Voltage-Dependent Gating: Novel Insights from KCNQ1 Channels

14 Biophysical Journal Volume 110 January 2016 14–25 Biophysical Perspective

Jianmin Cui1,* 1Department of Biomedical Engineering, Cardiac Bioelectricity and Arrhythmia Center and Center for the Investigation of Membrane Excitability Disorders, Washington University, St. Louis, Missouri

ABSTRACT Gating of voltage-dependent cation channels involves three general molecular processes: voltage sensor activation, sensor-pore coupling, and pore opening. KCNQ1 is a voltage-gated potassium (Kv) channel whose distinctive properties have provided novel insights on fundamental principles of voltage-dependent gating. 1) Similar to other Kv channels, KCNQ1 voltage sensor activation undergoes two resolvable steps; but, unique to KCNQ1, the pore opens at both the intermediate and activated state of voltage sensor activation. The voltage sensor-pore coupling differs in the intermediate-open and the activated-open states, resulting in changes of open pore properties during voltage sensor activation. 2) The voltage sensor-pore coupling and pore opening require the membrane lipid PIP2 and intracellular ATP, respectively, as cofactors, thus voltage- dependent gating is dependent on multiple stimuli, including the binding of intracellular signaling molecules. These mechanisms underlie the extraordinary KCNE1 subunit modiﬁcation of the KCNQ1 channel and have signiﬁcant physiological implications.

þ þ 2þ Voltage-gated K ,Na , and Ca ion channels (KV,NaV, (Fig. 1). During voltage sensor activation, S4 moves in the 2þ and CaV) are responsible for electric activities and Ca extracellular direction in response to depolarization of the signaling in excitable cells such as neurons and cardiac membrane potential (5,6), while during pore opening, myocytes, as well as in various nonexcitable cells. Since the cytoplasmic lower halves of S6 in the four subunits Hodgkin and Huxley discovered voltage-dependent changes move apart (7,8). VSD-PGD coupling is less understood. of membrane conductance of Naþ and Kþ ions in the We know that the interaction between the peptide linking 1950s (1), functional and structural studies of voltage-gated S4 and S5 and the cytoplasmic end of S6 is important for ion channels have revealed the general principles of voltage- the coupling (9,10). Other interactions, such as those be- dependent gating. It is known that the voltage sensors and tween S4 and S5 (11-13) and between S1 and the outer ves- the ionic pore of these channels are distinct structural do- tibule of the pore (14), may also contribute to the VSD-PGD mains (2,3)(Fig. 1, A and B). The voltage-dependent move- coupling. ments of voltage sensors regulate the opening of the pore. The KCNQ1 channel, also known as KvLQT1 or Kv7.1, Thus, voltage-dependent gating of these channels involves was discovered to be a novel Kv channel, and its gene was three general molecular processes: 1) voltage-dependent associated with type 1 long QT syndrome (LQT1) (15), movements in the voltage sensor domain (VSD), known as which predisposes inﬂicted patients to cardiac arrhythmia. voltage sensor activation; 2) activation gate movements in Besides having a critical function in the physiology of the the pore-gate domain (PGD), known as pore opening; heart, KCNQ1 is also involved in the functions of other or- and 3) interactions between the VSD and PGD that propa- gans, including the brain, kidney, and gastrointestinal system gate the movements from one to the other, known as (16,17). In different tissues, members of the KCNE auxiliary VSD-PGD coupling. Voltage sensor activation should not subunit family (KCNE1–5) associate with KCNQ1 to modify be confused with voltage-dependent activation of the chan- channel properties, creating the various phenotypes impor- nel, which is a term historically used to describe channel tant for physiological functions (17). KCNQ1 is the founding opening in response to voltage changes, encompassing all member of the KCNQ channel family (KCNQ1–5). three molecular processes. KCNQ2–5 form Kþ channels that are important for neural The VSD and the PGD, respectively, of voltage-gated ion functions and share important properties with KCNQ1 channels share common structural features. The VSD is (18,19), but because of the scope of this review, these chan- comprised of transmembrane helices S1–S4. In this domain, nels will not be discussed in this article. The mechanisms the primary voltage sensitive element is S4, which contains of voltage-dependent gating are central to the physiological the positively charged residues arginine (R) or lysine (K). and pathophysiological roles of KCNQ1 channels, high- The PGD is comprised of S5, S6, and the pore loop between lighted by the fact that KCNE subunits and various disease- the two helices, which contains the ion selectivity ﬁlter associated mutations alter voltage-dependent activation. Structural models of KCNQ1 based on sequence homology Submitted July 30, 2015, and accepted for publication November 17, 2015. and experimental results indicate that KCNQ1 has a canoni- *Correspondence: [email protected] cal Kv channel structure (4,20-23): the channel is formed by four KCNQ1 subunits, with the individual VSD from each Editor: Brian Salzberg Ó 2016 by the Biophysical Society 0006-3495/16/01/0014/12 http://dx.doi.org/10.1016/j.bpj.2015.11.023 Voltage-Dependent Gating of KCNQ1 15

