The functional properties of KCNQ1 channels are highly dependent on associated KCNE β subunits. Mutations in KCNQ1 or KCNE subunits can cause congenital channelopathies, such as deafness, cardiac arrhythmias, and epilepsy. The mechanism by which KCNE1 beta subunits slow the kinetics of KCNQ1 channels is a matter of current controversy. Here we show that KCNQ1/KCNE1 channel activation occurs in two steps: first, mutually independent voltage sensor movements in the four KCNQ1 subunits generate the main gating charge movement and underlie the initial delay in the activation time course of KCNQ1/KCNE1 currents. Second, a slower and concerted conformational change of all four voltage sensors and the gate, which opens the KCNQ1/KCNE1 channel. Our data show that KCNE1 divides the voltage sensor movement into two steps with widely different voltage dependences and kinetics. The two voltage sensor steps in KCNQ1/KCNE1 channels can be pharmacologically isolated and further separated by a disease-causing mutation.

Voltage-gated proton (Hv) channels play an essential role in phagocytic cells by generating a hyperpolarizing proton current that electrically compensates for the depolarizing current generated by the NADPH oxidase during the respiratory burst, thereby ensuring a sustained production of reactive oxygen species by the NADPH oxidase in phagocytes to neutralize engulfed bacteria. Despite the importance of the voltage-dependent Hv current, it is at present unclear which residues in Hv channels are responsible for the voltage activation. Here we show that individual neutralizations of three charged residues in the fourth transmembrane domain, S4, all reduce the voltage dependence of activation. In addition, we show that the middle S4 charged residue moves from a position accessible from the cytosolic solution to a position accessible from the extracellular solution, suggesting that this residue moves across most of the membrane electric field during voltage activation of Hv channels. Our results show for the first time that the charge movement of these three S4 charges accounts for almost all of the measured gating charge in Hv channels.

Voltage-gated proton (Hv1) channels are involved in many physiological processes, such as pH homeostasis and the innate immune response. Zn 2+ is an important physiological inhibitor of Hv1. Sperm cells are quiescent in the male reproductive system due to Zn 2+ inhibition of Hv1 channels, but become active once introduced into the low-Zn 2+ -concentration environment of the female reproductive tract. How Zn 2+ inhibits Hv1 is not completely understood. In this study, we use the voltage clamp fluorometry technique to identify the molecular mechanism of Zn 2+ inhibition of Hv1. We find that Zn 2+ binds to both the activated closed and resting closed states of the Hv1 channel, thereby inhibiting both voltage sensor motion and gate opening. Mutations of some Hv1 residues affect only Zn 2+ inhibition of the voltage sensor motion, whereas mutations of other residues also affect Zn 2+ inhibition of gate opening. These effects are similar in monomeric and dimeric Hv1 channels, suggesting that the Zn 2+ -binding sites are localized within each subunit of the dimeric Hv1. We propose that Zn 2+ binding has two major effects on Hv1: (i) at low concentrations, Zn 2+ binds to one site and prevents the opening conformational change of the pore of Hv1, thereby inhibiting proton conduction; and (ii) at high concentrations, Zn 2+ , in addition, binds to a second site and inhibits the outward movement of the voltage sensor of Hv1. Elucidating the molecular mechanism of how Zn 2+ inhibits Hv1 will further our understanding of Hv1 function and might provide valuable information for future drug development for Hv1 channels.Hv1 | voltage-gated proton channel | Zn2 + | inhibition | molecular model

