles), but left-shifts a large mechanism of how KCNE1 affects KCNQ1 is not completely un- fraction of the F(V) relation of KCNQ1-F351A (Fig. 2B,redcircles derstood. However, a recent study proposed that all of the effects of and red arrow). Note that the F(V) relation of KCNQ1-F351A/ KCNE1 are on the voltage sensor-to-gate coupling in KCNQ1 (31). KCNE3 channels exhibits two components: one fluorescence com- For example, it was shown that the KCNQ1 channel opens from an ponent that is similarly shifted to negative voltages as the F(V) in intermediate S4 position (after the first S4 step), whereas KCNQ1/ KCNQ1/KCNE3 channels and a second fluorescence component at KCNE1 channels open from a fully activated S4 position (31). positive voltages that concurs with the G(V) relation of KCNQ1- Using VCF, we here find that KCNE1 affects both the S4 F351A/KCNE3 channels (Fig. 2B). KCNE3 apparently affects S4 movement and the gate, independent of the S4-to-gate coupling, movement preceding the opening of the gate, but not gate opening

E7368 | www.pnas.org/cgi/doi/10.1073/pnas.1710335114 Barro-Soria et al. Downloaded by guest on September 29, 2021 due to indirect effects on S4 movement and the gate caused by PNAS PLUS effects of KCNE1 on the voltage sensor-to-gate coupling in KCNQ1, as suggested recently (31).

KCNE1 Acts Directly on the Voltage Sensor. We test these alternative possibilities by depleting phosphatidylinositol 4,5-bisphosphate (PIP2) in KCNQ1/KCNE1 channels. PIP2 depletion has been suggested to decouple S4 movement from gate opening in KCNQ1 channels because after PIP2 has been depleted, S4 movement still occurs but without opening the gate (34). There- fore, by depleting PIP2, we can measure any potential direct effect of KCNE1 on S4. We previously used this approach to show that KCNE3 directly affects the S4 movement in KCNQ1 (27). We coexpress KCNQ1 and KCNE1 with the voltage-sensing phosphatase from Ciona intestinalis (CiVSP) that, when activated, can deplete PIP2. We record the ionic current during (Fig. 3B) and the ionic current and fluorescence after (Fig. 3C) PIP2 has been depleted by repeated depolarizing pulses to +40 mV to activate CiVSP and compare these with the ionic current and fluorescence from cells expressing only KCNQ1/KCNE1 channels (Fig. 3A). Activation of CiVSP eliminates KCNQ1/KCNE1 ionic current, but does not eliminate the fluorescence change from the KCNQ1/KCNE1 voltage sensors (compare Fig. 3 A and C, red and blue traces). The F(V) relations from KCNQ1/ KCNE1 channels are similar in the presence and absence of PIP2: The first components of both F(V) relations are left-shifted to a similar extent compared with the F(V) relation of KCNQ1 channels alone (Fig. 3D). We also measured the effect of depleting PIP2 in KCNQ1- Fig. 2. KCNE1 shifts both S4 and gate, and KCNE3 shifts only the S4 move- F351A/KCNE1 channels. Similar to wild-type (wt) KCNQ1/ ment in F351A mutants. Representative current (black) and fluorescence (red) KCNE1, activation of CiVSP by a train of depolarizing pulses to traces from KCNQ1-F351A/KCNE3 (A; Q1-F351A/E3) and KCNQ1-F351A/KCNE1 +40 mV eliminates the ionic current of KCNQ1-F351A/KCNE1 (C; Q1-F351A/E1) channels for the indicated voltage protocol are illustrated. − − channels, but does not eliminate the fluorescence changes Currents are only shown for voltage steps down to 140 mV in A and 160 mV – in C. The dashed-dotted line represents zero current. (B) Normalized G(V) reporting on S4 movement (Fig. S1 A C). The KCNE1-induced (black circles) and F(V) (red circles) of KCNQ1-F351A/KCNE3 channels (mean ± left shift of the first fluorescence component and right shift of SEM; n = 7).Fig.2B reprinted with permission from ref. 27. (D) Relative G(V) the second fluorescence component in the KCNQ1-F351A mu- (black squares) and F(V) (red squares) of KCNQ1-F351A/KCNE1 (mean ± SEM; tant are maintained after PIP2 depletion (Fig. S1D, red and blue n = 5). The G(V) is scaled so that the slope of the G(V) is similar to the slope of arrows, respectively). the G(V) of KCNQ1-F351A/KCNE3 in B, and the F(V) is scaled to make the first The persisted shifts in the F(V) relations even in the absence component similar to the F(V) of KCNQ1-F351A/KCNE3 in B. For comparison, of PIP2 show that KCNE1 does not act on the S4 movement the G(V) (black) and F(V) (red) curves of KCNQ1/KCNE3 (Q1/E3) channels indirectly by acting on the voltage sensor-to-gate coupling or (dashed-dotted lines) and KCNQ1-F351A (Q1-F351A) channels expressed alone (triangles) are shown. Black and red arrows indicate the shifts in the G(V) and through a mechanism dependent on the voltage sensor-to-gate

