Mohamad, Fatin Hilyani
(2016)
The response of synthetic 4-hydroxybenzoic acid on kv1.4 potassium channel subunit expressed in xenopus laevis oocytes.
Masters thesis, Universiti Sains Malaysia.
Abstract
Extensive researches are being made on natural products especially herbs and plants
that have long been practiced in traditional medicines such as Chinese and
Ayurvedic that have been used to treat neuronal disorders such as convulsive,
dizziness and headaches. 4-hydroxybenzoic acid is a non-flavonoid phenol found
abundantly in Dendrocalamus asper shoots (bamboo), fruits (strawberries and
apples) and flowers. In this study, the response of synthetic 4-hydroxybenzoic acid
was tested on Kv1.4 potassium channel that was expressed in Xenopus laevis oocytes
as the model system. Kv1.4 is a rapidly inactivating Shaker-related member of the
voltage-gated potassium channels with two inactivation mechanisms; the fast N-type
and slow C-type. It plays vital roles in repolarization, hyperpolarization and
signaling the restoration of resting membrane potential through the regulation of the
movement of K+ across the cellular membrane. cRNA of Kv1.4 prepared during
molecular work was injected into viable oocytes that was extracted through surgery
at the lower abdomen of X.laevis. The current produced from K ions were detected
by the two-microelectrode voltage clamp (TEVC) method, holding potential starting
from -80mV with 20mV step-up until +80mV. Readings of treatments with 0.1%
DMSO, 4-Hba concentrations and K channel blockers were taken at +60mV. The
ratio of tail / peak amplitude is the index of the activity of the Kv1.4 channels with n≥ 6 (number of oocytes tested). Lower ratio signifies lower membrane potential and
enhancement of Kv1.4 channel inactivation. The decreases of the ratios of 5
different concentrations (1μM, 10μM, 100μM, 1mM and 2.5mM) were compared
with 0.1% DMSO as the control. All concentration showed statistically significant
results with p < 0.05 except for 100μM. The normalized current of the 4-hba
concentrations were compared with potassium channel blockers (TEA and 4-AP) and
all groups showed statistically significant results. This study also showed that time
taken for each concentration to affect Kv1.4 does not play any significant roles. In
conclusion, 4-hydroxybenzoic acid was found to be able to enhance the inactivation
of Kv1.4 by lowering the membrane potential so that the abnormal neuronal firing
can be inhibited which can be seen through comparison of DMSO and potassium
channel blockers. With IC50 slightly higher than 10μM, increasing concentrations
(100μM, 1mM and 2.5mM) had shown to exhibit toxicity effects. The best
concentration from this study is 10μM with Hill slope of 0.1799.
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