|Session Assignment: 1602|
|PRE-CRYSTALLIZATION AND FUNCTIONAL ANALYSIS OF HUMAN VOLTAGE-GATED PROTON CHANNEL|
|Author: Amruta Agharkar||Presenter: Amruta Agharkar|
|Department: Pharmacology & Neuroscience|
|Research Area: Receptor Pharmacology & Drug Delivery|
|(1) Hv1 proton channel, (2) Baculovirus expression system, (3) Chromatography|
|1) Amruta Agharkar Dept. of Pharmacology and Neuroscience UNT Health Science Center Fort Worth, TX-76107 2) Eric B. Gonzales, Ph.D. Assistant Professor, Dept. Pharmacology and Neuroscience UNT Health Science Center. Institute for Aging and Alzheimer's Disease Research Cardiovascular Research Institute|
|Short Description: The research focuses on pre-crystallization analysis of human voltage-gated proton channel (Hv1) towards determining the three-dimensional crystal structure. The Hv1 proton channel plays an important role in male fertility, stroke and respiratory burst. Recently, it has been found that Hv1 proton channel is over-expressed in breast cancer cells and helps them to survive and proliferate. The Hv1 proton channel pumps out the excessive acidity (protons) form cancer cells and maintains the intracellular pH optimal for survival. The study discusses various techniques used for isolation and purification of proton channel and detergents used to solubilize and test the channel's stability outside the plasma membrane. Determining the three-dimensional crystal structure of Hv1 proton channel will provide us with the template for designing of new drugs.|
Purpose: The human voltage-gated proton channel (Hv1) is a proton-selective channel implicated in the efflux of protons and is found as a homodimer. Each subunit of the Hv1 proton channel forms the proton-selective pore. Recently, it has been shown that Hv1 is overexpressed in metastaic breast cancer cells and helps in cancer cell proliferation and migration. Although the voltage-sensing domain of the Hv1 proton channel is similar to the voltage sensor of voltage gated channels that have determined three-dimensional structures, the Hv1 proton channel structure remains elusive. Determining the structure of Hv1 channel will serve as a template for designing new therapeutic strategies. Here, we present our progress towards solving the three-dimensional structure of the Hv1 proton channel.
Methods: The full length Hv1 proton channel gene was combined with a carboxyl-terminal 6Xhis tag within a baculovirus insect expression vector. Small-scale expression experiments for detergent screening followed by metal affinity chromatography and size exclusion chromatography (SEC) are used to assess protein stability. Our purification and stability assays are coupled with whole cell patch-clamp electrophysiology technique to determine Hv1 construct functionality.
Results: The Hv1 channel gene was subcloned into a vector for insect cell expression. The construct obtained produced a histidine tagged protein with a molecular weight comparable to Hv1 protein. The generated Hv1 proton channel has been isolated and assessed for stability and monodispersity using size exclusion chromatography.
Conclusions: Based on our preliminary data, the current Hv1 proton channel construct has potential as a protein crystallographic construct. Future experiments will focus on generating large quantities of purified and stable Hv1 proton channel and initiating protein crystallization trials.
|UNT Health Science Center Internal Seed Grant|