F(V), respectively, relative to those of KCNQ1-F351A alone. coupling (Fig. 3E). These data also show that the second fluo- PHYSIOLOGY rescence component in KCNQ1/KCNE1 channels is not dependent on gate opening or on voltage sensor-to-gate coupling. or a minor component of S4 movement associated with gate We propose that the second fluorescence component in KCNQ1/ opening in KCNQ1-F351A (Fig. 2B). KCNE1 channels reports on a second S4 movement that pre- We here similarly test whether KCNE1 affects the S4 move- cedes and triggers channel opening, as we and others have pre- ment, the gate, or both in the mutant F351A, which clearly sepa- viously suggested for KCNQ1/KCNE1 channels (30, 31) and rates the S4 movement from the ionic currents. In contrast to Shaker K channels (35). Therefore, we propose that the observed KCNE3, KCNE1 has effects on both the F(V) and the G(V) in KCNE1-induced right shift of the G(V) is, at least partly, due to KCNQ1-F351A channels (Fig. 2 C and D). KCNE1 right-shifts the the KCNE1-induced right shift of the second fluorescent com- G(V) (Fig. 2D, black arrow) and left-shifts the main component of ponent. All these data show that the effects of KCNE1 on the the F(V) (Fig. 2D, red arrow) compared with homomeric KCNQ1- S4 movement are preserved in the absence of an intact voltage F351A channels. Because KCNE1 shifts the G(V) and the small sensor-to-gate coupling and gate opening, as if KCNE1 acts di- component of the F(V) to the right and the main component of the rectly on the S4 movement. F(V) to the left, this suggests that KCNE1 has multiple effects on KCNE1 Acts Directly on the Gate. Does KCNE1 also directly affect KCNQ1 channels. Three alternatives may explain the effects of the gate of KCNQ1 channels? We test this by using the KCNQ1- KCNE1: (i) KCNE1 directly affects both S4 and the gate of I268A mutation that causes large constitutive currents at negative KCNQ1 channels, but with opposite effects on their voltage de- voltages (Fig. 4A,redarrowandFig.4E, black circles) in the pendences, and that the effect on the gate indirectly shifts the absence of KCNE1 (36). At these negative voltages, KCNQ1- second, smaller F(V) component; (ii) KCNE1 directly shifts the I268A channels have their voltage sensors (S4s) in their resting main component of the F(V) to the left and the second component position (32). Two alternatives may explain why the I268A mu- of the F(V) to the right, and that the shift of the second component tation causes channel opening at these negative voltages: The of the F(V) then indirectly shifts the voltage dependence of the I268A mutation might (i) directly affect the gate or (ii) affect the gate opening; or (iii) the shifts of the F(V) and G(V) relations of S4-to-gate coupling, so that the gate can open even if the S4s are KCNQ1/KCNE1 channels relative to KCNQ1 alone (Fig. 1D)are in their resting position. To distinguish between these alternatives,

Barro-Soria et al. PNAS | Published online August 14, 2017 | E7369 Downloaded by guest on September 29, 2021 Fig. 3. Decoupling S4 and gate by PIP2 depletion: KCNE1 still left-shifts the S4 movement. (A) Representative current (black) and fluorescence (red) traces from KCNQ1/KCNE1 channels for the indicated voltage protocol. (B) Currents from KCNQ1/KCNE1 channels during PIP2 depletion by activation of the CiVSP by stepping 18-fold to +40 mV for 5 s from a holding voltage of −80 mV, followed by a pulse to −40 mV. (C) Representative current (black) and fluorescence (blue) traces from KCNQ1/KCNE1 channels after activation of VSP. In A and C, currents are only shown for voltage steps down to −160 mV. (D) Normalized

G(V) and F(V) in the presence of VSP after PIP2 depletion (filled black and blue squares) compared with G(V) and F(V) in the absence of VSP (open black and red circles) (mean ± SEM; n = 5). The midpoints of the first component of the F(V) fits of KCNQ1/KCNE1 with (red) and without (blue) PIP2 are −103.01 ± 4mV and −102.8 ± 3 mV, respectively (n = 8). Dashed lines show the G(V) (black) and F(V) (red) curves of KCNQ1 expressed alone for comparison. (E) Cartoon

showing that even in the case where S4 movement is decoupled from gate opening by removing PIP2 (represented by the interrupted connection between S4 and S6), KCNE1 still shifts the first S4 movement to very negative voltages. S4 (red), KCNE1 (pink), S6 (purple), and Alexa Fluor 488 5-maleimide (green) are illustrated. For simplicity, only two of the four subunits in the tetrameric channel are shown.

we decouple the S4 movement from the gate in KCNQ1-I268A KCNQ1-I268A removes the constitutive currents of KCNQ1- channel by PIP2 depletion. We record the ionic current of I268A channels at negative voltages (i.e., KCNE1 closes the KCNQ1-I268A channel after PIP2 has been depleted by CiVSP channel) (Fig. 4 C and E, blue). KCNE1 could do this either by (Fig. 4 B and E, green) and compare this with the ionic current directly acting on the channel gate or by indirectly acting on the from cells expressing only KCNQ1-I268A channel without CiVSP gate through the S4-to-gate coupling. To distinguish between (Fig. 4 A and E, black). In wt KCNQ1, PIP2 depletion removes the these alternatives, we decouple the S4 movement from the gate in ionic currents (i.e., closes the channel) without altering the KCNQ1-I268A/KCNE1 channel by PIP2 depletion (Fig. 4 D and S4 movement, as if PIP2 depletion removes the coupling between E, maroon) and compare the ionic current with the ionic current S4 movement and channel opening (34, 37). In KCNQ1-I268A from cells expressing only KCNQ1-I268A channels (Fig. 4 B and channels, PIP2 depletion eliminates the voltage-dependent part of E, green). Coexpression with KCNE1 closes KCNQ1-I268A the currents (Fig. 4A, Inset, black arrow) without eliminating the channels even in the case when S4 movement is decoupled from constitutive currents (Fig. 4A, red arrow) at negative voltages the gate, as if KCNE1 directly affects the gate of KCNQ1 channels (compare Fig. 4 A and B and compare Fig. 4E, black and green). (Fig. 4 D and E, maroon and Fig. 4F, Bottom Right). These data show that the I268A mutation (in the absence of KCNE1) destabilizes the closed state relative to the open state “KCNE1-Like” KCNE3 also Affects the Second Component of S4 when S4 is in the resting state and when S4 is decoupled from the Movement and the Gate. It has previously been shown that gate (Fig. 4F, Top Right). substituting three residues in the TM segment of KCNE3 by the ho- We therefore reason that if KCNE1 directly affects the gate of mologous KCNE1 residues (i.e., the triple mutation T71F-V72T- KCNQ1 channels, it might modify these constitutive currents at G73L,whichwewillrefertoasKCNE3-FTL)convertsapparently negative voltages in KCNQ1-I268A. Coexpressing KCNE1 with constitutively conducting KCNQ1/KCNE3 channels into voltage-gated