FIGURE 1 Models of the KCNQ1 channel. (A) Top view of the tetrameric KCNQ1 homology model. Four subunits are shown in different colors. Every subunit consists S1–S6 transmembrane segments as indicated. The S1–S4 form the voltage sensor domain, and the S5-S6 from all four subunits form the pore-gate domain. This open state KCNQ1 structural model is originated from Van Horn et al. (4). (B) Side view of the KCNQ1 channel homology model; the selectivity filter is labeled as indicated. (C) Membrane topology of full length KCNQ1. Each circle represents an amino acid residue with the one-letter symbol. The S1–S6 and cytoplasmic C terminus helices (A–D) are labeled as indicated. The residues important for voltage-dependent gating, including the gating charges in S4 (R1, R2, and R4), E160 in S2, F351 in the cytoplasmic end of S6, and the PIP2 and ATP binding sites are colored as indicated. subunit located at the periphery of the central pore that is General mechanisms of voltage-dependent formed by the PGD of all four subunits (Fig. 1 A). The mech- gating in KCNQ1 anism of voltage-dependent gating of KCNQ1 has been Voltage sensor activation investigated mostly within a framework that has been estab- lished mainly by studies of the Shaker-related subfamily of The KCNQ1 S4 contains arginine residues that are þ Kv channels, and, in general, the mechanism of KCNQ1 conserved in other Kv channels (Fig. 1 C). In Shaker K gating closely follows the same principles. However, channels, the first four arginines (R1–R4) in the N-terminal KCNQ1 shows unique properties in voltage-dependent acti- half of S4 serve as gating charges for sensing membrane vation. Although these properties could reflect mechanisms potential (24-26), but in the KCNQ1 S4 the canonical R3 of gating specific to KCNQ1 channels, I argue in this mini re- is replaced by a neutral glutamine (Q234). The C-terminal view that they provide opportunities to reveal novel aspects half of S4 in KCNQ1 also has fewer net positive charges of the general mechanism of voltage-dependent gating. I first than Shaker. Mutations of S4 residues in KCNQ1 change summarize results suggesting that mechanisms common to the voltage dependence of channel activation (27-29); voltage-gated ion channels also apply to KCNQ1 voltage- particularly, neutralization of R231 (R2) with various dependent gating. Then I focus on the distinctive features amino acids (27-29), and mutation R228E (R1E) (29) elim- of KCNQ1 gating and how these features underlie the mod- inate voltage dependence and make the channel constitu- ulation of KCNQ1 by its auxiliary subunit, KCNE1. In the tively open. end, I discuss how the features of KCNQ1 gating can bring Gating currents resulting from movements of voltage novel insights into the general principles of voltage-depen- sensors in the membrane were recorded recently from dent gating, especially into the mechanism of VSD-PGD KCNQ1 channels (30). Fluorescence signals from a fluoro- coupling. phore attached to the S3-S4 linker or the N-terminus of S4,

Biophysical Journal 110(1) 14–25 16 Cui measured using voltage clamp fluorometry (VCF), showed channel. Nonetheless, the structural motifs in KCNQ1 that kinetics and voltage dependence that were similar to those restrict ionic flow in the closed channel and that move dur- of gating charge movements measured by gating currents ing pore opening are not known. (30,31), indicating that S4 moves during VSD activation. S4 movements were also demonstrated by the accessibility VSD-PGD coupling of cysteine mutations in S4 by extracellular 2-(tri- methylammonium) ethyl methanethiosulfonate (MTSET); KCNQ1 channels show a constitutive open component that R228C, G229C, and I230C are accessible only at depolar- cannot be turned off, even at very negative (~120 mV) volt- ized voltages where the VSD of KCNQ1 is activated, indi- ages. Many mutations increase the fraction of this constitu- cating that S4 moves toward the extracellular side and tive open conductance versus the total conductance, and a exposes these residues during voltage-dependent activation large-scale mutational scanning study showed that such an (32,33). Another piece of evidence for S4 movements was increase was correlated with the shift of the G-V relation provided by the study of interactions of arginine residues in of the voltage-dependent component to more negative volt- S4 with E160, located at the extracellular end of S2. There, ages (40). This relation could be quantitatively explained by R228 (R1) and R237 (R4) were found to make electrostatic a model assuming an allosteric VSD-PGD coupling, accord- interactions with E160 in the resting state (at hyperpolar- ing to which the pore can open without voltage sensor acti- ized voltages) and the activated state (at depolarized volt- vation, but activation of any of the four VSDs increased the ages) of the VSD, respectively, indicating an outward probability of pore opening (40). The same allosteric model movement of S4 during VSD activation (34). These studies has been proposed to describe voltage-dependent gating of also revealed specific characteristics of VSD activation in the BK and hyperpolarization-activated cyclic nucleotide- KCNQ1. First, S4 movements in KCNQ1 are slow, result- gated channels (41,42). In another study, VCF was used to ing in gating currents with small amplitudes and slow de- measure voltage sensor movements and the pore opening caying kinetics (time constant >20 ms) (30), which of channels composed of mutant KCNQ1 subunits with accounts for the slower kinetics of ionic current onset as different voltage dependences. The results suggested that compared with Shaker Kþ channels. Second, the paucity the VSD of each subunit moved independently and that of positive charges in S4 may explain why the channel pore opening was not tightly correlated with VSD move- can be turned to constitutively open by S4 mutations ments of any of the subunits; rather, the results could be (27). Supporting this idea, the addition of a positive charge well fitted by the same model of allosteric VSD-PGD by the mutation Q234R (Q3R) recovered voltage depen- coupling (43). The allosteric VSD-PGD coupling was also dence of R228E (R1E) (29). directly shown by locking the pore in an open state that did not prevent