The neuronal choline transporter CHT1 is regulated by immunosuppressor-sensitive pathways

Area of Science:

• Neuroscience
• Pharmacology
• Molecular Biology

Background:

• Cyclosporin A (CsA) is an immunosuppressor that inhibits cyclophilins and calcineurin.
• Calcineurin, a phosphatase, plays roles in cellular signaling pathways.
• Cholinergic neurotransmission relies on efficient choline uptake.

Purpose of the Study:

• To investigate the effect of immunosuppressors on choline uptake in Torpedo electric organ nerve endings.
• To elucidate the molecular mechanisms underlying CsA-induced inhibition of choline transport.
• To identify the role of calcineurin in regulating the high-affinity choline transporter 1 (CHT1).

Main Methods:

• Isolation of peripheral nerve endings from Torpedo electric organ.
• Measurement of choline uptake and hemicholinium-3 binding.
• Cloning and functional reconstitution of Torpedo CHT1 (tCHT1) in Xenopus oocytes.
• Inhibition studies using CsA, FK506, and rapamycin.

Main Results:

• Cyclosporin A specifically inhibited choline uptake by up to 40% without affecting choline transporter binding.
• FK506 inhibited the rate of choline transport, while rapamycin reduced choline transporter affinity.
• Calcineurin activity inhibition, via CsA, FK506, or calcineurin autoinhibitory domain, reduced tCHT1 activity.
• Rapamycin's effect on affinity suggests an immunophilin-mediated mechanism involving proline isomerization in tCHT1.

Conclusions:

• The phosphatase calcineurin regulates the activity of the high-affinity choline transporter 1 (CHT1).
• Cyclosporin A and FK506 likely target calcineurin to inhibit choline transport rate.
• Rapamycin may inhibit choline uptake by altering tCHT1 affinity through an immunophilin-dependent pathway.