Angela R. Schubert

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.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu. Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability. The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques. In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest(1). This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

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.

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.