the voltage sensor from moving between the resting and activated state (44). Pore opening The interaction between the S4-S5 linker and the cyto- In Kv channels, S6 lines the inner pore. Scanning mutations plasmic end of S6 is important for VSD-PGD coupling in of the KCNQ1 S6 by substituting alanine or tryptophan for Kv channels (9,10). In a study of LQT-associated muta- each residue showed that many mutations changed macro- tions in the KCNQ1 S4-S5 linker, R243C and W248R scopic current amplitudes and altered the voltage depen- were found to reduce the opening rate and to shift the dence of channel activation (35,36). Particularly, a cluster voltage dependence of channel opening to more positive of mutations, including P343A, G345A, and I346A, abol- voltages, whereas E261K abolished channel function, sug- ished the current but not channel expression in the surface gesting the importance of the S4-S5 linker in voltage- membrane (35,37), whereas F340W made the channel dependent gating of KCNQ1 (45). Likewise, a mutation constitutively open and insensitive to voltage (36). Muta- in the cytoplasmic end of S6 associated with LQT, tions associated with LQT, F339S, A341V, A341E, and Q357R, also reduced channel function (46). Subsequent G345E were also found to abolish KCNQ1 currents studies of scanning mutations of the S4-S5 linker (47) (38,39). Interestingly, P343, A344, and G345 are aligned and the cytoplasmic end of S6 (48) found many mutations with proline-valine-proline in the Shaker S6 that are impor- that had high impact on voltage-dependent gating of tant for the motion of S6 during pore opening (7). A glycine KCNQ1. The effects of these mutations from the two residue conserved in the S6 of many Kþ channels is thought different domains share some phenotypical similarities. to also act as a hinge for S6 movements (8). The correspond- For instance, one group of mutations, including T247A, ing residue in the KCNQ1 S6 is A336, and mutations of L251A, V255W, H258A, and R259A in the S4-S5 linker A336 altered the voltage dependence of channel gating and S349A, F351A, A352W, and V355W in the cyto- (37). These results are consistent with the idea that muta- plasmic end of S6, reduced the opening rate and shifted tions in the KCNQ1 S6 around the PAG region may alter the voltage dependence of channel opening to more posi- the conformation or the motion of the activation gate, result- tive voltages. Another group of mutations, including ing in either a constitutively closed or a constitutively open V254A, L, or E in the S4-S5 linker, and L353A in the

Biophysical Journal 110(1) 14–25 Voltage-Dependent Gating of KCNQ1 17 cytoplasmic end of S6, led to an increased constitutively where the pore opens only at the activated state of the open component, and L353E and L353K KCNQ1 even VSD, the pore in KCNQ1 opens at both the intermediate lost voltage dependence entirely, becoming constitutively and activated states, where the voltage dependence of open. Interestingly, the double mutation V254L/L353A conductance, the G-V relation, nearly overlaps with that eliminated the constitutively open component, suggesting of Fmain (30,31,52). Consistently, the double charge that the S4-S5 linker and the cytoplasmic end of S6 inter- reversal mutations, E160R/R231E (E1R/R2E) and acted to close the channel and that the volume of the side E160R/R237E (E1R/R4E), which arrest the VSD at an in- chain in residues V254 and L353 was important for the termediate and the fully activated state, respectively, make interaction (47). Consistent with this idea, a parallel study the channel constitutively open (21,29,52,59). Interest- showed that isolated peptides corresponding to the S4-S5 ingly, it was found that the E1R/R2E and E1R/R4E linker inhibited the KCNQ1 current, whereas peptides cor- mutant channels showed different relative Rbþ/Kþ perme- responding to the cytoplasmic end of S6 increased the cur- abilities or sensitivities to the inhibitor XE991. Compared rent (49). These results were taken to suggest that the with E1R/R4E, E1R/R2E exhibited a larger ratio of in- S4-S5 linker in the channel acted as a ligand, binding to ward currents in high Rbþ versus high Kþ extracellular the cytoplasmic end of S6 to stabilize the closed state of solutions; 5 mM XE991 inhibited E1R/R2E currents, but the channel. Voltage sensor activation prevented this inter- not E1R/R4E (52). The wild-type (WT) KCNQ1 channel action, thereby promoting pore opening. The increased showed a larger ratio of inward currents in high Rbþ binding of isolated S4-S5 linker peptide to the channel versus high Kþ extracellular solutions (60). The larger helped stabilize the closed states, whereas the competition Rbþ current was attributed to a reduced ﬂickering block for the native S4-S5 linker by the isolated S6 peptide desta- of the pore upon Rbþ binding (60,61). Consistent with bilized the closed states (49,50). this idea, many mutations in the PGD of KCNQ1 modulated Rbþ/Kþ permeability, presumably by altering the PGD conformation (62). Thus, the results showing that Unique properties of voltage-dependent gating of E1R/R2E and E1R/R4E mutations in the VSD modulate þ þ KCNQ1 channels Rb /K permeability suggest that when the VSD is at either the intermediate or activated state, the pore is in Pore opening and VSD-PGD coupling at different stages of different open conformations. The mechanism of XE991 voltage sensor activation inhibition is not clear. The mutations E1R/R2E and In a study of the interaction between S4 arginine residues E1R/R4E may alter XE991 inhibition allosterically, which with E160 in S2 (Fig. 1 C), it was found that the VSD moves is also consistent with the idea that the conformation of in two resolvable steps. Measured in a mutant E160R/ the channel protein differs at the intermediate-open and R228E/Q234R KCNQ1 coexpressed