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Convergence of Voltage Gated Ion Channel Location and Activity in Mammalian Neurons


Voltage gated ion channels underlie the electrical activity of cells. Here I present three data chapters addressing a central question, where does voltage gated ion channel activity occur in neurons? The voltage-gated sodium channel Nav1.7 plays a key role in action potential propagation of C-fiber nociceptors and is an established molecular target for pain therapy. In chapter 2 of this thesis, I find that non-peptidergic nociceptors expressed detectable Nav1.7 protein. I identify that application of a designed peptide, PTx2-3127, inhibits NaV1.7 currents and reduces the number of action potentials fired in mouse non-peptidergic nociceptors. These results providing a mechanism for analgesic activity of the designed peptide.

The two members of the Kv2 family of voltage-gated potassium channels Kv2.1 and Kv2.2 play important roles in brain neurons to mediate action potential firing and to organize specialized membrane contact sites. However, little is known about the cellular expression and subcellular localization of Kv2.1 and Kv2.2 in somatosensory neurons. In chapter 3 of this thesis, I use a panel of Kv2.1 and Kv2.2 specific antibodies and a set of genetically modified mouse lines to define the cellular expression and subcellular localization of Kv2.1 and Kv2.2 protein in dorsal root ganglion (DRG) neurons. I find that Kv2 channels have enriched subcellular localization at the plasma membrane distinct from other ion channel proteins in the DRG. While most DRG neurons co-express Kv2.1 and Kv2.2, distinct neuron subpopulations show preferential expression of one or the other. I show that proprioceptors express Kv2.2 robustly but have little detectable Kv2.1. In contrast to Kv2.1, higher levels of Kv2.2 were observed in DRG neurons relative to spinal cord neurons. Kv2.1 expression decreases in older mice while Kv2.2 does not. I show that while both Kv2.1 and Kv2.2 are localized to the soma and stem axon of DRG neurons, Kv2.2 is distinct in being found at juxtaparanodes and paranodes of myelinated DRG neuron axons. Both Kv2 subtypes adopt clustered subcellular patterns in DRG neurons that are similar but distinct from those in central neurons. I find that the overall patterns of Kv2 channel protein expression and subcellular localization are similar between mouse and human DRG neurons.

A primary goal of molecular physiology is to understand how conformational changes of proteins affect the function of cells, tissues, and organisms. In chapter 4 of this thesis, I describe an imaging method for measuring the conformational changes of the voltage sensors of endogenous ion channel proteins within live tissue, without genetic modification. GxTX-594 was synthesized from a variant of the peptidyl tarantula toxin guangxitoxin-1E and conjugated to a fluorophore optimal for two-photon excitation imaging through light-scattering tissue. 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. I 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. These findings show that EVAP imaging methods enable the identification of conformational changes of endogenous Kv2 voltage sensors in tissue.

Together the chapters of this thesis investigate the intersection of localization and activity of voltage gated ion channel subtypes. The work presented here identifies where functional Nav1.7 and Kv2 channels are and how the channels are uniquely positioned in specific anatomical contexts.

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