Triad interaction stabilizes the voltage sensor domains in a constitutively 1 open KCNQ 1-KCNE 3 channel 2

Tetrameric voltage-gated K+ channels have four identical voltage sensor domains, and they regulate channel gating. KCNQ1 (Kv7.1) is a voltage-gated K+ channel, and its auxiliary subunit KCNE proteins dramatically regulate its gating. For example, KCNE3 makes KCNQ1 a constitutively open channel by affecting the voltage sensor movement. However, how KCNE proteins regulate the voltage sensor domain is largely unknown. In this study, by utilizing the recently determined KCNQ1-KCNE3-calmodulin complex structure, we identified amino acid residues on KCNE3 facing the S1 segment of KCNQ1 that are required for constitutive activity. In addition, we found that the interaction of these amino acid residues of KCNE3 and the S1 segment affects the voltage sensor movement via M238 and V241 residues of the S4 segment. This triad interaction shifts the voltage sensor domain’s equilibrium, leading to stabilization of the channel’s open state.

Introduction 18 KCNQ1 (Kv7.1) is a voltage-gated K + channel. Its gating behavior depends mainly on its auxiliary 19 subunit KCNE proteins (Wang et al., 2020). There are five KCNE genes in the human genome, and 20 all of them are known to modify KCNQ1 channel gating behavior, at least in Xenopus oocytes or 21 mammalian cell lines (Bendahhou et al., 2005). Therefore, the physiological functions of the 22 KCNQ1 channel are determined by the type of KCNE proteins that are co-expressed in a tissue. The 23 most well-known example is the cardiac KCNQ1-KCNE1 channel, which underlies the slow cardiac 24 delayed-rectifier K + current (IKs) (Barhanin et al., 1996;Sanguinetti et al., 1996;Takumi et al., 1988). 25 Another example is the KCNQ1-KCNE3 channel, a constitutively open channel expressed in 26 epithelial cells in the trachea, stomach, and intestine. This channel complex couples with some ion 27 transporters to facilitate ion transport by recycling K + ions (Abbott, 2016;Grahammer et al., 2001;28 Preston et al., 2010;Schroeder et al., 2000). The mechanisms by which KCNE proteins modify 29 KCNQ1 channel gating behavior have been a central question of this ion channel. Since KCNQ1 is a 30 classic shaker-type tetrameric voltage-gated K + channel, it has four independent voltage-sensing 31 domains (VSDs), one from each α subunit (Long et al., 2005). Each VSD consists of four 32 transmembrane segments, S1-S4. Each S4 segment bears several positively-charged amino acid 33 residues. When the membrane potential is depolarized, each S4 segment moves upward (towards the 34 extracellular side). That upward movement eventually triggers pore opening (Jensen et al., 2012;35 Larsson et al., 1996;Mannuzzu et al., 1996). Therefore, the S4 segment is considered to be an 36 essential part of voltage-sensing (Aggarwal and MacKinnon, 1996;Catterall, 1988;Fedida and 37 Hesketh, 2001;Liman and Hess, 1991;Logothetis et al., 1992;Noda et al., 1984;Papazian et al., 38 1991). As in the Shaker K + channel, the S4 segment of the KCNQ1 channel also moves upward with 39 depolarization, as proved by scanning cysteine accessibility mutagenesis (SCAM) (Nakajo and Kubo, 40 2007;Rocheleau and Kobertz, 2008)  ( Barro-Soria et al., 2017, 2015Taylor et al., 2020). 49 Early studies by Melman et al. revealed "the triplet" of amino acid residues in the middle of the 50 transmembrane segment ("FTL" in KCNE1 and "TVG" in KCNE3) as structural determinants of 51 KCNE modulation properties (Melman et al., 2002(Melman et al., , 2001. Exchanging the triplet (or one of the 52 three amino acid residues) between KCNE1 and KCNE3 could introduce the other's modulation 53 properties, at least partially. For example, introducing "FTL" into KCNE3 transforms it into a 54 KCNE1-like protein. Therefore, it has been long considered that "the triplet" is a functional 55 interaction site between KCNQ1 and KCNE proteins. Possible interaction sites of the KCNQ1 side 56 have also been explored and are believed to be located between the pore domain and the VSD 57 (Chung et al., 2009;Kang et al., 2008;Nakajo and Kubo, 2007;Van Horn et al., 2011;Xu et al., 58 2008). By utilizing the KCNQ1 ortholog from ascidian Ciona intestinalis, which lacks KCNE genes, 59 we previously found that F127 and F130 of the S1 segment are required for KCNE3 to make 60 KCNQ1 a constitutively open channel (Nakajo et al., 2011). A recent computational model and the 61 cryo-EM structure of the KCNQ1-KCNE3-calmodulin (CaM) complex clearly showed that KCNE3 62 interacts with the S1 segment and the pore domain (Kroncke et al., 2016;Sun and MacKinnon, 63 2020). However, the mechanism by which KCNE3 retains the KCNQ1 VSD at a specific position is 64 Submitted Manuscript: Confidential -9 -by Phyre2 server (Kelley et al., 2015), we tested the idea with two candidate pairs (KCNQ1 150 F127-KCNE3 G73 and KCNQ1 F130-KCNE3 A69) ( Figure 3A). We introduced the G73L mutation 151 because KCNE1 has a leucine residue at the same position ( Figure 1C). KCNE3 G73L showed 152 voltage-dependent gating, meaning that KCNQ1 is no longer constitutively active ( Figure 3B, 153 C-figure supplement 2). We next tested the pair of KCNQ1 F127A-KCNE3 G73L. The pair 154 successfully rescued the G-V relationship comparable to that of KCNQ1 WT-KCNE3 WT ( Figure  155 3B, C-figure supplement 2). Furthermore, ΔF-160mV/ΔF60mV of KCNQ1vcf F127A-KCNE3 G73L 156 (0.80 ± 0.04, n=5) showed significant recovery compared to that of KCNQ1vcf WT-KCNE3 G73L 157 (0.22 ± 0.03, n=5) and that of KCNQ1vcf F127A-KCNE3 WT (0.31 ± 0.04, n=5), suggesting that 158 KCNE3 G73L can stabilize the S4 segment of KCNQ1 F127A in the intermediate position to some 159 extent ( Figure 3D, E-figure supplement 2). In contrast, the pair of KCNQ1 F130A-KCNE3 A69F did 160 not recover the channel modulation by KCNE3 ( Figure 3F, G-figure supplement 2), implying that 161 F130 (or A69) may have another crucial role in KCNE3 modulation (discussed later). Overall, the 162 results demonstrate the importance of the interaction between the S1 segment and KCNE3. 163 Appropriate side-chain volumes at the interaction interface are necessary for functional interaction in 164 the KCNE3 modulation. 165 166 KCNE3 and the S1 segment modulate VSD movement via M238 and V241 of the S4 167