with the auxiliary sub- activated-open states. unit KCNE1, VSD movements in the voltage ranges of The presence of two different open states during KCNQ1 100 ~ 50 mV and 50 ~ þ50 mV showed stepwise in- gating was suggested to account for the properties of inacti- crements, suggesting that, during activation, the voltage vation (62,63). Two open states were also shown by a study sensor traversed a stable intermediate state at ~50 mV of Naþ block of the instantaneous KCNQ1 current at the before reaching the activated state at high voltages (34). beginning of voltage pulses following a prepulse that acti- Also, for the triple mutant C214A/G219C/C331A vated the channel (64). The currents were blocked more KCNQ1þKCNE1, stepwise VSD movements were shown by Naþ when the prepulse duration was longer, suggesting using VCF, and the two VSD movements were consistently that the open state visited at early times during the prepulse separated at ~50 mV (51). More recently, it was found that was less sensitive to Naþ block than the open state visited in KCNQ1 alone, without KCNE1 association, the VSD also later. Whether these different open states correlate with showed stepwise movements during activation. The ﬂuores- the intermediate-open and activated-open states remains to cence signal measured in VCF experiments showed that be tested. the movement at negative voltages (Fmain) shifted to less It was shown that a mutation at the cytoplasmic end of negative voltages than in KCNQ1þKCNE1, whereas the S6, F351A, could abolish the intermediate-open state so movement at more positive voltages (Fhigh) was not that the channel contained predominantly the activated- changed (52). open state (52). F351 (Fig. 1 C) is highly conserved in Stepwise activation of the VSD has been described pre- many Kv channels and is important for VSD-PGD viouslyinShakerKþ channels (53,54). In these channels, coupling (48,65). The mutation selectively suppressed mutations such as the isoleucine-leucine-threonine muta- one open state but not the other, suggesting that the tioninS4(11) and mutations in the gating charge transfer VSD-PGD coupling may differ when the VSD is at the in- center (55), also known as the hydrophobic plug (56-58), termediate or activated state. This is consistent with results make the separation in voltage ranges between the two showing that the conformation of the open pore differs movements more prominent. However, unlike in Shaker, when the VSD is at either the intermediate-open or

Biophysical Journal 110(1) 14–25 18 Cui activated-open state, so that different VSD-PGD couplings lix C, including R359W, R555C, R555H, K557E, and result in different open pore conformations. R562M (74,77,78), also alter the responses of KCNQ1 to PIP2 depletion. Thus, these residues may be also important for PIP2-dependent activation. These residues may form in- PIP2 is required for VSD-PGD coupling dependent PIP2 binding sites to affect channel activation Phosphatidylinositol 4,5-bisphosphate (PIP2) is an anionic with mechanisms that are not yet known, or may contribute lipid found in the inner leaﬂet of the plasma membrane. directly or allosterically to the site at the interface between PIP2 is a second messenger for cell signaling and known the VSD and PGD (76). to directly bind to and regulate a wide variety of ion channels, including KCNQ1 (66). KCNQ1 was found to require Intracellular ATP is required for pore opening PIP2 to function; depletion of PIP2 from the membrane sup- presses KCNQ1. The suppression can be prevented or During patch clamp studies, ATP was required in the intracellular bath solution to maintain the KCNQ1 current in reversed by replenishing PIP2 through cellular metabolisms or exogenous application (44,67-70). excised inside-out membrane patches (67,79). ATP was also required for the native KCNQ1 channels (in association VCF measurements showed that PIP2 is required for with KCNE1) in cardiac myocytes to activate, with an EC VSD-PGD coupling. PIP2 depletion by a voltage-activated 50 lipid phosphatase, CiVSP (71), abolished pore opening but at ~1.6 mM. This concentration is close to the cytoplasmic left voltage sensor activation intact (44). This result suggests concentration of ATP in physiological conditions, which in- that either the VSD-PGD coupling or pore opening was dis- dicates that ATP-dependent activation of the channel plays a physiological role (79). rupted in the absence of PIP2. PIP2 depletion was experi- mentally shown to actually disrupted VSD-PGD coupling It was shown that ATP activates the channel by directly (44). Because of VSD-PGD coupling, constitutive opening binding to KCNQ1. Mutations of residues W379, R380, of the pore, held by a mutation L353K, shifted the voltage K393, and R397 in the cytoplasmic helix A and the A-B dependence of voltage sensor activation to more negative linker (Fig. 1 C) abolished not only channel opening but also the binding of an ATP analog (79), suggesting that these voltages. After PIP2 depletion, the pore of the mutant KCNQ1 was still constitutively open, but the voltage depen- residues form the ATP binding site. In the absence of ATP dence of voltage sensor activation was no longer altered, binding, voltage sensors activated normally, although the indicating that the VSD-PGD coupling was disrupted. channel was not open. The voltage dependence of voltage sensor activation was shifted to more negative voltages in Consistent with the role of PIP2 in mediating VSD-PGD the constitutively open L353K KCNQ1, in both the presence coupling, a PIP2 binding site was found at the interface between each VSD and the PGD, which consists of residues in and absence of ATP. These results indicate that ATP is not the S2-S3 linker, S4-S5 linker, and the cytoplasmic end of required for voltage sensor activation or VSD-PGD coupling; rather, ATP is required