Document Type

Open Access Thesis

Embargo Period

2-24-2017

Degree Program

Molecular & Cellular Biology

Degree Type

Master of Science (M.S.)

Year Degree Awarded

2017

Month Degree Awarded

May

Advisor Name

John

Advisor Middle Initial

M.

Advisor Last Name

Clark

Abstract

Microtransplantation of mammalian neurolemma into Xenopus laevis oocytes has been used to study ion channels in terms of their structure and function in the central nervous system. Use of microtransplanted neurolemma is advantageous in that tissue can be obtained from various sources, ion channels and receptors are present in their native configuration and they can be used to evaluate numerous channelpathies caused by environmental toxicants. Here we show that Xenopus oocytes injected with fragments of rat brain neurolemma successfully express functional native ion channels that are assembled in their own plasma membrane. Using a high throughput two electrode voltage clamp (TEVC) electrophysiological system, currents that were sensitive to tetrodotoxin (TTX), omega-conotoxin MVIIC, and tetraethylammonium (TEA) were detected, indicating the presence of multiple voltage-sensitive ion channels (voltage-sensitive sodium, calcium and potassium channels, respectively). In this current research, a “proof-of-principle” experiment was conducted where TTX-sensitive voltage-sensitive sodium channel (VSSC) currents were measured. VSSCs are a well-established site of action for 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) but not for its non-toxic metabolite 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene (DDE). A differential sensitivity of DDT versus DDE on TTX-sensitive sodium current in neurolemma-injected oocytes was determined. DDT elicited an increase in depolarization-dependent, TTX-sensitive sodium current while DDE had no significant effect. Additionally, DDT resulted in a slowing of sodium channel inactivation kinetics whereas DDE has no similar effect. These results are consistent with the findings obtained using heterologous expression of single isoforms of rat brain VSSCs by injecting cRNA into Xenopus oocytes. By demonstrating the classic structural activity relationship of DDT and DDE on mammalian voltage-gated sodium channels isolated in rat brain neurolemma, this study supports the use of automated high-throughput electrophysiology to study the effects of various environmental toxicants on multiple mammalian cellular targets. More importantly, using rat brain neurolemma ensures that the proteins of interest have been transcribed and have undergone all the necessary post-translational modifications before they were injected and expressed in the Xenopus oocytes which is not the case for traditional heterologous expression.

First Advisor

John M. Clark