Huntingtin-associated Protein 1 Participates In Transport Of Potassium Ion Channel In PC12 Cell

Huntingtin-associated protein-1 (HAP1) is the first protein identified to interact with the N-terminus of huntingtin(htt). Hap1's binding capacity with mutant huntingtin(mHtt), which leads to neurodegenerative disorder Huntington's disease(HD), is much stronger than normal Htt. Earlier studies proved that, HAP1 widely expressed in the central and peripheral nervous system, especially in the olfactory bulb, hypothalamus, brain stem, spinal cord dorsal horn and other parts where HAP1 expresses in a high level. Later studies also found that, HAP1 not only expresses in the nervous system, but also in gonads and the endocrine system. HAP1 selective expression in endocrine cells, that secrete proteins, peptides and other nitrogen-containing monoamine hormones et al. These cells are widely distributed in the pituitary, thyroid, adrenal medulla, pancreatic islets, gastrointestinal tract and other organs. Rat adrenal pheochromocytoma cells (PC12) cells also express HAP1. HAP1 has no transmembrane domain and nuclear localization signal, so it's a cytoplasmic protein.Although the function of HAP1 is not so clear at present, series of studies show that HAP1 relates to the transport of organelles and molecules and also the transfer and recycle of membrane receptor in neurons. Especially HAP1 plays an important role in intracellular transport based on microtubules. Hap1 regulates or participates in the transport of vesicles in neurons,neurotrophic factors,receptors based on microtubules by the interactions with the Kinesin related motor protein including kinesin light chain (KLC) and kinesin heavy chain (KHC), P150Glued, a subunit of dynactin (a protein inducing the binding of dynactin to microtubules and two motor protein) and kinesin family motor protein 5 (KIF5).Ion channels are important functional proteins on membrane. Ion channels are transported to the Golgi complex through microtubule after synthesis in the rough endoplasmic reticulum and then transported to the cell surface through vesicles. PC12 cells express voltage-gated potassium channels(Kv),Ca2+-activated potassium channels(KCa),ATP-sensitive potassium channels(KATP),inward rectifier potassium channels(Kir) et al,that play a important role in stabling the resting membrane potential, regulating action potential amplitude, duration and frequency, and influcing vesicles secretory. To prove that HAP1 participates in the transport of potassium channels from the rough endoplasmic reticulum /Golgi complex to the cell surface.We observed the influence on membrane electrophysiological properties and contents of potassium channels, when HAP1 was silenced in PC12 cells.Silenceing HAP1 inhibited the delayed rectifier potassium current in cytoplasmic membrane of PC12 cell If HAP1 participates in the transport of potassium channels from the rough endoplasmic reticulum / Golgi complex to the cell surface, when HAP1 was silenced, the number of potassium channels on membrane would reduce and then possium current also would decrease. In voltage-clamp mode, we can record the synthetic current in PC12, including inward Na+ and Ca2+ current and outward K+ currents. And we discoveried Na+ and Ca2+ current are relatively small, while the potassium current is large. When HAP1 was silenced, K+ current significantly reduced in PC12 cells.Silencing HAP1 reduced Kv2.1 on cytoplasmic membrane of PC12 cell To prove that the reduction of potassium currents on the cell membrane of PC12 is related to the silence of HAP1, we observed the effect of HAP1 silencing on the mRNA expression of Kv2.1 in PC12 cells. The slightly up-regulation of the Kv2.1 mRNA expression, instead of down- regulation, indicates the supposition that HAP1 has no inhibitory effect of the expression of Kv2.1, from which we can infer that the decrease of the membrane potassium current in PC12 cells is not the result of the potassium channel expression reduction.To prove that the reduction of potassium currents on the cell membrane of PC12 is related to the silence of HAP1, We observated the effect of HAP1 silencing on the transport of Kv2.1 from cytoplasm to cell membrane in PC12 cells. The immunofluorescence shows the increase of HA immunoreactivity in the cytoplasm and the decrease of that in the plasma membrane after co-transfection of HAP-siRNA and GFP-Kv2.1-HA, which indicates that the silenceing HAP1 results in the decrease transport of Kv2.1 from the cytoplasm to the cell membrane.