for pore opening (79). S6 (Fig. 1 C)(44,72). The location of the PIP2 binding site is consistent with mutational and biochemical results that reveal the importance of some residues in PIP binding 2 KCNE1 alters VSD-PGD coupling of KCNQ1 to to the isolated peptides from the KCNQ1 protein (73) and on selectively modulate different open states KCNQ1 function (74) (for a review, see Zaydman and Cui (19)). This PIP2 binding site is a major physiologically KCNQ1 is associated with the auxiliary subunit KCNE1, important feature of the KCNQ channels that has been pre- which was discovered before KCNQ1 (80,81). Together, served during channel evolution (75). they form the slowly activating IKs channel in the heart, Molecular dynamics simulation and experimental results which is important for action potential repolarization showed that both protein-protein interactions between the (82,83). Mutations in either KCNQ1 or KCNE1 are associ- VSD and PGD and protein-PIP2 interactions are involved ated with long QT syndrome (LQT), which predisposes pa- in VSD-PGD coupling (76). The protein-protein interac- tients to cardiac arrhythmias, resulting in syncope and tions are weakened in KCNQ1 channels by electrostatic in- sudden death (15,84,85). KCNE1 is a peptide of only 129 teractions such as that between R249 in the S4-S5 linker and amino acids and a single transmembrane helix (80,86,87), K358 in the cytoplasmic end of S6. The negative charges in but its association with KCNQ1 drastically alters every the PIP2 head group make salt bridges with basic residues in aspect of channel function. The most prominent changes the S2-S3 linker, S4-S5 linker, and the cytoplasmic end of include an increase in the total current amplitude, a shift S6; the interaction of PIP2 with each individual residue de- in the voltage-dependence of activation toward more depo- pends on the open and closed state of the channel to mediate larized potentials, a prolonged activation and deactivation VSD-PGD coupling. time course (82,83), the removal of inactivation (63,88), Besides the above site at the interface between the VSD a decreased Rbþ/Kþ selectivity (60), altered effects of drugs and PGD, mutations of basic amino acids located in other on channel activity (89-93), and increased effects of protein parts of the KCNQ1 protein, such as in the cytoplasmic he- kinase A (PKA) phosphorylation on channel function

Biophysical Journal 110(1) 14–25 Voltage-Dependent Gating of KCNQ1 19

(94,95). These KCNE1 associated changes in channel the IKs channel, suggested that residues in the transmem- gating, ion selectivity, pharmacology, and posttranslational brane helix of KCNE1 might not be physically close to those modulation are essential to the proper physiological role in the KCNQ1 S6. Thus the functional interactions among of the IKs channels in the heart, and they are fundamental these sites might derive from an allosteric connection to the understanding and therapeutic treatment of diseases (23). Further studies are needed to resolve whether a associated with IKs. For instance, in natural fight or flight re- physical interaction between the transmembrane helix of sponses b-adrenergic stimulation increases the rate and con- KCNE1 and S6 of KCNQ1 is important for KCNE1 to tractile force of the heart; at the same time, the b-adrenergic modulate KCNQ1 function. stimulation reduces action potential duration, in part by KCNE1 alters the environment of S4 during activation enhancing IKs currents to facilitate proper diastolic filling because KCNE1 association alters the accessibility of extra- at faster rates. The b-adrenergic stimulation enhances IKs cellular MTS reagents to engineered cysteine residues in the currents via PKA dependent phosphorylation of KCNQ1 VSD during voltage sensor activation. E160C in S2 is not only in association with KCNE1 (94,95). The interaction be- accessible in KCNQ1 but can be modulated by extracellular tween KCNQ1 and KCNE1 also confers kinetic properties MTS reagents in the presence of KCNE1 (34). T224C in S4 on IKs that make it suitable for participation in rate-depen- is accessible at voltages above 100 mV in the absence of dent adaptation of the cardiac action potential (96,97). KCNE1 but is accessible only in the presence of KCNE1 Corroborating with the importance of IKs in rate-dependent at voltages above 0 mV (51). Tryptophan scanning showed adaptation of the cardiac action potential, patients with IKs- that the same mutations of the S4 residues alter voltage- associated LQT syndrome often have lethal arrhythmia dependent gating differently with or without KCNE1 coex- events during exercise or emotion, when the b-adrenergic pression, which also suggest interactions altered between S4 pathway was stimulated (98,99). LQT mutations that and its environment by KCNE1 association (28,29). abolish the response of IKs to b-adrenergic stimulation Although these differences are prominent for mutations pose a much higher risk of death than other mutations in located over the entire S4, the differences at the lower end KCNQ1 (100-102). How can such a small KCNE1 subunit of S4 were particularly striking. Some of the mutations there alter the channel function so broadly and radically? Is there caused shifts in the voltage dependence of activation in an overarching mechanism for all these changes? These KCNQ1 alone, but almost a total loss of currents in the pres- questions have been a major driving force in studies of the ence of KCNE1 (M238, L239, H240, D242, and R243) (29). KCNQ1 and IKs channels. Recently, it was reported that upon KCNE1 association the Structurally, the transmembrane helix of KCNE1 is interaction between a specific pair of residues, F232 in S4 located in the cleft formed by two adjacent VSDs and the and F279 in S5, hindered activation of the channel (119). PGD (Fig. 1 A)(4,22,23,87,103). This location of KCNE1 KCNE1 also altered the effects of mutations S140G and in the channel complex was identified from disulfide bonds V141M in S1, which are associated with atrial fibrillation, or ion bridges formed between engineered or native cysteine on voltage-dependent gating (59,107,120,121). Consistent residues in KCNE1 and KCNQ1 proteins at extracellular with these changes, VSD movements measured by VCF (23,104-107), transmembrane (108), and cytosolic (109) shifted to more negative voltages in association with (also see (110,111)) domains. The location of KCNE1 in KCNE1 (30,31,52). Nevertheless, KCNE1 association the channel suggests that it may interact with the PGD, does not alter the rate of voltage sensor activation (30- VSD, or both to modulate channel functions. 