KCNE β-subunits assemble with and modulate the properties of voltage-gated K + channels. In the heart, KCNE1 associates with the α-subunit KCNQ1 to generate the slowly activating, voltagedependent potassium current (I Ks ) in the heart that controls the repolarization phase of cardiac action potentials. By contrast, in epithelial cells from the colon, stomach, and kidney, KCNE3 coassembles with KCNQ1 to form K + channels that are voltageindependent K + channels in the physiological voltage range and important for controlling water and salt secretion and absorption. How KCNE1 and KCNE3 subunits modify KCNQ1 channel gating so differently is largely unknown. Here, we use voltage clamp fluorometry to determine how KCNE1 and KCNE3 affect the voltage sensor and the gate of KCNQ1. By separating S4 movement and gate opening by mutations or phosphatidylinositol 4,5-bisphosphate depletion, we show that KCNE1 affects both the S4 movement and the gate, whereas KCNE3 affects the S4 movement and only affects the gate in KCNQ1 if an intact S4-to-gate coupling is present. Further, we show that a triple mutation in the middle of the transmembrane (TM) segment of KCNE3 introduces KCNE1-like effects on the second S4 movement and the gate. In addition, we show that differences in two residues at the external end of the KCNE TM segments underlie differences in the effects of the different KCNEs on the first S4 movement and the voltage sensor-togate coupling.V oltage-gated K + (Kv) channels are mainly expressed in excitable cells, where changes in the voltage across the membrane, such as action potentials, demand rapid channel activation and deactivation. Among Kv channels, the KCNQ1 channel (also called Kv7.1 or KvLQT1) differs from most other Kv channels in that KCNQ1 plays key physiological roles in nonexcitable cells, such as in epithelia, in addition to its roles in excitable cells, such as cardiomyocytes. The KCNQ1 channels display dramatically different biophysical properties in various cell types, differences that are thought to be mainly due to the KCNQ1 channel's ability to associate with five tissue-specific KCNE β-subunits to form different K + channel complexes (1-7).KCNQ1 subunits expressed by themselves form voltage-dependent K + channels that open at negative voltages (1, 2) ( Fig. 1 A and D, black squares). However, coassembly of KCNQ1 with KCNE1 (also called MinK) produces a much slower activating potassium current (I Ks ) in the heart that activates at positive voltages ( Fig. 1 B and D, black triangles) and shapes the repolarization phase of cardiac action potentials (1, 2). Mutations in the KCNQ1/KCNE1 complex are linked to life-threatening cardiac arrhythmias, such as torsade de pointes (8, 9). Association of KCNQ1 with KCNE3 (also called MiRP2) produces channels that are voltage-independent in the physiological voltage range (Fig. 1 C and D, black circles) and that are crucial in regulating the transport of water and salt in several epithelial tissues, including the colon, small intestine, and airways (3, 10, 1...

KCNE β-subunits assemble with and modulate the properties of voltage-gated K + channels. In the colon, stomach, and kidney, KCNE3 coassembles with the α-subunit KCNQ1 to form K + channels important for K + and Cl − secretion that appear to be voltageindependent. How KCNE3 subunits turn voltage-gated KCNQ1 channels into apparent voltage-independent KCNQ1/KCNE3 channels is not completely understood. Different mechanisms have been proposed to explain the effect of KCNE3 on KCNQ1 channels. Here, we use voltage clamp fluorometry to determine how KCNE3 affects the voltage sensor S4 and the gate of KCNQ1. We find that S4 moves in KCNQ1/KCNE3 channels, and that inward S4 movement closes the channel gate. However, KCNE3 shifts the voltage dependence of S4 movement to extreme hyperpolarized potentials, such that in the physiological voltage range, the channel is constitutively conducting. By separating S4 movement and gate opening, either by a mutation or PIP 2 depletion, we show that KCNE3 directly affects the S4 movement in KCNQ1. Two negatively charged residues of KCNE3 (D54 and D55) are found essential for the effect of KCNE3 on KCNQ1 channels, mainly exerting their effects by an electrostatic interaction with R228 in S4. Our results suggest that KCNE3 primarily affects the voltage-sensing domain and only indirectly affects the gate.proteins with a variety of crucial physiological roles. Most Kv channels are expressed in excitable cells where, e.g., they regulate and modulate the resting potential and the threshold and duration of the action potential (1). The KCNQ1 channel (also called Kv7.1 or KvLQT1) differs from most other Kv channels in that it has key physiological roles in both excitable cells, such as cardiomyocytes (2, 3) and pancreatic β-cells (4, 5), and in nonexcitable cells, such as in epithelia (3, 6). The KCNQ1 channels display diverse biophysical properties in different cell types, a diversity thought to be mainly due to the KCNQ1 channel's association with five tissue-specific, single-transmembrane segment KCNE β-subunits (KCNE1-5) (7-13). KCNQ1 α-subunit expressed by itself forms a voltage-dependent K + channel that opens at negative voltages ( Fig. 1 A and D). However, coexpression of KCNQ1 with KCNE1 slows the kinetics of activation and shifts the voltage dependence of activation to positive voltages ( Fig. 1 B and D) (7, 8), thereby generating the slowly activating, voltage-dependent I Ks current that controls the repolarization phase of cardiac action potentials. In contrast, coexpression of KCNQ1 with KCNE3 results in a constitutively conducting channel in the physiological voltage range of -80 to +40 mV ( Fig. 1 C and D), which is important for transport of water and salt in epithelial tissues, including those of the colon, small intestine, and airways (9,14,15). In addition, mutations of KCNE3 have been associated with cardiac arrhythmia (16,17) and diseases in the inner ear, such as Meniere's disease and tinnitus (18,19). Because KCNQ1/KCNE3 channels are necessary for water and salt secretion...

