Pharmacological Screening of Kv7.1 and Kv7.1/KCNE1 Activators as Potential Antiarrhythmic Drugs in the Zebrafish Heart.

Long QT syndrome (LQTS) can lead to ventricular arrhythmia and sudden cardiac death. The most common congenital cause of LQTS is mutations in the channel subunits generating the cardiac potassium current IKs. Zebrafish (Danio rerio) have been proposed as a powerful system to model human cardiac diseases due to the similar electrical properties of the zebrafish heart and the human heart. We used high-resolution all-optical electrophysiology on ex vivo zebrafish hearts to assess the effects of IKs analogues on the cardiac action potential. We found that chromanol 293B (an IKs inhibitor) prolonged the action potential duration (APD) in the presence of E4031 (an IKr inhibitor applied to drug-induced LQT2), and to a lesser extent, in the absence of E4031. Moreover, we showed that PUFA analogues slightly shortened the APD of the zebrafish heart. However, PUFA analogues failed to reverse the APD prolongation in drug-induced LQT2. However, a more potent IKs activator, ML-277, partially reversed the APD prolongation in drug-induced LQT2 zebrafish hearts. Our results suggest that IKs plays a limited role in ventricular repolarizations in the zebrafish heart under resting conditions, although it plays a more important role when the IKr is compromised, as if the IKs in zebrafish serves as a repolarization reserve as in human hearts. This study shows that potent IKs activators can restore the action potential duration in drug-induced LQT2 in the zebrafish heart.

Similar voltage-sensor movement in spHCN channels can cause closing, opening, or inactivation.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to the rhythmic firing of pacemaker neurons and cardiomyocytes. Mutations in HCN channels are associated with cardiac arrhythmia and epilepsy. HCN channels belong to the superfamily of voltage-gated K+ channels, most of which are activated by depolarization. HCN channels, however, are activated by hyperpolarization. The mechanism behind this reversed gating polarity of HCN channels is not clear. We here show that sea urchin HCN (spHCN) channels with mutations in the C-terminal part of the voltage sensor use the same voltage-sensor movement to either close or open in response to hyperpolarizations depending on the absence or presence of cAMP. Our results support that non-covalent interactions at the C-terminal end of the voltage sensor are critical for HCN gating polarity. These interactions are also critical for the proper closing of the channels because these mutations exhibit large constitutive currents. Since a similar voltage-sensor movement can cause both depolarization- and hyperpolarization-activation in the same channel, this suggests that the coupling between the voltage sensor and the pore is changed to create channels opened by different polarities. We also show an identical voltage-sensor movement in activated and inactivated spHCN channels and suggest a model for spHCN activation and inactivation. Our results suggest the possibility that channels open by opposite voltage dependence, such as HCN and the related EAG channels, use the same voltage-sensor movement but different coupling mechanisms between the voltage sensor and the gate.

A second S4 movement opens hyperpolarization-activated HCN channels.

Rhythmic activity in pacemaker cells, as in the sino-atrial node in the heart, depends on the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. As in depolarization-activated K+ channels, the fourth transmembrane segment S4 functions as the voltage sensor in hyperpolarization-activated HCN channels. But how the inward movement of S4 in HCN channels at hyperpolarized voltages couples to channel opening is not understood. Using voltage clamp fluorometry, we found here that S4 in HCN channels moves in two steps in response to hyperpolarizations and that the second S4 step correlates with gate opening. We found a mutation in sea urchin HCN channels that separate the two S4 steps in voltage dependence. The E356A mutation in S4 shifts the main S4 movement to positive voltages, but channel opening remains at negative voltages. In addition, E356A reveals a second S4 movement at negative voltages that correlates with gate opening. Cysteine accessibility and molecular models suggest that the second S4 movement opens up an intracellular crevice between S4 and S5 that would allow radial movement of the intracellular ends of S5 and S6 to open HCN channels.

A general mechanism of KCNE1 modulation of KCNQ1 channels involving non-canonical VSD-PD coupling.

Voltage-gated KCNQ1 channels contain four separate voltage-sensing domains (VSDs) and a pore domain (PD). KCNQ1 expressed alone opens when the VSDs are in an intermediate state. In cardiomyocytes, KCNQ1 co-expressed with KCNE1 opens mainly when the VSDs are in a fully activated state. KCNE1 also drastically slows the opening of KCNQ1 channels and shifts the voltage dependence of opening by >40 mV. We here show that mutations of conserved residues at the VSD-PD interface alter the VSD-PD coupling so that the mutant KCNQ1/KCNE1 channels open in the intermediate VSD state. Using recent structures of KCNQ1 and KCNE beta subunits in different states, we present a mechanism by which KCNE1 rotates the VSD relative to the PD and affects the VSD-PD coupling of KCNQ1 channels in a non-canonical way, forcing KCNQ1/KCNE1 channels to open in the fully-activated VSD state. This would explain many of the KCNE1-induced effects on KCNQ1 channels.