33,51,52) (for a review, see Liin et al. (122)). Mutations of residues in the transmembrane helix of The interaction of KCNE1 with the PGD and the conse- KCNE1, particularly those of F57, T58, and L59, alter the quent changes in VSD movements cannot directly explain effects of KCNE1 on voltage-dependent gating (112,113). the effects of KCNE1 on voltage-dependent gating. These residues were found to have functional interactions KCNE1 does not reduce the rate of VSD activation, with S338, F339, F340, and A341 located in S6 of although it prolongs the opening and closing time course; KCNQ1, because the effects on voltage-dependent gating KCNE1 shifts the voltage dependence of channel opening by the mutations in KCNE1 were altered by the mutations to more positive voltages, which is opposite to the direction in KCNQ1, and vice versa (36,103,114). These functional of KCNE1-induced change in the voltage dependence of interactions were cited as evidence for physical interaction voltage sensor activation. Furthermore, little attention was between S6 and the transmembrane helix of KCNE1 paid to whether these interactions were important for (87,103,115,116). However, recent studies suggested that KCNE1 modulation of other properties such as ion selec- interactions between the extracellular domain of KCNE1 tivity, pharmacology, or modulation. Therefore, the question with that of the PGD might affect voltage-dependent activa- of how KCNE1 alters such a broad spectrum of channel tion as well (23,117). A homology model and molecular dy- functions so radically has remained unanswered. However, namics simulation of the IKs channel structure based on the building on these results and experimental approaches, crystal structure of the KV1.2/Kv2.1 chimera (118), the more recent studies suggested a novel mechanism that NMR structure of KCNE1 (87), and experimental data on may explain the effects of KCNE1 on channel function.

Biophysical Journal 110(1) 14–25 20 Cui

It was found that, although voltage sensor activation (123). This result indicates that the active-open state showed two resolvable steps in either the absence or pres- observed via macroscopic current and fluorescence record- ence of KCNE1, pore opening at both the intermediate ings is actually quite complex. The various subconductance and activated states of the VSD was observed only in levels may be related to a nonconcerted gating of the four KCNQ1 alone; in the presence of KCNE1 the pore opens KCNQ1 subunits wherein voltage sensor activation in only at the activated state (52). Thus, KCNE1 appeared to each subunit promotes pore opening to a subconductance suppress the intermediate-open state but spare the acti- level, whereas the pore opens fully only when all four vated-open state. This mechanism was further examined voltage sensors are activated (123). A similar mechanism by the coexpression of KCNE1 with the mutant KCNQ1 was also proposed for gating of KCNQ1 channels in the channels E1R/R2E and E1R/R4E, which were constitutively absence of KCNE1 based on time and voltage dependence open because of the VSD being arrested at an intermediate of voltage sensor activation and channel opening (31). In and active state, respectively. Coexpression with KCNE1 a study of channels with different numbers of the KCNQ1 eliminated the currents of E1R/R2E but enhanced the cur- subunit that contained a mutation in the voltage sensor, rents of E1R/R4E, although KCNE1 did not alter the expres- the results suggested that the voltage sensor in each subunit sion of the channel proteins in the cell membrane (52). of both the KCNQ1 and KCNQ1þKCNE1 channels inde- These results suggest that KCNE1 not only suppressed inter- pendently contributed to channel opening (124). Therefore, mediate opening but also enhanced the activated opening. although association with KCNE1 alters VSD-PGD KCNE1 selectively modulates the two open states by coupling, it may not alter the interactions among different altering the VSD-PGD coupling, because mutation F351A subunits. mimicked KCNE1 to suppress the intermediate-open state, The above mechanism explains many effects of KCNE1 producing currents with similar voltage dependence and on channel function. First, a kinetic model depicting the slow opening/closing kinetics. Similar to KCNE1 associa- mechanism through which KCNE1 modulates VSD-PGD tion, F351A also altered the Rbþ/Kþ conductance ratio, coupling to suppress the intermediate-open state and response to XE991, and PIP2 sensitivity of the channel enhance the activated-open state could recapitulate the (48,52). F351 is located at the cytoplasmic end of S6, highly voltage dependence of current onset and of the time course conserved in Kþ channels and important for VSD-PGD of voltage sensor activation/deactivation (52). Second, the coupling (48,65). Likewise, mutations in the S4-S5 linker finding that the channels arrested at the intermediate-open of KCNQ1 that may interact with the cytoplasmic end of (E1R/R2E) and activated-open (E1R/R4E) states show S6 for VSD-PGD coupling, including