Wandi Zhu

Direct Measurement of Cardiac Na+ Channel Conformations Reveals Molecular Pathologies of Inherited Mutations.

Background—Dysregulation of voltage-gated cardiac Na+ channels (NaV1.5) by inherited mutations, disease-linked remodeling, and drugs causes arrhythmias. The molecular mechanisms whereby the NaV1.5 voltage-sensing domains (VSDs) are perturbed to pathologically or therapeutically modulate Na+ current (INa) have not been specified. Our aim was to correlate INa kinetics with conformational changes within the 4 (DI–DIV) VSDs to define molecular mechanisms of NaV1.5 modulation. Method and Results—Four NaV1.5 constructs were created to track the voltage-dependent kinetics of conformational changes within each VSD, using voltage-clamp fluorometry. Each VSD displayed unique kinetics, consistent with distinct roles in determining INa. In particular, DIII-VSD deactivation kinetics were modulated by depolarizing pulses with durations in the intermediate time domain that modulates late INa. We then used the DII-VSD construct to probe the molecular pathology of 2 Brugada syndrome mutations (A735V and G752R). A735V shifted DII-VSD voltage dependence to depolarized potentials, whereas G752R significantly slowed DII-VSD kinetics. Both mutations slowed INa activation, although DII-VSD activation occurred at higher potentials (A735V) or at later times (G752R) than ionic current activation, indicating that the DII-VSD allosterically regulates the rate of INa activation and myocyte excitability. Conclusions—Our results reveal novel mechanisms whereby the NaV1.5 VSDs regulate channel activation and inactivation. The ability to distinguish distinct molecular mechanisms of proximal Brugada syndrome mutations demonstrates the potential of these methods to reveal how inherited mutations, post-translational modifications, and antiarrhythmic drugs alter NaV1.5 at the molecular level.

Mechanisms of noncovalent β subunit regulation of NaV channel gating

Voltage-gated Na+ (NaV) channels comprise a macromolecular complex whose components tailor channel function. Key components are the non-covalently bound &bgr;1 and &bgr;3 subunits that regulate channel gating, expression, and pharmacology. Here, we probe the molecular basis of this regulation by applying voltage clamp fluorometry to measure how the &bgr; subunits affect the conformational dynamics of the cardiac NaV channel (NaV1.5) voltage-sensing domains (VSDs). The pore-forming NaV1.5 &agr; subunit contains four domains (DI–DIV), each with a VSD. Our results show that &bgr;1 regulates NaV1.5 by modulating the DIV-VSD, whereas &bgr;3 alters channel kinetics mainly through DIII-VSD interaction. Introduction of a quenching tryptophan into the extracellular region of the &bgr;3 transmembrane segment inverted the DIII-VSD fluorescence. Additionally, a fluorophore tethered to &bgr;3 at the same position produced voltage-dependent fluorescence dynamics strongly resembling those of the DIII-VSD. Together, these results provide compelling evidence that &bgr;3 binds proximally to the DIII-VSD. Molecular-level differences in &bgr;1 and &bgr;3 interaction with the &agr; subunit lead to distinct activation and inactivation recovery kinetics, significantly affecting NaV channel regulation of cell excitability.

Molecular motions that shape the cardiac action potential: Insights from voltage clamp fluorometry

Very recently, voltage-clamp fluorometry (VCF) protocols have been developed to observe the membrane proteins responsible for carrying the ventricular ionic currents that form the action potential (AP), including those carried by the cardiac Na(+) channel, NaV1.5, the L-type Ca(2+) channel, CaV1.2, the Na(+)/K(+) ATPase, and the rapid and slow components of the delayed rectifier, KV11.1 and KV7.1. This development is significant, because VCF enables simultaneous observation of ionic current kinetics with conformational changes occurring within specific channel domains. The ability gained from VCF, to connect nanoscale molecular movement to ion channel function has revealed how the voltage-sensing domains (VSDs) control ion flux through channel pores, mechanisms of post-translational regulation and the molecular pathology of inherited mutations. In the future, we expect that this data will be of great use for the creation of multi-scale computational AP models that explicitly represent ion channel conformations, connecting molecular, cell and tissue electrophysiology. Here, we review the VCF protocol, recent results, and discuss potential future developments, including potential use of these experimental findings to create novel computational models.