E7370 | www.pnas.org/cgi/doi/10.1073/pnas.1710335114 Barro-Soria et al. Downloaded by guest on September 29, 2021 KCNE1 channels, the first fluorescence component correlates PNAS PLUS with the gating currents (29). In KCNQ1/KCNE3-FTL, the first fluorescence component is left-shifted compared with KCNQ1/ KCNE1 channels (KCNQ1/KCNE3-FTL: FV1/2 = −127 ± 2mV and KCNQ1/KCNE1: FV1/2 = −103 ± 7mV;Fig.5B, compare red dashed and solid lines), and the F(V) relation is somewhere between that of KCNQ1/KCNE3 and KCNQ1/KCNE1 channels (Fig. 5B, red circles between red dashed-dotted and dashed lines). In contrast, the G(V) relation of KCNQ1/KCNE3-FTL channels is shifted toward that of KCNQ1/KCNE1 channels (KCNQ1/KCNE3- FTL: GV1/2 = −15 ± 3 mV and KCNQ1/KCNE1: GV1/2 =+26 ± 6mV;Fig.5B, black arrow, compare black solid and dashed lines). Similar results are obtained from coexpression of KCNE3-FTL with KCNQ1-F351A (Fig. 5 C and D): The G(V) and both components of the F(V) relation of KCNQ1-F351A/KCNE3 are right-shifted by the FTL mutation (Fig. 5D, black and blue arrows). The effect of the mutated β-subunit KCNE3-FTL on KCNQ1-F351A is very similartotheeffectofKCNE1onKCNQ1-F351A(compare dashed arrows in Figs. 2D and 5D). The KCNE3-FTL–induced right shift of the second fluorescence component in KCNQ1 and KCNQ1-F351A is maintained after PIP2 depletion (Fig. S2,blue arrows), just as for the KCNE1-induced shifts of the second fluorescence component. These results show that the FTL residues introduce an effect on the voltage sensor movement of KCNQ1 channels, even when the coupling of the voltage sensor and the gate Fig. 4. KCNE1 directly affects the gate. Representative current traces from are eliminated by PIP2 depletion. KCNQ1-I268A channels without (A) and with (C) KCNE1 for the indicated Do FTL residues also introduce an effect on the gate? Coex- voltage protocol are shown. Currents from KCNQ1-I268A channels without pressing KCNE3-FTL with KCNQ1-I268A removes the constitu- (B)andwith(D) KCNE1 for the indicated voltage protocol after activation of VSP are shown. We stepped 18-fold to +40 mV for 5 s from a holding voltage of tive currents of KCNQ1-I268A channels at negative voltages (i.e., KCNE3-FTL closes the channel) (Fig. 5 E and F, circles). KCNE3- −80 mV to deplete PIP2.(A, Inset) Tail currents during channel closing at higher magnification. Red-dotted lines represent zero current. Experiments are per- FTL closes KCNQ1-I268A channels similar to KCNE1, as if + formed in 98 mM extracellular potassium (high K )(Methods); cells are held at KCNE3-FTL directly affects the gate of KCNQ1 channels (com- −80 mV and pulsed to voltages between −160 and +60 mV for 2 s, followed by pare Figs. 4E and 5F). 2-s pulses to −100 mV, to measure tail currents. Red arrows in A and B indicate Together, these results show that KCNE1-like effects on the the constitutive currents at negative voltages. (E) Normalized G(V)s from tail gate and second component of the S4 movement have been in- currents of KCNQ1-I268A (filled black circles, n = 4) and KCNQ1-I268A/KCNE1 = troduced by the FTL triple mutation and that both of these effects (open blue circles, n 5) in the presence of PIP2 (without CiVSP) and relative G(V)s are independent of an intact voltage sensor-to-gate coupling, as of KCNQ1-I268A (filled green squares, n = 4) and KCNQ1-I268A/KCNE1 (filled these effects are preserved in PIP2-depleted conditions. maroon squares, n = 5) after PIP2 depletion (after activation of CiVSP). The G(V)s of KCNQ1-I268A and KCNQ1-I268A/KCNE1 after PIP2 depletion were scaled by normalizing the currents to the maximum tail current amplitude in A and C, Converting KCNE3 into KCNE1. The FTL mutation right-shifts the respectively. (F) Cartoon of KCNQ1-I268A channel with and without KCNE1 in G(V) and the F(V) of KCNQ1/KCNE3 toward those of KCNQ1/

