Functional Characterization of the Human β2b I354T Mutation Associated with Epilepsy

Epilepsy is characterized by spontaneous seizures that can cause brain damage. In epilepsy, the normal pattern of neuronal activity becomes disturbed, causing convulsions, muscle spasms, and loss of consciousness. Epilepsy may develop because of an abnormality in brain wiring, an imbalance in neurotransmitters, changes in ion channels, or some combination of these factors.

Ion channels are cell membrane proteins that allow the passage of ions, such as calcium, into or out of the cell, which generates the electrical signals of neural networks. My research project will focus on the involvement of a mutation in a voltage-gated calcium channel in epilepsy. Voltage-gated calcium channels have a pore through which calcium passes, and one or more auxiliary subunits that regulate pore opening and closing. The auxiliary subunit that my research will focus on is the β subunit. The β subunit functions in delivering the calcium channel to the cell membrane and regulating activation and inactivation kinetics of the ion channel. The mutation in the β subunit that I will be studying is the I354T mutation, which was found in a cohort of epileptic patients, but not unaffected individuals.

In order to study how the I354T mutation alters β subunit function and thus voltage-gated calcium channel function, site-directed mutagenesis was performed with QuickChange II XL kit to introduce the desired mutation into the wild-type β subunit. Now that the desired mutation is obtained, RNA is going to be synthesized for the I354T mutant. The RNA will then be injected into frog oocytes, which allows for the wild-type and mutant β subunit to be studied. Then, two-electrode voltage clamp will be performed by inserting two glass microelectrodes into the oocyte. One electrode applies the voltage to activate the voltage-gated calcium channels while the other electrode records the resulting currents. This is done to compare the functions of the wild-type and mutant subunits.

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