segment. 168
As we have seen, the interactions between the S1 segment of KCNQ1 and KCNE3 are required for 169 the channel's constitutive activity (Figures 1-3). However, how these interactions regulate the 170 movement of the S4 segment is still not clear. We again closely looked at the KCNQ1-KCNE3-CaM 171 complex structure and noticed that KCNE3 (F68, V72, and I76) and F130 of the S1 segment face the 172 bottom half of the S4 segment, especially the side chains of M238 and V241 ( Figure 4A) (Sun and 173 Submitted Manuscript: Confidential -10 -MacKinnon, 2020). Therefore, we created alanine-substituted mutants of the S4 segment (M238A 174 and V241A) one at a time and found that both KCNQ1 M238A and V241A co-expressed with 175 KCNE3 WT attenuated the shift of the G-V curves in the negative direction ( Figure 4B, C). We next 176 applied VCF to analyze the VSD movement of M238A and V241A. Both M238A and V241A 177 without KCNE3 showed normal F-V curves and almost a 0 value of ΔF-160mV/ΔF60mV ( Figure 4D, This mechanism may be required to prevent the S4 segment from going to the down position by 184 KCNE3. 185 186 M238 and V241 of the S4 segment are also involved in VSD modulation by KCNE1. 187 We also co-expressed KCNQ1 M238A and V241A with KCNE1 to investigate whether these S4 188 mutants influence the VSD modulation by KCNE1 since previous experiments demonstrated that 189 KCNE1 affects S4 movement (Osteen et al., 2012(Osteen et al., , 2010. In the presence of KCNE1, M238A and 190 V241A mutants showed the typical IKs-like current and positively-shifted G-V curves ( Figure 5A, B). 191 We then examined the VSD movements of these mutants with KCNE1.  with KCNE1 WT showed ΔF-160mV/ΔF60mV (0.98 ± 0.04, n=5) similar to that of n=5), suggesting that many of the S4 segments of the suggesting that both M238A and V241A mutations affect the S4 movement modulated by KCNE1. 199 These results indicate that M238 and V241 in the S4 segment are involved in the VSD modulation 200 for both KCNE1 and KCNE3. 201 202 Discussion 203 In this work, we conducted site-directed mutational analyses using TEVC and VCF, inspired by the 204 recently determined cryo-EM structures of the KCNQ1-KCNE3-CaM complex (Sun and 205 MacKinnon, 2020). Our main finding is triad interaction formed by KCNE3 with the S1 and S4 206 segments of KCNQ1. The triad is a key component for the channel modulation by KCNE3, which 207 prevents the S4 segment of the VSD from going to the down position at resting membrane potential. 208 Another interesting finding is that M238 and V241 of the S4 segment in the triad may also be 209 involved in the VSD modulation by KCNE1. 210 Previous studies demonstrated that "the triplet" of amino acid residues in the middle of the 211 transmembrane segment ("FTL" for KCNE1 and "TVG" for KCNE3) is a structural determinant of 212 KCNE modulation properties (Barro-Soria et al., 2017;Melman et al., 2002Melman et al., , 2001. However, why 213 the triplets determine the modulation type is still not well understood. Besides "the triplet," our 214 current work showed that a broader range of amino acid residues (F68, V72, and I76), which forms 215 three helical turns in total in the middle of the transmembrane segment of KCNE3, were involved in 216 the interactions among KCNE3 and S1 and S4 segments and were required for maintaining the 217 constitutive activity of the KCNQ1-KCNE3 channel (Figure 1). For the KCNQ1 side, we previously 218 demonstrated that two phenylalanine residues, F127 and F130, in the S1 segment are important for 219 the channel modulation by KCNE3, but how these mutations affect the channel modulation has been 220 unknown (Nakajo et al., 2011). The cryo-EM KCNQ1-KCNE3-CaM structure (PDB: 6V00) clearly 221 Submitted Manuscript: Confidential -12 -shows that F127 and F130 of the S1 segment face KCNE3 (F68, V72, and I76). We therefore 222 hypothesized that the interaction of these amino acid residues is essential for the KCNE3 function. 223 Our electrophysiology and VCF studies confirmed that the interaction of these amino acid residues is 224 required for proper KCNE3 function, which maintains the opening of the KCNQ1 channel. 225 Although we mainly introduced alanine mutations to assess the amino acid residue's importance for 226 the interactions (Figure 2), the introduction of bulky amino acid residues at A69 or G73 also 227 disrupted the KCNE3 function, suggesting that an appropriate distance between the S1 segment and 228 KCNE3 is required ( Figure 3). The rescue of KCNE3 G73L by KCNQ1 F127A also supports that 229 idea ( Figure Figure 6B). On the other hand, in the VSD of the cryo-EM structure (Sun and MacKinnon, 2020), 246 R237 is located over F167, and the VSD is likely to be in the up position ( Figure 6A). In this study, 247 we utilized the cryo-EM structure to confirm that the interaction of KCNE3 and the S1 segment is 248 necessary for the modulation. Subsequently, we identified M238 and V241 of the S4 segment, again 249 by utilizing the cryo-EM structure. In the intermediate state structure, M238 and V241 are still 250 directed towards F130, although M238 is slightly closer to F130 and V241 is slightly away from 251 F130 ( Figure 6B We showed that M238 and V241 of the S4 segment also play a pivotal role in channel modulation by 255 KCNE1 ( Figure 5). According to the ionic currents and the G-V curves of M238A and V241A 256 mutants, alanine-substituted mutants of these residues (M238A and V241A) showed more severe 257 effects on the pore gating modulation by KCNE3 than that by KCNE1. However, they showed 258 similar effects on the ΔF-160mV/ΔF60mV values ( Figure 4D On the other hand, the KCNQ1-KCNE3 channel is conductive at the IO state; therefore, ionic 264 currents appear to be significantly affected by M238A and V241A. Thus, the modulation of VSD 265 movement by KCNE1 and KCNE3 might share a common mechanism of stabilization of the 266 intermediate state through M238 and V241. 267 One drawback of this kind of mutation study is that the impairment of mutations could occur at 268 different stages, such as expression, binding, or modulation efficacy. We confirmed that the 269 under the protocol no. 18027-03 and were performed following the institutional guidelines. 297