Polyunsaturated fatty acid analogues differentially affect cardiac NaV, CaV, and KV channels through unique mechanisms.

The cardiac ventricular action potential depends on several voltage-gated ion channels, including NaV, CaV, and KV channels. Mutations in these channels can cause Long QT Syndrome (LQTS) which increases the risk for ventricular fibrillation and sudden cardiac death. Polyunsaturated fatty acids (PUFAs) have emerged as potential therapeutics for LQTS because they are modulators of voltage-gated ion channels. Here we demonstrate that PUFA analogues vary in their selectivity for human voltage-gated ion channels involved in the ventricular action potential. The effects of specific PUFA analogues range from selective for a specific ion channel to broadly modulating cardiac ion channels from all three families (NaV, CaV, and KV). In addition, a PUFA analogue selective for the cardiac IKs channel (Kv7.1/KCNE1) is effective in shortening the cardiac action potential in human-induced pluripotent stem cell-derived cardiomyocytes. Our data suggest that PUFA analogues could potentially be developed as therapeutics for LQTS and cardiac arrhythmia.

Gating mechanism of hyperpolarization-activated HCN pacemaker channels.

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.

Polyunsaturated fatty acids produce a range of activators for heterogeneous IKs channel dysfunction.

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.

ω-6 and ω-9 polyunsaturated fatty acids with double bonds near the carboxyl head have the highest affinity and largest effects on the cardiac IKs potassium channel.

AIM: The IKs channel is important for termination of the cardiac action potential. Hundreds of loss-of-function mutations in the IKs channel reduce the K+ current and, thereby, delay the repolarization of the action potential, causing Long QT Syndrome. Long QT predisposes individuals to Torsades de Pointes which can lead to ventricular fibrillation and sudden death. Polyunsaturated fatty acids (PUFAs) are potential therapeutics for Long QT Syndrome, as they affect IKs channels. However, it is unclear which properties of PUFAs are essential for their effects on IKs channels. METHODS: To understand how PUFAs influence IKs channel activity, we measured effects on IKs current by two-electrode voltage clamp while changing different properties of the hydrocarbon tail. RESULTS: There was no, or weak, correlation between the tail length or number of double bonds in the tail and the effects on or apparent binding affinity for IKs channels. However, we found a strong correlation between the positions of the double bonds relative to the head group and effects on IKs channels. CONCLUSION: Polyunsaturated fatty acids with double bonds closer to the head group had higher apparent affinity for IKs channels and increased IKs current more; shifting the bonds further away from the head group reduced apparent binding affinity for and effects on the IKs 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 IKs channels in Long QT Syndrome.

KCNE1 and KCNE3 modulate KCNQ1 channels by affecting different gating transitions.

KCNE ß-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, voltage-dependent potassium current (IKs) 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 voltage-independent 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-to-gate coupling.

KCNE3 acts by promoting voltage sensor activation in KCNQ1.

KCNE ß-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 voltage-independent. 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 PIP2 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.

KCNE1 divides the voltage sensor movement in KCNQ1/KCNE1 channels into two steps.

The functional properties of KCNQ1 channels are highly dependent on associated KCNE-ß subunits. Mutations in KCNQ1 or KCNE subunits can cause congenital channelopathies, such as deafness, cardiac arrhythmias and epilepsy. The mechanism by which KCNE1-ß 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.

Molecular mechanism of voltage sensing in voltage-gated proton channels.

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.

CE-108, a new macrolide tetraene antibiotic.

In the search for strains producing antifungal compounds, a new tetraene macrolide CE-108 (3) has been isolated from culture broth of Streptomyces diastaticus 108. In addition, the strain also produces the previously described tetraene rimocidin (1) and also the aromatic polyketide oxytetracycline. Both tetraene compounds, structurally related, are produced in a ratio between 25 to 35% (CE-108 compared to rimocidin), although it can be inverted toward CE-108 production by changing the composition of the fermentation medium. This paper deals with the characterization of the producer strain, fermentation, purification, structure determination and biological properties of the new macrolide tetraene CE-108.