Repolarization and termination of the ventricular cardiac action potential is highly dependent on the activation of the slow delayed-rectifier potassium IKs channel. Disruption of the IKs current leads to the most common form of congenital long QT syndrome (LQTS), a disease that predisposes patients to ventricular arrhythmias and sudden cardiac death. We previously demonstrated that polyunsaturated fatty acid (PUFA) analogues increase outward K+ current in wild type and LQTS-causing mutant IKs channels. Our group has also demonstrated the necessity of a negatively charged PUFA head group for potent activation of the IKs channel through electrostatic interactions with the voltage-sensing and pore domains. Here, we test whether the efficacy of the PUFAs can be tuned by the presence of different functional groups in the PUFA head, thereby altering the electrostatic interactions of the PUFA head group with the voltage sensor or the pore. We show that PUFA analogues with taurine and cysteic head groups produced the most potent activation of IKs channels, largely by shifting the voltage dependence of activation. In comparison, the effect on voltage dependence of PUFA analogues with glycine and aspartate head groups was half that of the taurine and cysteic head groups, whereas the effect on maximal conductance was similar. Increasing the number of potentially negatively charged moieties did not enhance the effects of the PUFA on the IKs channel. Our results show that one can tune the efficacy of PUFAs on IKs channels by altering the pKa of the PUFA head group. Different PUFAs with different efficacy on IKs channels could be developed into more personalized treatments for LQTS patients with a varying degree of IKs channel dysfunction.

Polyunsaturated fatty acids with double bonds closer to the head group had higher apparent affinity for I channels and increased I current more; shifting the bonds further away from the head group reduced apparent binding affinity for and effects on the I current. Interestingly, we found that ω-6 and ω-9 PUFAs, with the first double bond closer to the head group, left-shifted the voltage dependence of activation the most. These results allow for informed design of new therapeutics targeting I channels in Long QT Syndrome.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential for rhythmic activity in the heart and brain, and mutations in HCN channels are linked to heart arrhythmia and epilepsy. HCN channels belong to the family of voltage-gated K + (Kv) channels. However, why HCN channels are activated by hyperpolarization whereas Kv channels are activated by depolarization is not clear. Here we reverse the voltage dependence of HCN channels by mutating only two residues located at the interface between the voltage sensor and the pore domain such that the channels now open upon depolarization instead of hyperpolarization. Our data indicate that what determines whether HCN channels open by hyperpolarizations or depolarizations are small differences in the energies of the closed and open states, due to different interactions between the voltage sensor and the pore in the different channels.