The voltage-gated L-type Ca2+ channel CaV1.2 is crucial for initiating heartbeat and control of a number of neuronal functions such as neuronal excitability and long-term potentiation. Mutations of CaV1.2 subunits result in serious health problems including arrhythmia, autism spectrum disorders, immunodeficiency, and hypoglycemia. Thus precise control of CaV1.2 surface expression and localization is essential. We previously reported that α-actinin associates and colocalizes with neuronal CaV1.2 channels and that shRNA-mediated depletion of α-actinin significantly reduces localization of endogenous CaV1.2 in dendritic spines in hippocampal neurons. Here we investigated the hypothesis that direct binding of α-actinin to CaV1.2 supports its surface expression. Using two-hybrid screens and pull-down assays we identified three point mutations (K1647A, Y1649A, and I1654A) in the central, pore-forming α11.2 subunit of CaV1.2 that individually impaired α-actinin binding. Surface biotinylation and flow cytometry assays revealed that CaV1.2 channels composed of the corresponding α-actinin-binding-deficient mutants results in a 35–40% reduction in surface expression compared to wild-type channels. Moreover, the mutant CaV1.2 channels expressed in HEK293 cells exhibit a 60–75% reduction in current density. The larger decrease in current density as compared to surface expression imparted by these α11.2 subunit mutations hints at the possibility that α-actinin not only stabilizes surface localization of CaV1.2 but also augments its ion conducting activity.

1A primary goal of molecular physiology is to understand how conformational changes of 2 proteins affect the function of cells, tissues, and organisms. Here, we describe an imaging 3 method for measuring the conformational changes of a voltage-sensing protein within tissue. We 4 synthesized a fluorescent molecular probe, compatible with two-photon microscopy, that targets 5 a resting conformation of Kv2-type voltage gated K + channel proteins. Voltage-response 6 characteristics were used to calibrate a statistical thermodynamic model relating probe labeling 7 intensity to the conformations adopted by unlabeled Kv2 proteins. Two-photon imaging of rat 8 brain slices labeled with the probe revealed fluorescence consistent with conformation-selective 9 labeling of endogenous neuronal Kv2 proteins. In principle, this method of quantifying 10 endogenous protein conformational change from fluorescence images is generalizable to other 11 proteins labeled with conformation-selective probes. 12

A primary goal of molecular physiology is to understand how conformational changes of proteins affect the function of cells, tissues, and organisms. Here, we describe an imaging method for measuring the conformational changes of the voltage sensors of endogenous ion channel proteins within live tissue, without genetic modification. We synthesized GxTX-594, a variant of the peptidyl tarantula toxin guangxitoxin-1E, conjugated to a fluorophore optimal for two-photon excitation imaging through light-scattering tissue. We term this tool EVAP (Endogenous Voltage-sensor Activity Probe). GxTX-594 targets the voltage sensors of Kv2 proteins, which form potassium channels and plasma membrane–endoplasmic reticulum junctions. GxTX-594 dynamically labels Kv2 proteins on cell surfaces in response to voltage stimulation. To interpret dynamic changes in fluorescence intensity, we developed a statistical thermodynamic model that relates the conformational changes of Kv2 voltage sensors to degree of labeling. We used two-photon excitation imaging of rat brain slices to image Kv2 proteins in neurons. We found puncta of GxTX-594 on hippocampal CA1 neurons that responded to voltage stimulation and retain a voltage response roughly similar to heterologously expressed Kv2.1 protein. Our findings show that EVAP imaging methods enable the identification of conformational changes of endogenous Kv2 voltage sensors in tissue.

The voltage-gated sodium NaV1.7 channel plays a key role as a mediator of action potential propagation in C-fiber nociceptors and is an established molecular target for pain therapy. ProTx-II is a potent and moderately selective peptide toxin from tarantula venom that inhibits human NaV1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human NaV1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with IC50s of 7 nM and 4 nM for hNaV1.7 and more than 1,000-fold selectivity over human NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.8, and NaV1.9 channels. PTx2-3127 inhibits NaV1.7 currents in mouse and human sensory neurons and shows efficacy in rat models of chronic and thermal pain when administered intrathecally. Rationally-designed peptide inhibitors of human NaV1.7 channels have transformative potential to define a new class of biologics to treat pain.

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