L251A, V255W, different Rbþ/Kþ permeation ratios and different responses H258A, and T247A, also produced KCNE1-like effects on to XE991 suggests that modulation of the VSD-PGD inter- ionic currents, with a right-shifted voltage dependence of actions by KCNE1 can alter ion selectivity and pharma- activation and reduced opening/closing rates (47). KCNE1 cology. This idea is substantiated by findings that a single association altered the effects of some mutations in the mutation, F351A, which affects VSD-PGD interactions S4-S5 linker (45), suggesting that KCNE1 affects the directly, can also alter ion selectivity and pharmacology. conformation and protein interactions in this region. However, the changes in ion selectivity and drug effects That KCNE1 alters VSD-PGD coupling is also supported by this mutation were not quantitatively the same as by by the findings that KCNE1 association increases the affinity KCNE1, indicating that the effects of KCNE1 on VSD- of KCNQ1 to PIP2 (52,69), which is required to mediate VSD- PGD interactions are probably more complex than those PGD coupling (44). Applying PIP2 to the intracellular solution of the single-point mutation F351A (52). Third, the mecha- rescued currents from run-down in inside-out patch record- nism also contributes to the increase in current amplitude ings, and it was found that the EC50 for KCNQ1þKCNE1 upon association with KCNE1, because of enhanced VSD- was more than 100 times lower than for KCNQ1 alone (69). PGD coupling in the activated-open state (52). Besides PIP2 depletion inhibited currents from KCNQ1 channels but this mechanism, previous studies also suggested other did not inhibit the current from open KCNQ1þKCNE1 chan- mechanisms that may contribute to the increase in current nels; however, PIP2 depletion prevented the closed amplitude by KCNE1. With an increased PIP2 affinity KCNQ1þKCNE1 channels from reopening (52). These re- upon association with KCNE1, the native PIP2 level in the sults indicate that PIP2 affinity for the open KCNQ1þKCNE1 membrane is high enough for most KCNQ1þKCNE1 chan- channel is so high that the bound PIP2 did not dissociate from nels to open. Conversely, for KCNQ1 channels expressed the channel until the channel was closed. Thus, association of alone, given the low PIP2 affinity, the level of PIP2 in the KCNE1 enhances PIP2 affinity for the activated-open state, membrane is not sufficient to saturate the binding sites, which suggests that KCNE1 enhances the VSD-PGD thus limiting channel opening (69). Previous studies also coupling for this state. suggested that the difference in single-channel conductance A recent study found that the single KCNQ1þKCNE1 between KCNQ1 and KCNQ1þKCNE1 might explain the channel exhibited long-lived subconductance levels: the difference in macroscopic current amplitude between the channel could open with five different conductance states two channels (125-127). Using nonstationary noise analysis,

Biophysical Journal 110(1) 14–25 Voltage-Dependent Gating of KCNQ1 21 these studies found that the single-channel conductance was Rbþ concentrations the flicker-open state was favored and smaller in KCNQ1 than in KCNQ1þKCNE1. However, the flickering rate was slowed (60). On the other hand, more recent studies suggest that single-channel behaviors some mutations in the pore domain were found to alter of KCNQ1 and KCNQ1þKCNE1 channels are complex. both inactivation and Rbþ/Kþ conductance ratio, and the ef- These channels may have multiple open states (52,63,64) fects on both were positively correlated (62). The Rbþ/Kþ and subconductance open states (123). There is also a fast conductance ratio also changes upon KCNE1 association flickering process in the KCNQ1 pore that is associated (60). These studies suggest that fast inactivation involves with inactivation (60,62). These data were not available at two open states with different Rbþ/Kþ conductance ratios. the time of the studies using noise analysis, a method that Consistent with the idea that inactivation may involve assumes only a single open state and that the recording conformational changes in the pore domain, it was found bandwidth is sufficient to capture all relevant timescales. that many mutations in the pore domain altered inactivation The observed difference in single-channel conductance (62,128,129). Whether the two open states that are impor- may reflect the combined effects of all these complications. tant for KCNQ1 inactivation are related to the intermedi- In sum, the above results suggest that multiple mechanisms, ate-open and activated-open states, and whether the including the ability of KCNE1 to selectively modulate the association of KCNE1 eliminates inactivation by elimi- two open states, may contribute to the KCNE1 effect on cur- nating one of the open states, are questions yet to be rent amplitude. answered. Another remaining question is whether and how Whether the selective modulation of the two open states the mechanism through which KCNE1 selectively modu- by KCNE1 also underlies the elimination of inactivation re- lates the intermediate-open and activated-open states under- mains to be determined. KCNQ1 channels exhibit an incom- lies the changes in channel modulation by PKA plete inactivation that is masked by the activation process; phosphorylation. inactivation only becomes apparent upon repolarization, when inactivated channels first traverse to an open state CONCLUSIONS before closing, resulting in a hook in the tail current (63,88). Inactivation can be also measured using a triple KCNQ1 is a bona fide voltage-gated Kþ channel, with pulse voltage protocol where a brief hyperpolarizing pulse conserved Kv channel structure, classical properties, and after depolarization allows the channels to recover from general mechanisms of voltage-dependent gating that are