the presence and absence of PIP2.(Top Left) In the presence of PIP2,KCNQ1- KCNE1 channels (Fig. 5B, arrows). However, the G(V) and the I268A channels are leaky when S4 is in its resting position at negative voltages, F(V) of KCNQ1/KCNE3-FTL channels are still more left- PHYSIOLOGY but open more at depolarized voltages. (Top Right) In the absence of PIP2, shifted compared with the F(V) of KCNQ1/KCNE1 (Fig. 5B, KCNQ1-I268A channels are not voltage-dependent and are leaky at both neg- compare solid and dashed lines). We have previously shown that ative and positive voltages. (Bottom Left) In the presence of PIP2, KCNQ1-I268A/ the D54N-D55N mutation in KCNE3 shifts the G(V) and F(V) KCNE1 channels are closed when S4 is in its resting position at negative voltages, of KCNQ1/KCNE3 channels to more depolarized voltages by but open more at depolarized voltages. (Bottom Right) In the absence of PIP , 2 removing an electrostatic interaction between D54–D55 and the KCNQ1-I268A/KCNE1 channels are closed at both negative and positive voltages. – S4 (red), KCNE1 (pink), and S6 (purple) are illustrated. For simplicity, only two of positive S4 (27). Therefore, we reason that the D54N D55N the four subunits in the tetrameric channel are shown. mutation (Fig. 1E) will further shift the G(V) and F(V) curves of KCNQ1/KCNE3-FTL toward those of KCNQ1/KCNE1. The G(V) and F(V) curves of KCNQ1/KCNE3-FTL-D54N-D55N channels with ionic currents similar to those in KCNQ1/ channels are shifted toward those of KCNQ1/KCNE1 channels KCNE1 channels (38). The mechanism underlying these changes (Fig. 5H, red and black arrows). The midpoints of the major in channel gating caused by this triple mutation is not completely F(V) and G(V) fits for KCNQ1/KCNE1 and KCNQ1/KCNE3- understood. It is not known whether the FTL mutations affect FTLNN channels are similar (the FV1/2 and GV1/2 of KCNQ1/ S4 movement, gate movement, or the coupling between S4 and KCNE3-FTLNN are −116 ± 7 mV and +8 ± 4 mV, respectively; the gate. the FV1/2 and GV1/2 of KCNQ1/KCNE1 are −103 ± 7 mV and We measure ionic current and fluorescence of cells expressing +26 ± 6 mV, respectively). Therefore, neutralizing D54 and KCNQ1/KCNE3-FTL channels (Fig. 5 A and B)todetermine D55 at the external end of the TM segment of KCNE3 more whether the effects of KCNE3-FTL on voltage sensor movement completely converts the effect of KCNE3 into that of KCNE1. and gate opening resemble the effects of KCNE3 or KCNE1. Similar to the F(V) of KCNQ1/KCNE1 channels (29) (Fig. 5B, FTL Residues Interact with F339 in S6. Previous studies showed that dashed red line), the F(V) relation in KCNQ1/KCNE3-FTL chan- mutations in the S6 of KCNQ1 mimic or restore the function of nels comprises two components, with a first, major fluorescence mutations in the FTL residues in KCNE1, suggesting that FTL of component at more negative voltages and a second, minor fluo- KCNE1 interacts with S6 of KCNQ1 channels (38–41). Espe- rescence component at more depolarized voltages, in a similar cially, F339 in S6 of KCNQ1 (Fig. 1E) has been suggested to voltage range as gate opening (Fig. 5B, red circles). In KCNQ1/ interact with the FTL residues (41). We therefore introduce the

Barro-Soria et al. PNAS | Published online August 14, 2017 | E7371 Downloaded by guest on September 29, 2021 Fig. 5. Interconversion of KCNE3 into KCNE1. Representative current (black) and fluorescence (red) traces from KCNQ1/KCNE3-FTL (A; Q1/E3-FTL), KCNQ1- F351A/KCNE3-FTL (C; Q1-F351A/E3-FTL), KCNQ1-I268A/KCNE3-FTL (E; Q1-I268A/E3-FTL), and KCNQ1/KCNE3-FTLNN (G; Q1/E3-FTLNN) channels are shown. Cells are held at −80 mV and stepped between −200 and +100 mV for 5 s, followed by a pulse to −40 mV to record tail currents. Currents are only shown for voltage steps down to −120 mV (A), −160 mV (C and E), and −140 mV (G). The dashed line in E depicts zero current. Normalized G(V) (black) and F(V) (red) of KCNQ1/KCNE3-FTL (B; n = 7), KCNQ1-F351A/KCNE3-FTL (D), KCNQ1-I268A/KCNE3-FTL (F; n = 5), and KCNQ1/KCNE3-FTLNN (H; n = 5) are shown (mean ± SEM). For comparison, the G(V) (black) and F(V) (red) curves of KCNQ1/KCNE3 (Q1/E3) channels (dashed-dotted lines in B and H), KCNQ1/KCNE1 (Q1/E1) channels (dashed lines in B and H), and KCNQ1/KCNE3-FTL (Q1/E3-FTL) channels (solid thin lines in H) are shown. Thick black and red arrows indicate the shifts of the G(V) and F(V) induced by the mutations FTL (B) and NN (H), relative to the G(V) and F(V) of KCNQ1/KCNE3 (B) and KCNQ1/KCNE3-FTL (H). Thin arrows in H show the shifts induced by the mutation FTL relative to KCNQ1/KCNE3 as in B.InD, the G(V) is scaled so that the slope of the G(V) is similar to the slope of the G(V) of KCNQ1-F351A/KCNE3, and the F(V) is scaled to make the first component similar to the F(V) of KCNQ1-F351A/KCNE3. For comparison, the G(V) (black) and F(V) (red) curves of KCNQ1-F351A channels (open triangles) and KCNQ1-F351A/KCNE3 (Q1-F351A/E3) channels (solid thin lines) are shown in D. Solid arrows in D indicate the shifts of the G(V) (black), the major component of the F(V) (red), and the minor component of the F(V) (blue) induced by the mutation FTL, relative to the G(V) and F(V) of KCNQ1-F351A/KCNE3. Black and red dashed arrows indicate the shifts in the G(V) and F(V), respectively, induced by the mutated β-subunit KCNE3-FTL relative to KCNQ1-F351A. (F) Normalized G(V) from tail currents of KCNQ1-I268A/KCNE3-FTL (filled black circles; n = 6).