Regulation of Na+ channel inactivation by the DIII and DIV voltage-sensing domains

Functional eukaryotic voltage-gated Na+ (NaV) channels comprise four domains (DI–DIV), each containing six membrane-spanning segments (S1–S6). Voltage sensing is accomplished by the first four membrane-spanning segments (S1–S4), which together form a voltage-sensing domain (VSD). A critical NaV channel gating process, inactivation, has previously been linked to activation of the VSDs in DIII and DIV. Here, we probe this interaction by using voltage-clamp fluorometry to observe VSD kinetics in the presence of mutations at locations that have been shown to impair NaV channel inactivation. These locations include the DIII–DIV linker, the DIII S4–S5 linker, and the DIV S4-S5 linker. Our results show that, within the 10-ms timeframe of fast inactivation, the DIV-VSD is the primary regulator of inactivation. However, after longer 100-ms pulses, the DIII–DIV linker slows DIII-VSD deactivation, and the rate of DIII deactivation correlates strongly with the rate of recovery from inactivation. Our results imply that, over the course of an action potential, DIV-VSDs regulate the onset of fast inactivation while DIII-VSDs determine its recovery.

Depolarization of the conductance-voltage relationship in the NaV1.5 mutant, E1784K, is due to altered fast inactivation

E1784K is the most common mixed long QT syndrome/Brugada syndrome mutant in the cardiac voltage-gated sodium channel NaV1.5. E1784K shifts the midpoint of the channel conductance-voltage relationship to more depolarized membrane potentials and accelerates the rate of channel fast inactivation. The depolarizing shift in the midpoint of the conductance curve in E1784K is exacerbated by low extracellular pH. We tested whether the E1784K mutant shifts the channel conductance curve to more depolarized membrane potentials by affecting the channel voltage-sensors. We measured ionic currents and gating currents at pH 7.4 and pH 6.0 in Xenopus laevis oocytes. Contrary to our expectation, the movement of gating charges is shifted to more hyperpolarized membrane potentials by E1784K. Voltage-clamp fluorimetry experiments show that this gating charge shift is due to the movement of the DIVS4 voltage-sensor being shifted to more hyperpolarized membrane potentials. Using a model and experiments on fast inactivation-deficient channels, we show that changes to the rate and voltage-dependence of fast inactivation are sufficient to shift the conductance curve in E1784K. Our results localize the effects of E1784K to DIVS4, and provide novel insight into the role of the DIV-VSD in regulating the voltage-dependencies of activation and fast inactivation.

Mechanisms and models of cardiac sodium channel inactivation

ABSTRACT Shortly after cardiac Na+ channels activate and initiate the action potential, inactivation ensues within milliseconds, attenuating the peak Na+ current, INa, and allowing the cell membrane to repolarize. A very limited number of Na+ channels that do not inactivate carry a persistent INa, or late INa. While late INa is only a small fraction of peak magnitude, it significantly prolongs ventricular action potential duration, which predisposes patients to arrhythmia. Here, we review our current understanding of inactivation mechanisms, their regulation, and how they have been modeled computationally. Based on this body of work, we conclude that inactivation and its connection to late INa would be best modeled with a “feet-on-the-door” approach where multiple channel components participate in determining inactivation and late INa. This model reflects experimental findings showing that perturbation of many channel locations can destabilize inactivation and cause pathological late INa.