inactivation but not to deactivate (88). Subsequent depolar- applicable to all Kv channels. On the other hand, voltage- ization does not activate more channels but elicits a larger dependent gating of KCNQ1 channels reveals novel and current because of the recovery of open channels from inac- unique properties. These include the requirement of intra- tivation. This additional current decays rapidly as the chan- cellular signaling molecules, PIP2 and ATP, for VSD-PGD nels inactivate again. One hallmark of KCNE1 association coupling and pore opening, respectively, and pore opening with KCNQ1 is the suppression of inactivation (63,88). at both the intermediate and activated state of VSD activa- The currents of KCNQ1þKCNE1 do not exhibit a tail tion. The study of these unique properties of KCNQ1 gating hook or a current decay during the triple pulse protocol. provides an excellent opportunity to reveal novel principles Based on the delay of the onset and the voltage depen- for voltage-dependent gating that may be generally appli- dence of inactivation, it was suggested that the model ac- cable to all voltage-gated channels but hidden in the pheno- counting for the fast inactivation of KCNQ1 must contain type of most. at least two open states (63). The existence of two open Voltage-dependent gating is subject to modulations by states in KCNQ1 was also suggested by the studies of posttranslational modification and the binding of signaling Naþ block (64). In Shaker-like Kv channels, inactivation molecules. For instance, phosphorylation of KCNQ1 by occurs when open channels spontaneously enter a noncon- protein kinase A affects voltage-dependent gating in the ducting state at voltages that activate the channel. However, presence of KCNE1 association (94,95), which underscores the behavior of KCNQ1 that was taken as ‘‘fast inactiva- the critical physiological role of b-adrenergic regulation of tion’’ was related to a fast flicker of the open channel that IKs channels in cardiac function. In another example, intra- changed the open probability of the pore, but not to an cellular Ca2þ binds to BK type Kþ channels to increase explicit nonconducting inactivation state (62). The flickers channel activation (130). These modulations, although were initially suggested by a study of single-channel cur- altering voltage-dependent gating, are clearly not a required rents using noise analysis, in which the single-channel cur- part of the mechanism of voltage-dependent gating; i.e., the rent amplitude depended on the frequency bandwidth of channels can be activated by voltage without these modula- recordings (60). The flickers were then related to the fast tions. PIP2 and ATP binding, on the other hand, are required inactivation because the ratio of Rbþ/Kþ conductance in for voltage-dependent gating of KCNQ1. In the absence of KCNQ1 correlated both to the flickers (60) and to the fast PIP2 or ATP binding, the channels cannot open (44,79). It is inactivation (62). In KCNQ1 channels, a large Rbþ/Kþ still not clear if ATP is part of the voltage-dependent gating conductance ratio was attributed to the fact that at high mechanism or is simply required for setting the PGD

Biophysical Journal 110(1) 14–25 22 Cui conformation ready for opening. However, PIP2 binding is a rare opportunity to assess this framework of channel func- critical step during voltage-dependent gating; PIP2 acts as a tion. Remarkably, the intermediate-open and activated- cofactor to mediate VSD-PGD coupling (44,76). These find- open states had different permeation and pharmacological ings reveal that voltage-dependent gating is a molecular pro- properties, and in these two open states the VSD-PGD cou- cess involving not only the ion channel protein alone, but plings were different (52). These results revealed that VSD- also involving other molecules. These findings also provide PGD interactions determine both the open probability and insights into the physiological and pathophysiological roles open conformation. VSD-PGD coupling does not just of the channel. For instance, more than 300 mutations in happen at the end of VSD movements; changes in VSD KCNQ1 are associated with LQT syndrome, of which conformation may alter the conformation of PGD during some were found to reduce PIP2 or ATP binding the entire activation transitions via VSD-PGD interactions. (44,74,77-79). Thus, the modulation of voltage-dependent Therefore, VSD activation and pore opening are not gating by cellular signaling is critical to the physiologic independent, as assumed in models descending from the functions of these channels, and the dependence of activa- Hodgkin and Huxley formulism. Dynamic VSD-PGD inter- tion on multiple stimuli is one of the reasons for the vulner- actions may influence VSD movements during activation ability of channel function to so many mutations. ATP even for those channels that the pore opens only when the dependence of the channel also provides a direct link be- VSD is in the final activated state. tween the electric activities and energetic metabolism of the heart. ACKNOWLEDGMENTS The VSD and PGD in voltage-gated ion channels are distinct structural domains. Functional channels can be I would like to thank Mark Zaydman, Jiajing Xu, Smiruthi Ramasubrama- formed naturally by a PGD without a VSD (131,132)or nian, Panpan Hou, Ling Zhong, and Yoram Rudy for reading the manuscript by a PGD artificially isolated from Kv (133,134) or Nav and providing helpful comments. Ling Zhong and Panpan Hou made Fig. 1. (135) channels. Likewise, the VSD identified in CiVSP This work was supported by NIH Grants R01-HL70393 and R01- and voltage-sensor only proteins (VSOP) (71,136,137) and NS092570. isolated from Kv channels adopt structures similar to the VSD in voltage-gated ion channels (138-140). 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