Phuong T. Nguyen

In vivo optophysiology reveals that G-protein activation triggers osmotic swelling and increased light scattering of rod photoreceptors

Significance Complete activation of the phototransduction G-protein cascade of dark-adapted rod photoreceptors causes outer segments to undergo 10% elongation and large local increases in backscattering, as measured in vivo with noninvasive, high-resolution optical coherence tomography. Maximal elongation is caused by a potentially harmful 20% increase in internal osmotic pressure generated by excess osmolytes arising from phototransduction. The light-stimulated elongation and backscattering responses can be explained by an osmo-elastic model of cytoplasmic swelling, combined with changes in refractive index consequent to the swelling and translocation of the G-protein subunits into the cytosol. Disease conditions that affect the structural integrity of rods may cause them to be especially vulnerable to osmotic stress caused by bright light. The light responses of rod and cone photoreceptors have been studied electrophysiologically for decades, largely with ex vivo approaches that disrupt the photoreceptors’ subretinal microenvironment. Here we report the use of optical coherence tomography (OCT) to measure light-driven signals of rod photoreceptors in vivo. Visible light stimulation over a 200-fold intensity range caused correlated rod outer segment (OS) elongation and increased light scattering in wild-type mice, but not in mice lacking the rod G-protein alpha subunit, transducin (Gαt), revealing these responses to be triggered by phototransduction. For stimuli that photoactivated one rhodopsin per Gαt the rod OS swelling response reached a saturated elongation of 10.0 ± 2.1%, at a maximum rate of 0.11% s−1. Analyzing swelling as osmotically driven water influx, we find the H2O membrane permeability of the rod OS to be (2.6 ± 0.4) × 10−5 cm⋅s−1, comparable to that of other cells lacking aquaporin expression. Application of Van’t Hoff’s law reveals that complete activation of phototransduction generates a potentially harmful 20% increase in OS osmotic pressure. The increased backscattering from the base of the OS is explained by a model combining cytoplasmic swelling, translocation of dissociated G-protein subunits from the disc membranes into the cytoplasm, and a relatively higher H2O permeability of nascent discs in the basal rod OS. Translocation of phototransduction components out of the OS may protect rods from osmotic stress, which could be especially harmful in disease conditions that affect rod OS structural integrity.

A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel

Voltage‐gated sodium channels (NaVs) are activated by transiting the voltage sensor from the deactivated to the activated state. The crystal structures of several bacterial NaVs have captured the voltage sensor module (VSM) in an activated state, but structure of the deactivated voltage sensor remains elusive. In this study, we sought to identify peptide toxins stabilizing the deactivated VSM of bacterial NaVs. We screened fractions from several venoms and characterized a cystine knot toxin called JZTx‐27 from the venom of tarantula Chilobrachys jingzhao as a high‐affinity antagonist of the prokaryotic NaVs NsVBa (nonselective voltage‐gated Bacillus alcalophilus) and NaChBac (bacterial sodium channel from Bacillus halodurans) (IC50 = 112 nM and 30 nM, respectively). JZTx‐27 was more efficacious at weaker depolarizing voltages and significantly slowed the activation but accelerated the deactivation of NsVBa, whereas the local anesthetic drug lidocaine was shown to antagonize NsVBa without affecting channel gating. Mutation analysis confirmed that JZTx‐27 bound to S3–4 linker of NsVBa, with F98 being the critical residue in determining toxin affinity. All electrophysiological data and in silico analysis suggested that JZTx‐27 trapped VSM of NsVBa in one of the deactivated states. In mammalian NaVs, JZTx‐27 preferably inhibited the inactivation of NaV1.5 by targeting the fourth transmembrane domain. To our knowledge, this is the first report of peptide antagonist for prokaryotic NaVs. More important, we proposed that JZTx‐27 stabilized the NsVBa VSM in the deactivated state and may be used as a probe to determine the structure of the deactivated VSM of NaVs.—Tang, C., Zhou, X., Nguyen, P. T., Zhang, Y., Hu, Z., Zhang, C., Yarov‐Yarovoy, V., DeCaen, P. G., Liang, S., Liu, Z. A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel. FASEB J. 31, 3167–3178 (2017). www.fasebj.org

Structural Basis for Antiarrhythmic Drug Interactions with the Human Cardiac Sodium Channel

The human voltage-gated sodium channel, hNav1.5, is responsible for the rapid upstroke of the cardiac action potential and is target for antiarrhythmic therapy. Despite the clinical relevance of hNav1.5 targeting drugs, structure-based molecular mechanisms of promising or problematic drugs have not been investigated at atomic scale to inform drug design. Here, we used Rosetta structural modeling and docking as well as molecular dynamics simulations to study the interactions of antiarrhythmic and local anesthetic drugs with hNav1.5. These calculations revealed several key drug binding sites formed within the pore lumen that can simultaneously accommodate up to two drug molecules. Molecular dynamics simulations identified a hydrophilic access pathway through the intracellular gate and a hydrophobic access pathway through a fenestration between domains III and IV. Our results advance the understanding of molecular mechanisms of antiarrhythmic and local anesthetic drug interactions with hNav1.5 and will be useful for rational design of novel therapeutics.

Gain-of-function mutation of a voltage-gated sodium channel NaV1.7 associated with peripheral pain and impaired limb development

Dominant mutations in voltage-gated sodium channel NaV1.7 cause inherited erythromelalgia, a debilitating pain disorder characterized by severe burning pain and redness of the distal extremities. NaV1.7 is preferentially expressed within peripheral sensory and sympathetic neurons. Here, we describe a novel NaV1.7 mutation in an 11-year-old male with underdevelopment of the limbs, recurrent attacks of burning pain with erythema, and swelling in his feet and hands. Frequency and duration of the episodes gradually increased with age, and relief by cooling became less effective. The patients sister had short stature and reported similar complaints of erythema and burning pain, but with less intensity. Genetic analysis revealed a novel missense mutation in NaV1.7 (2567G>C; p.Gly856Arg) in both siblings. The G856R mutation, located within the DII/S4-S5 linker of the channel, substitutes a highly conserved non-polar glycine by a positively charged arginine. Voltage-clamp analysis of G856R currents revealed that the mutation hyperpolarized (−11.2 mV) voltage dependence of activation and slowed deactivation but did not affect fast inactivation, compared with wild-type channels. A mutation of Gly-856 to aspartic acid was previously found in a family with limb pain and limb underdevelopment, and its functional assessment showed hyperpolarized activation, depolarized fast inactivation, and increased ramp current. Structural modeling using the Rosetta computational modeling suite provided structural clues to the divergent effects of the substitution of Gly-856 by arginine and aspartic acid. Although the proexcitatory changes in gating properties of G856R contribute to the pathophysiology of inherited erythromelalgia, the link to limb underdevelopment is not well understood.