For comparison, the G(V)s (dashed lines) of KCNQ1-I268A (gray) and KCNQ1-I268A/KCNE1 (blue) in the presence of PIP2 (without CiVSP) and KCNQ1-I268A (green) and KCNQ1-I268A/KCNE1 (maroon) after PIP2 depletion (after activation of CiVSP) are shown.

mutation F339A into KCNQ1-I268A channels (KCNQ1-I268A- channels (Fig. 7 A and B, black circles). However, both the G(V) F339A) to test whether the introduction of the FTL residues in and the F(V) relations of KCNQ1/KCNE1-TVG are shifted to- KCNE3 closes the KCNQ1 gate at negative voltages (Fig. 5 E and ward those of KCNQ1/KCNE3 (Fig. 7B, black and red arrows). F) by interacting with F339. KCNQ1-I268A-F339A channels have If we also make the double mutations G40D and K41D in similar constitutive currents at negative voltages as KCNQ1-I268A KCNE1-TVG to introduce aspartate residues in KCNE1 at the channels (Fig. 6 A and C, black circles). We reason that if FTL corresponding positions of D54 and D55 in KCNE3 (Fig. 1E), residues interact with F339 to close the gate of KCNQ1, the the G(V) is further shifted toward that of KCNQ1/KCNE3 (Fig. KCNE3-FTL mutation will not be able to reduce the constitutive 7 C and D, black circles and black arrow) and the separation of currents of KCNQ1-I268A-F339A channels at negative voltages. the F(V) and G(V) is decreased and approaching that in Coexpression of KCNQ1-I268A-F339A with KCNE3-FTL does KCNQ1 (Fig. 1D) and KCNQ1/KCNE3 channels (Fig. 7D, cor- not reduce the large constitutive currents at negative voltages (Fig. respondence of black and red circles). KCNE1 restricts KCNQ1 6 B and C, red circles), as if FTL in KCNE3 affects the KCNQ1 opening so that KCNQ1/KCNE1 channels open only from the gate through interactions with F339 in S6. fully activated S4 conformation (i.e., after the second S4 movement), whereas KCNQ1 expressed alone is thought to open from Converting KCNE1 Toward KCNE3. To see whether we can also an intermediate S4 conformation (31). However, the G40D/ convert the effect of KCNE1 into that of KCNE3, we substitute K41D mutation allows gate opening at voltages more negative the corresponding triple residues in KCNE1 into their KCNE3 than those necessary for reaching the fully activated S4 confor- counterparts (i.e., the triple mutation F57T-T58V-L59G, which mation (Fig. 7D). This suggests that these mutations in KCNE1 we will refer to as KCNE1-TVG) in an effort to remove the alter the voltage sensor-to-gate coupling, thereby allowing gate effect of KCNE1 on the second fluorescence component and the opening from an intermediate S4 conformation. gate (Fig. 7 A and B). As previously reported (40), the triple To test whether KCNE1-TVGDD alters the voltage sensor-to- + + mutation KCNE1-TVG fails to completely convert KCNQ1/ gate coupling, we measure the permeability ratios of K and Rb KCNE1 channels into constitutively open KCNQ1/KCNE3-like for KCNQ1/KCNE1 and KCNQ1/KCNE1-TVGDD channels

E7372 | www.pnas.org/cgi/doi/10.1073/pnas.1710335114 Barro-Soria et al. Downloaded by guest on September 29, 2021 and KCNQ1/KCNE1-TVGDD channels look very similar in a PNAS PLUS physiological voltage range, if the holding potential is slightly more positive (Fig. S4). However, additional mutations are clearly necessary to completely convert KCNE1 into KCNE3. Together, these results show that the effect of KCNE1 on the major component of S4 movement persists in KCNE1-TVGDD, whereas the additional KCNE1-like effects on the second S4 movement and the gate have been removed by the TVGDD mutation. Discussion Our results suggest that the main difference between the effects of KCNE1 and KCNE3 on KCNQ1 is that KCNE1 affects both the voltage-sensing domain and the gate, whereas KCNE3 primarily affects the voltage-sensing domain and only indirectly affects the gate through an intact voltage sensor-to-gate coupling. Furthermore, our data suggest that three residues in the Fig. 6. FTL residues interact with F339 in S6. Representative current traces middle of the TM segments of KCNE1 and KCNE3 determine from KCNQ1-I268A-F339A (A) and KCNQ1-I268A-F339A/KCNE3-FTL (B)chan- whether these KCNE β-subunits affect the gate and the second nels for the indicated voltage protocol. Dotted lines represent zero current. + S4 movement that triggers gate opening, but does not signifi- Experiments are performed in 98 mM extracellular potassium (high K ) cantly control the effects of these two KCNEs on the first, major (Methods), and cells are held at −80 mV and pulsed to voltages between −160 and +60 mV for 2 s, followed by 2-s pulses to −100 mV, to measure tail S4 movement of KCNQ1 (Figs. 5 and 7). We also show that two currents. (C) Normalized G(V)s from tail currents of KCNQ1-I268A-F339A (filled residues at the external end of the TM segment of KCNE1 are black circles, n = 6) and KCNQ1-I268A-F339A/KCNE3-FTL (filled red circles, n = necessary for the KCNE1-induced changes in voltage sensor-to- 5). Black arrows indicate the constitutive currents at negative voltages. For gate coupling that only allow for KCNQ1/KCNE1 channel comparison, the G(V)s of KCNQ1-I268A-F339A (dashed line) and KCNQ1-I268A/ opening from the fully activated S4 position (Fig. 7). KCNE3-FTL (solid line) are shown. We propose the following model to explain our data. KCNE3 shifts the S4 movement to more negative voltages via an interaction between D54/D55 in KCNE3 and R228 in KCNQ1. (Fig. 7 E and F). It has been previously shown that KCNQ1 + + The shift in the voltage dependence of S4 movement indirectly channels have a higher Rb /K permeability ratio than KCNQ1/ + + shifts the G(V) to more negative voltages via the S4-to-gate KCNE1 channels (42). This higher Rb /K permeability ratio in coupling (27, 28) (Fig. 7G). In contrast, KCNE1 has several ef- KCNQ1 channels has been suggested to be due to channel opening fects on KCNQ1 (Fig. 7G): from an intermediate S4 position, which puts the KCNQ1 pore in a + + conformation that permeates Rb better than K (31). We find that i) We propose that G40/K41 in KCNE1 interacts with residues + + KCNQ1/KCNE1-TVGDD channels have a similar Rb /K per- at the top of S1 in KCNQ1 (e.g., V141; Fig. S5) and that this meability ratio as previously found for KCNQ1 (31) (Fig. 7E, Middle interaction alters the S4-to-gate coupling in KCNQ1, such that and Fig. 7F, white and hashed bars). However, KCNQ1/KCNE1- KCNQ1/KCNE1 channels only open from a fully activated + + TVGDD channels have a significantly increased Rb /K perme- S4 conformation. ability ratio compared with KCNQ1/KCNE1 channels (Fig. 7E, Top ii) In addition, the FTL residues in KCNE1 interact with and Middle and Fig. 7F,blackandwhitebars),consistentwiththat F339 in KCNQ1 to reduce the open probability at negative of KCNQ1/KCNE1-TVGDD channels open from an intermediate voltages. Both of these effects would contribute to the