Two-electrode voltage-clamp 298
cRNA-injected oocytes were incubated for 2-3 days. Ionic currents were recorded with a 299 two-electrode voltage clamp using an OC-725C amplifier (Warner Instruments) at room temperature. 300 The bath chamber was perfused with Ca 2+ -free ND96 solution (96 mM NaCl, 2 mM KCl, 2.8 mM 301 MgCl2, 5 mM HEPES, pH 7.6) supplemented with 100 µM LaCl3 to block endogenous 302 hyperpolarization-activated currents (Osteen et al., 2010). The microelectrodes were drawn from 303 borosilicate glass capillaries (Harvard Apparatus, GC150TF-10) using a P-1000 micropipette puller 304 (Sutter Instrument) to a resistance of 0.2-1.0 MΩ and filled with 3 M KCl. Currents were elicited 305 Submitted Manuscript: Confidential -16 -from the holding potential of -90 mV to steps ranging from -100 to +60 mV in +20-mV steps each 306 for 2 sec with 5-sec intervals (or 10-sec intervals for KCNQ1-KCNE1 currents). Generation of 307 voltage-clamp protocols and data acquisition were performed using a Digidata 1550 interface 308 (Molecular Devices) controlled by pCLAMP 10.7 software (Molecular Devices). Data were sampled 309 at 10 kHz and filtered at 1 kHz. 310 Voltage dependence analysis 311 G-V relationships were taken from tail current amplitude at -30 mV fitted using pCLAMP 10.7 312 software (Molecular Devices) to a two-state Boltzmann equation: 313 where Gmax and Gmin are the maximum and minimum tail current amplitudes, respectively, z is the 315 effective charge, V1/2 is the half-activation voltage, T is the temperature in degrees Kelvin, F is 316 Faraday's constant, and R is the Boltzmann constant. G/Gmax, which is the normalized tail current 317 amplitude, was plotted against membrane potential for presentation of the G-V relationships. Gating 318 parameters obtained in this study are listed in Table supplement 1. 319