DIII of Voltage-Gated Na+ Channels Interacts With Inactivation in the Time Domain of Intermediate Inactivation

Background: Dysregulation of human cardiac voltage-gated Na+ channel (hNaV1.5) inactivation predisposes patients to sudden death. hNaV1.5 comprises a requisite α-subunit that contains four domains (DI-DIV); each of these contains a voltage sensing domain (VSD). We have shown that prolonged depolarizing pulses uniquely immobilize the DIII-VSD. However, the significance of this immobilization and its potential connection to inactivation has not been explored. Here, we observe activation of the VSDs in the presence of mutations that disrupt inactivation.Methods: Previously, we created four DNA constructs with a cysteine introduced into the DI-DIV VSDs. Channels encoded by these constructs were expressed in Xenopus oocytes, and cysteine-labeled with TAMRA-MTS fluorophores. Ionic current and fluorescence emission that reflected changes in VSD conformation were simultaneously recorded using the cut-open oocyte configuration.Results: A hydrophobic motif (IFM) on the DIII-DIV linker is essential for inactivation, and we disrupted it with the F1486Q mutation. With F1486Q, activation of the DI-, DII- and DIII-VSDs was unaffected, while DIV-VSD activation was shifted to depolarized potentials (DIV-VSD WT-V1/2=-61.8±3.5 mV, IQM-V1/2=-48.9±3.5 mV, p=0.03), stabilizing its rested stated. Previously, we observed that DIII-VSD deactivation was significantly slowed after prolonged depolarizing pulses (t10-90%=11.6±1.9 ms after 1 ms, 18.6±1.6 ms after 200 ms, p=0.02). In contrast, F1486Q caused faster DIII-VSD deactivation (t10-90%=16.3±2.2 ms after 1 ms, 8.8±0.9 ms after 200 ms, p=0.01), implying the IFM motif also stabilizes the DIII-VSD activated state, but only after prolonged pulses.Conclusions: Our results show a clear interaction of the DIII-VSD with the IFM motif in the 100 ms time domain, which is essential for regulating late Na+ current (INa,L). Our results indicate that INa,L, whose enhancement predisposes patients with heart-failure, diabetes and ischemia to arrhythmia, might be regulated by therapeutically targeting the DIII-VSD.

Class I Anti-Arrhythmics Differentially Regulate Cardiac Sodium Channel Voltage-Sensing Domains

Background: Class I anti-arrhythmics target cardiac voltage-gated Na+ channels (NaV1.5). The NaV1.5 α-subunit contains four domains (DI-DIV) with six membrane-spanning segments (S1-S6). S1-S4 form voltage-sensing domains (VSDs), and S5-S6 create the ion-conducting pore. The distinct therapeutic action of subclasses Ia, Ib, and Ic have been traditionally attributed to differences in NaV1.5 access and pore-binding rate. However, others have shown that lidocaine, a local anesthetic and Class Ib anti-arrhythmic interacts with the muscle Na+ channel, NaV1.4, DIII-VSD. Thus, we tested the hypothesis that Class I drug interaction with the NaV1.5 VSDs significantly determines the therapeutic phenotype.Methods: Previously, we created four NaV1.5 DNA constructs with a cysteine introduced to the extracellular S4 of individual VSDs. Channels encoded by these constructs are expressed in Xenopus oocytes and cysteine-labeled with the TAMRA-MTS fluorophore. Ionic current and fluorescence emission corresponding to changes in VSD conformation are then simultaneously recorded using the cut-open oocyte configuration. After control recordings, anti-arrhythmics are administered to the internal solution. When currents are 80% blocked, VSD kinetics are measured.Results: We have not observed significant interaction of Class I drugs with the DI, DII, or DIV VSDs. In contrast, quinidine, lidocaine, and ranolazine all uniquely shift DIII-VSD activation. During control recordings, we observe DIII-VSD activation has V1/2=-108.96±2.72mV. Lidocaine and ranolazine both induced a hyperpolarizing DIII-VSD activation shift (V1/2=-147.28±4.17mV, S.E.M., p=0.003, V1/2=-143.09±1.94mV, S.E.M, p=0.001, respectively) while quinidine caused a large depolarizing shift (V1/2=-80.67±4.39mV, S.E.M, p=0.007).Conclusions: Drug interaction with the DIII-VSD has been tightly linked to use-dependent block of the late Na+ current, a hallmark class Ib drugs. In contrast, class Ia drugs typically reduce peak Na+. Because the DIII-VSD is tightly coupled to NaV channel gating, we propose that the differences in DIII-VSD interaction are determining their unique therapeutic phenotypes.