S4 conformation. This suggests that KCNQ1/KCNE1 and KCNQ1/ KCNE1-induced G(V) shift to more positive voltages. PHYSIOLOGY KCNE1-TVGDD channels open from different S4 conformations, iii) FTL residues also have an effect on the second S4 move- consistent with the finding that the G40D/K41D mutation alters ment that shifts the second fluorescence component to more the voltage sensor-to-gate coupling. positive voltages and indirectly shifts the G(V) to more positive voltages. This effect of FTL on S4 could be direct or To further test whether the effect of the G40D/K41D mutation is – due to the removal of the G and K amino acids at positions 40 and indirect through effects on S6 (but not via the S4 S5 linker). iv) KCNE1 also has an effect on the first component of S4 41 or the introduction of the two D amino acids at positions 40 and movement through a yet unknown mechanism. 41, we measure the effect of the mutation G40N/K41N. Similar to KCNQ1/KCNE1-TVGDD, KCNQ1/KCNE1-NN channels have a This model of the effects of KCNE1 and KCNE3 on KCNQ1 + + significantly increased Rb /K permeability ratio compared with can explain most of the data we have presented here (Fig. S6) KCNQ1/KCNE1 channels, as if G40/K41 is necessary for the and is consistent with the recent cryo-EM structure of the Xen- KCNE1-induced change in voltage sensor-to-gate coupling of opus KCNQ1 channel (43) (Fig. S5). KCNQ1 (Fig. 7E, Top and Bottom and Fig. 7F, black and gray bars). A previous study suggested that the effects of KCNE1 on We also measured the effects of coexpressing KCNE1-TVGDD KCNQ1 could be explained solely by effects on the voltage sensor-to- with KCNQ1-F351A (Fig. S3) to see whether KCNE1-TVGDD gate coupling (31). However, both KCNE1 and KCNE3 appear to acts similar to KCNE1 or KCNE3 on KCNQ1-F351A channels. directly affect the S4 movement of KCNQ1, because separation of The G(V) and the second component of the F(V) relation of S4 movement and gate opening by the mutation F351A or by re- KCNQ1-F351A/KCNE1 are both left-shifted by the TVGDD mu- moval of PIP2 still allows KCNE1 and KCNE3 to induce a substantial tation (Fig. S3B, black and blue arrows) compared with KCNQ1- left shift of the first component of the F(V) relation to negative F351A/KCNE1 channels. The G(V) of KCNQ1-F351A/KCNE1- voltages and allows KCNE1 to induce a substantial right shift of the TVGDD is more similar to the G(V) of KCNQ1-F351A/KCNE3 second component of the F(V) relation to positive voltages. So, in than to that of KCNQ1-F351A/KCNE1 (Fig. S3B, compare black addition to the previous suggested KCNE1-induced change in voltage circles and black dashed-dotted lines), suggesting that the KCNE1- sensor-to-gate coupling (31), KCNE1 must have an effect on the TVGDD acts more similar to KCNE3 than to KCNE1 on KCNQ1 S4 movement independent of the voltage sensor-to-gate coupling. channels. Furthermore, the currents from KCNQ1/KCNE3 channels Therefore, we suggest that the observed KCNE1-induced G(V) shift

