Abstract
There is increasing evidence that the fast-inactivating potassium current IA, encoded by KV3. 4 channels, plays an important role in Alzheimer's Disease (AD), since the neurotoxic β-amyloid peptide1-42 (Aβ1−42) increases the IA current triggering apoptotic processes. The specific inhibition of KV3.4 by the marine toxin extracted from Anemonia sulcata, named blood depressing substance-I (BDS-I), reverts the Aβ peptide-induced cell death. The aim of the present study was to identify the smallest fragments of BDS-I, obtained by peptide synthesis, able to inhibit KV3.4 currents. For this purpose, whole-cell patch clamp technique was used to evaluate the effects of BDS-I fragments on KV3.4 currents in CHO cells heterologously expressing KV3.4. We found that BDS-I[1-8] fragment, containing the N-terminal octapeptide sequence of full length BDS-I, was able to inhibit KV3.4 currents in a concentration dependent manner, whereas the scrambled sequence of BDS-I[1-8] and all the other fragments obtained from BDS-I full length were ineffective. As we demonstrated in a previous study, BDS-I full length is able to counteract Aβ1−42-induced enhancement of KV3.4 activity, preventing Aβ1−42-induced caspase-3 activation and the abnormal nuclear morphology in NGF-differentiated PC-12 cells. Similarly to BDS-I, we found that BDS-I[1-8] blocking KV3.4 currents prevented Aβ1−42-induced caspase-3 activation and apoptotic processes. Collectively, these results suggest that BDS-I[1-8] could represent a lead compound to be developed as a new drug targeting KV3.4 channels.
Highlights
Voltage-gated potassium (KV) channels are transmembrane proteins with strong selectivity for K+ ions and sensitivity to voltage changes
To identify the smallest blood depressing substance-I (BDS-I) amino acid sequence able to block Kv3.4 activity, we evaluated the effect of the newly synthesized blood depressing substance (BDS)-I fragments in Chinese hamster ovary (CHO) cells transiently transfected with Kv3.4/MinK-related peptide 2 (MiRP2), by patch clamp technique in whole cell configuration
Electrophysiological experiments performed in CHO cells transiently transfected with KV3.4/MiRP2 showed that BDS-I[1-8scr] at the same concentration of 100 nM was not able to modify KV3.4 activity (Figures 4A,B)
Summary
Voltage-gated potassium (KV) channels are transmembrane proteins with strong selectivity for K+ ions and sensitivity to voltage changes. KV channels are key regulators of membrane excitability, resting membrane potentials and spontaneous firing rate, action potential waveform and duration, neurotransmitter release, and apoptosis (Rudy et al, 1999). Several neuronal populations in the central nervous system (CNS) are required to generate action potentials with high frequency (Coetzee et al, 1999). KV channels of the KV3 family are prominently expressed in these neurons and have a central role in facilitating sustained and/or repetitive high frequency firing (Moreno et al, 2001). Many evidence have been provided revealing the increase of KV3.4 protein expression in several pathological conditions such as hypoxia (Kaab et al., 2005), oxidative stress (Song et al, 2017), and neurodegeneration (Baranauskas et al, 2003; Angulo et al, 2004; Boscia et al, 2017)
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