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

Abstract

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 four (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 (VCF). 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 two Brugada Syndrome (BrS) mutations (A735V and G752R). A735V shifted DII-VSD voltage-dependence to depolarized potentials, while G752R significantly slowed DII-VSD kinetics. Both mutations slowed INa activation, even though 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 ofINa activation and myocyte excitability. Conclusions Our results reveal novel mechanisms whereby the NaV1.5 VSDs regulate its activation and inactivation. The ability to distinguish distinct molecular mechanisms of proximal BrS mutations demonstrates the potential of these methods to reveal how inherited mutations, post-translational modifications and anti-arrhythmic drugs alter NaV1.5 at the molecular level.

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

“…Tracking DIV-VSD conformation during the steady-state inactivation protocol reveals a striking correlation between DIV-VSD activation and closed-state inactivation ( Fig. 2) (Varga et al, 2015), similar to previous findings in Nav1.4, where DIV-VSD activation was found to be the rate limiting step of channel inactivation (Capes et al, 2013).…”

“…We found that the A735V mutation dramatically shifted DII-VSD activation to depolarized potentials, while the G752R mutation significantly slowed DII-VSD kinetics ( Fig. 3B) (Varga et al, 2015). The ability of the channel to open at potentials negative to the DII-VSD activation in A735V and at times prior to activation in G752R, confirmed our initial suspicion that the DII-VSD is a facilitator of activation, but is not absolutely required for opening.…”

“…To probe similar phenomena in the cardiac channel, we recently developed VCF protocols to track each of the VSDs of Na V 1.5. Out of 16 positions attempted, we observed robust signals with DI-V215C, DII-S805C, DIII-M1296C, and DIV-S1618C (Varga et al, 2015 voltage-dependent kinetics. The DI, DII, and DIII VSDs each activated much more rapidly than DIV (Fig.…”

“…These steps were then followed by a 50 ms step to À120 mV before returning to the holding potential. G-V curve (black circles), SSI (black squares), and the corresponding fluorescence voltage relationship FeV curve (color circles and squares, respectively) (Varga et al, 2015 flux induces intracellular Ca 2þ release from the sarcoplasmic reticulum, which in turn causes myocyte contraction. Similar to Na V 1.5, the Ca v 1.2 a subunit contains four transmembrane domains (DI-DIV) connected by intracellular linkers, and each is composed of six transmembrane spanning segments S1eS6 with the VSDs formed by S1eS4 and the pore by S5eS6 (Catterall, 2011 /Camodulin binding to the C-terminus (Imredy and Yue, 1994;Zühlke et al, 1999).…”

“…B) Normalized fluorescence traces of DII-A735V (left) and DII-G752R (right) (green), compared to WT DII (black). G-V curve (black circles), SSI (black squares), and the corresponding fluorescence voltage relationship FeV curve (green circles and squares, respectively) of DII-A735V (left) and DII-G752R (right) (Varga et al, 2015). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64accessory subunits, including the a 2 d subunits, a g subunit, and multiple b subunits (b 1 e b 4 ).…”