Date of Award

2008

Document Type

Thesis

Degree Name

Bachelor of Science

Department

Biology

First Advisor

Dr. Linda Boland

Abstract

Potassium channel interacting proteins (KChIPs) co-assemble with Kv4 a subunits to form native complexes that encode considerable components of neuronal somatodendritic A- type K+current (Is,J in neurons, and transient outward current (ho) in cardiac myocytes. The binding of KChIPs to the cytoplasmic N-termini of Kv4 a subunits enhances surface expression by facilitating intracellular trafficking, stimulates subunit assembly, and regulates the functional gating properties of Kv4 channels. KChIPs, like many other neuronal calcium sensor proteins, contain four EF-hand calcium binding sites; two of which (EF-3 and 4) have particularly high affinity for calcium. Because Ca2+ is an important signaling molecule for cell physiology, it is suggested that Ca2+ may be an important factor for KChIP interactions with Kv4. In addition, it has been observed in many studies that polyunsaturated fatty acids (PUFAs), such as docosahexanoic acid (DHA) and arachidonic acid (AA) modulate A-type peak current and inactivation kinetics. In this study, we tested the hypothesis that PUFA modulation of Kv4/KChIP peak current and kinetics is sensitive to internal Ca2+concentration. Using 50 μM BAPTA-AM to lower intracellular Ca2+, results showed no difference of Kv4/KChlP kinetics or modulation by DHA among cells recorded under normal Ca2+ and low Ca2+. These results were confirmed in cells expressing mutant forms of KChIP2c, which lacked either high affinity Ca2+ binding site EF-3 or 4. Control cells injected with mSlo, a channel activated by intracellular Ca2+, exhibited a rightward shift of activation when treated with BAPTA-AM, suggesting that this treatment was sufficient. To test the effects of high intracellular Ca2+,cells were incubated in 10 μM A23187, a calcium ionophore. Under increasing Ca2+ conditions (0.1, 0.5, 1.0, and 1.8 mM), there was no apparent change inKv4/KChIP activation kinetics. mSlo- injected cells displayed a leftward shift of activation when treated with A23187 and were used as a positive control. Together, our results suggest that Kv4/KChIP kinetic properties and PUFA modulation are not directly sensitive to intracellular Ca2+levels.

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