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KCNE1 and KCNE3 Modulate KCNQ1 Channels by Affecting

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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.
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