Barro-Soria et al. PNAS | Published online August 14, 2017 | E7373 Downloaded by guest on September 29, 2021 Fig. 7. Interconversion of KCNE1 into KCNE3. Representative current (black) and fluorescence (red) from KCNQ1/KCNE1-TVG (A; Q1/E1-TVG) and KCNQ1/ KCNE1-TVG-G40D-K41D (C; Q1/E1-TVGDD) channels for the indicated voltage protocol. Normalized G(V) (black circles) and F(V) (red circles) of KCNQ1/KCNE1- TVG (B; n = 5) and KCNQ1/KCNE1-TVGDD (D; n = 8) channels (mean ± SEM). For comparison, the G(V) (black) and F(V) (red) curves of KCNQ1/KCNE3 (Q1/E3) (dashed-dotted lines), KCNQ1/KCNE1 (Q1/E1) (dashed lines), and KCNQ1/KCNE1-TVG (thin lines in D) are shown. Thick black and red arrows indicate the shifts of the G(V) and F(V) induced by the mutations TVG (B) and DD (D), relative to the G(V) and F(V) of KCNQ1/KCNE1 (B) and KCNQ1/KCNE1-TVG (D). Thin arrows in D show the shifts induced by the mutation TVG relative to KCNQ1/KCNE1 as in B.(E) Representative currents from cells expressing KCNQ1/KCNE1 (Top), KCNQ1/KCNE1-TVGDD (Middle), and KCNQ1/KCNE1-G40N-K41N (Bottom) in external solutions containing 100 mM Na+ (gray), K+ (black), or Rb+ (red). Cells + are held at −80 mV and pulsed to +60 mV for 2 s, followed by a 2-s pulse to −60 mV, to measure tail currents. For control, currents in regular 100 mM Na + + + ND96 solution (Na , gray) were measured. (F) Summary of Rb /K permeability ratios calculated by comparing the tail current amplitudes (mean ± SEM; n = 7; + + *P < 0.05). n.s., not significant. For comparison, the Rb /K permeability ratio of KCNQ1 channel is shown from an earlier study (31). (G) Proposed effects of KCNE3 and KCNE1 on different domains or residues of KCNQ1 and the functional consequences of these interactions.

is, at least partly, due to the KCNE1-induced shift of the second differences in the triplet of residues in the middle of the TM S4 movement (i.e., the second fluorescent component). segments of KCNE1 (i.e., FTL) and KCNE3 (i.e., TVG) de- We show that KCNE1 eliminates the constitutive currents at termine the differences in the effects of these two accessory negative voltages in KCNQ1-I268A channels, as if KCNE1 also subunits (38). However, another study concluded that the TM directly affects the gate. In addition, when decoupling the segment of KCNE3 plays an “active” role in KCNQ1 modulation S4 movement from the gate in KCNQ1-I268A channels by PIP2 and that the C terminus of KCNE3 is not important (45), depletion, KCNE1 completely removes the remaining constitutive whereas the TM segment of KCNE1 plays only a “passive” role currents at negative voltages (it closes the channel), showing that and that the C-terminal domain of KCNE1 controls KCNQ1 KCNE1 also directly affects the gate of KCNQ1 channels in- channel modulation (45). We here show that deleting the dependent of the voltage sensor-to-gate coupling. In contrast, C terminus of KCNE3 does not affect KCNQ1/KCNE3-FTL KCNE3 does not modify the G(V) relation of KCNQ1-F351A channel gating (Fig. S7), indicating that for a KCNQ1/KCNE1- channels (27) (Fig. 2B), suggesting that an intact coupling be- like channel, such as KCNQ1/KCNE3-FTL, the effect of the tween the voltage sensor and the gate is required for KCNE3 to KCNE β-subunit is not dependent on the C terminus. This is in affect the gate in KCNQ1 (27). We therefore conclude that contrast to studies showing that the C terminus is necessary for KCNE1 affects both the S4 movement and the gate movement of KCNE1 modulation of KCNQ1 (44, 45), but is consistent with KCNQ1, whereas KCNE3 mainly affects the S4 movement and another study showing that deletion of the C terminus had no only indirectly affects the gate movement of KCNQ1 through the effect (46). Note that even if our study suggests that the C ter- voltage sensor-to-gate coupling. Our findings that KCNE1 and minus is not necessary for a KCNE1-like effect on KCNQ1, KCNE3 modulate different regions of KCNQ1 and affect differ- mutations in the C termini of KCNE1 or KCNQ1 could still have ent gating transitions (i.e., KCNE3 affects the S4 segment, effects on KCNQ1/KCNE1 gating, as has been previously shown, whereas KCNE1 affects both the S4 segment and the gate) may for example, for LQTS mutations in these regions (47–49). explain the dramatically different gating properties conferred by Our results are consistent with previous studies that suggested these two related KCNE β-subunits on KCNQ1 channels (Fig. 1). interactions between the FTL residues (F57, T58, and L59) in Several previous studies have sought to determine the regions KCNE1 and S6 of KCNQ1 (38–40). One study showed that of KCNEs responsible for the effect on KCNQ1 channel func- mutating F340 in S6 of KCNQ1 increased the speed of activation tion. One study suggested that the C-terminal region of KCNE1 in a similar manner as in mutations of the FTL residues in was the main region responsible for KCNE1 modulation of KCNE1, whereas another study showed that the F340A mutation KCNQ1 gating (44). In contrast, another study suggested that restores the function of voltage-independent KCNQ1/KCNE1