Voltage-clamp fluorometry 320
Sample preparation, data acquisition, and data analysis were performed similarly as described 321 previously (Nakajo and Kubo, 2014 Apparatus, GC150TF-15). Currents were elicited from the holding potential of -90 mV to steps 328 ranging from +60 to -160 mV in -20-mV steps each for 2 sec with 20-sec intervals. Generation of 329  and R is the Boltzmann constant. 357

Statistical analysis 364
The data were expressed as mean±s.e.m. Differences between WT and mutants were evaluated by 365 Dunnett's test with EZR software (Kanda, 2013).

W K C F V Y H F V F L I V L C L I F S V L S T I V W K C F V Y H F V F L I V L C L I F S V L S T I T V W K C F V Y H F V F L I V L C L I F S V L S T I T I W K C F V Y H F V F L I V L C L I F S V L S T I
T I S1 2 3 0 2 4 0  (A) Close-up view of the interface between KCNQ1 and KCNE3 in the KCNQ1-KCNE3-CaM complex structure (PDB: 6V00). Three KCNQ1 subunits are colored in blue, green, and gray. A KCNE3 subunit is colored in red.

S4 C TM
The residues involved in the KCNQ1-KCNE3 interaction are depicted by stick models. The molecular graphics were illustrated with CueMol (http://www.cuemol.org/). (B, C) Sequence alignment around the S1 and S4 segments of KCNQ1 (B) and the TM segments of KCNE3 and KCNE1 (C). Amino acid sequences were aligned using Clustal Omega (Madeira et al., 2019) and are shown using ESPript3 (Robert and Gouet, 2014). KCNQ1 residues focused on in this work are highlighted with blue dots. KCNE3 residues focused on in this work and "the triplet" (Melman et al., 2002(Melman et al., , 2001        for n = 5 in (D, E). Statistical significance was determined by Dunnett's test. *** denotes P < 0.001 for F -160mV / F 60mV compared to KCNQ1 vcf WT-KCNE1 WT in (E). structures. Amino acid residues involved in the interaction between S1 and S4 segments as well as the charge transfer center (F167) and the fourth arginine (R237) of the S4 segment are depicted by stick models.  mutants used in this study were confirmed by confocal microscopy. mEGFP without KCNE3 was not detected at the surface. All KCNE3 constructs and mEGFP were co-expressed with KCNQ1 WT.      Maximum tail current amplitudes G max , tail current amplitudes at -100 mV G -100 , and parameters deduced from the Boltzmann fitting (V 1/2 and z) for the KCNQ1/KCNE3 mutants. n is the number of experiments. Same sample names appear in two lines (e.g. KCNQ1 WT + KCNE3 WT appears in Figure 1 and 3) because they are from different data sets.