E7374 | www.pnas.org/cgi/doi/10.1073/pnas.1710335114 Barro-Soria et al. Downloaded by guest on September 29, 2021 channels with an F57W mutation in KCNE1 (38–40). However, knowledge may help future specific drug designs to target PNAS PLUS another study showed interactions between the KCNE1-T58 and KCNQ1-, KCNE1-, and KCNE3-related channelopathies. KCNQ1-F339 using mutant cycle analysis (41). However, how these interactions alter the function of the KCNQ1 channel was Methods not clear. We find that the introduction of the FTL in the TM Molecular Biology. Mutations in human KCNQ1, KCNE3, and KCNE1 were segment of KCNE3 changes KCNQ1/KCNE3 ionic currents into introduced using the QuikChange Site-Directed Mutagenesis Kit (Qiagen). In KCNQ1/KCNE1-like currents mainly by introducing effects on vitro transcription of cRNA was performed using the mMessage mMachine T7 the second S4 movement and the gate. These effects are in- RNA Transcription Kit (Ambion). dependent of the voltage sensor-to-gate coupling (Fig. 5 and Fig. S2) and likely via F339 for the effects on the gate (Fig. 6). VCF Recordings. VCF experiments were carried out as previously reported (30). Other regions of KCNEs and KCNQ1 have also been proposed Fifty nanograms of KCNQ1 RNA and 10 ng of KCNE3 or KCNE1 RNA (or their mutated versions) were injected into Xenopus laevis oocytes. VCF experi- to affect the properties of KCNQ1/KCNEs, for example, interac- – – ments were performed 2 7 d after injection: Oocytes were labeled for tions between KCNEs and the external ends of S1, S5, and S6 (50 30 min with 100 μM Alexa Fluor 488 5-maleimide (Molecular Probes) in high + 52). KCNQ1/KCNE1 normally only opens from the fully activated K ND96 solution [100 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM Hepes S4 conformation, whereas KCNQ1 expressed alone opens from an (pH 7.6) with NaOH] at 4 °C. Following labeling, they were kept on ice to intermediate S4 conformation (31). We here show that mutating prevent internalization of labeled channels. Oocytes were placed into a re- + two residues at the external end of the KCNE1 TM segment allows cording chamber animal pole “up” in nominally Ca2 -free solution [96 mM gate opening at voltages more negative than those necessary for NaCl, 2 mM KCl, 2.8 mM MgCl2, 5 mM Hepes (pH 7.6) with NaOH] or in high + reaching the fully activated S4 conformation (Fig. 7 D–F). This K ND96 solution. One hundred micromolar LaCl3 is used to block endoge- 3+ suggests that these mutations alter the voltage sensor-to-gate cou- nous hyperpolarization-activated currents. At this concentration, La does pling, thereby allowing gate opening from an intermediate S4 not affect G(V) or F(V) curves from KCNQ1 (30). conformation. Our rubidium/potassium permeability experiments further suggest that these mutations in KCNE1 alter the voltage Data Analysis. Steady-state voltage dependence of current was calculated from exponential fits of tail currents following different test potentials. Tail sensor-to-gate coupling of KCNQ1 channels. It is not known how currents are measured at −40 mV following five test pulses to voltages be- KCNE1 normally alters the voltage sensor-to-gate coupling, thereby tween −180 mV and +60mV(orasspecifiedforeachfigure). For experiments in allowing KCNQ1/KCNE1 to only open from a fully activated high K+ solution (Figs. 4 and 6), tail currents are measured at −100 mV following + + S4 conformation, whereas KCNQ1 expressed alone opens from an a family of test pulses to voltages between −160 and +60 mV. For Rb /K per- intermediate S4 conformation (31). A recent study showed that meability experiments (Fig. 7 E and F), tail currents are measured at −60 mV + mutations at the external end of S1 in KCNQ1 also alter the voltage following a test pulse to +60 mV. The Rb ND96 solution contained 96 mM RbCl, sensor-to-gate coupling in a similar manner as the G40/K41 muta- 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM Hepes (pH 7.6) with NaOH. tions, thereby allowing gate opening from an intermediate S4 po- Each G(V) experiment was fit with a Boltzmann equation: sition (53). Residues at the external ends of the KCNQ1 S1 and À ÀÀ Á ÁÁ ð Þ = + ð − Þ + − KCNE1 TM segments have been shown to be close, because when G V A0 A1 A0 1 exp V V1=2 K , substituted for cysteines, they can form disulfide bonds (41, 50, 51, where A0 and A1 are the minimum and maximum, respectively; V1/2 is the voltage 54). We therefore propose that interactions between the external at which there is half-maximal activation; and K is the slope. Data were nor- ends of the KCNQ1 S1 and KCNE1 TM segments change the malized between the A0 and A1 values of the fit. Fluorescence signals were voltage sensor-to-gate coupling by altering interactions between the bleach-subtracted, and data points were averaged over tens of milliseconds at voltage-sensing domain and pore domain, thereby allowing the gate the end of the test pulse to reduce errors from signal noise. The steady-state to open from different S4 conformations (31). fluorescence data are fit with a single (or double) Boltzmann curve and nor- In summary, our results suggest that the main difference be- malized between the minimum and maximum fluorescence for each experiment. tween the effects of KCNE1 and KCNE3 on KCNQ1 gating is that KCNE1 affects both the voltage-sensing domain and the Statistics. All experiments were repeated four or more times from at least four batches of oocytes. Pairwise comparisons were achieved using a Stu-

gate of KCNQ1, whereas KCNE3 primarily affects the voltage- PHYSIOLOGY dent’s t test, and multiple comparisons were performed using an ANOVA sensing domain of KCNQ1 and only indirectly affects the gate. with a Tukey’s test. Data are represented as mean ± SEM, and n represents One molecular difference between KCNE1 and KCNE3 is in a the number of experiments. triplet of residues in the middle of the KCNEs’ TM segment that adds an effect on the second, minor S4 movement and the gate ACKNOWLEDGMENTS. We thank Dr. Fredrik Elinder for helpful comments of KCNQ1. Another molecular difference is in two residues at on the manuscript. We also thank Dr. Nicole Schmitt for her generous gift of the external end of the KCNE TM segments that shift the KCNE3 mutants. This work was supported by NIH Grants R01-GM109762 and S4 movement to very negative voltages in the presence of R01-HL131461 (to H.P.L.), American Heart Association Postdoctoral Fellow- ship 13POST17000057, Taking Flight Award 414889 from Citizens United for KCNE3 and that alter the coupling between S4 and the gate in Research in Epilepsy, NIH K01 Award 1K01NS096778-01A1 from the National the presence of KCNE1. This study shows that KCNE1 and Institute of Neurological Disorders and Stroke (to R.B.-S.), and Postdoctoral KCNE3 affect KCNQ1 in different structural domains. This Fellowship 2011-6806 from the Swedish Research Council (to S.I.L.).

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