Voltage-Gated Calcium Channels in Neurons Derived from Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

Abstract

Voltage-gated calcium channels couple pre-synaptic action potentials to transmitter release in neurons. In this study, we characterized voltage-activated Ca2+ channels expressed in neurons differentiated from human embryonic stem cell (hESC) or induced pluripotent stem cell (iPSC)-derived long-term self-renewing neuroepithelial-like stem cells (lt-NES) at one to three months of differentiation in vitro. Using barium (Ba2+) as the charge carrier, activation and inactivation of voltage-dependent calcium channels were investigated by the whole-cell configuration of the patch-clamp technique. In lt-NES-derived neurons, we found both non-inactivating high-voltage-activated (HVA) and inactivating low-voltage-activated (LVA) Ca2+ currents. Pharmacological characterization of high-threshold Ca2+ currents was attempted using nifedipine (10 μM ), ω-conotoxin-GVIA (ω-CgTx GVIA, 3μM) and ω-agatoxin-IVA (ω-Aga IVA, 200 nM), which block L, N, and P/Q-type Ca2+ currents, respectively. By analysing the averaged current in response to a square depolarizing voltage step protocol from a holding potential of −110 mV to 0 mV in the presence of these specific blockers, we could show that lt-NES cell-derived neurons express N and P/Q-type high-threshold Ca2+ channels. We also observed Ba2+ currents activated at voltages below −40 mV that were sensitive to NiCl2 (100 μM), indicating that T-type Ca2+ channels also contribute to Ca2+ currents in these neurons. Furthermore, the pharmacology of calcium channels involved in synaptic transmission was analyzed with specific agonists and antagonists of different high-voltage-activated calcium channels. Bath application of ω-CgTx GVIA (3μM) or ω-Aga IVA (200 nM) reduced the frequency of spontaneous postsynaptic currents (sPSCs). Thus, neurons differentiated from lt-NES express both high-threshold and low-threshold voltage-gated Ca2+ channels, and both N and P/Q-type Ca2+ channels mediate synaptic transmission in these neurons. Correction et al.Biophysical JournalApril 16, 2013In Brief2013. Voltage-Gated Calcium Channels Exist and Function as Fragment-Channels in Neuronal Plasma Membranes. Ioannis E. Michailidis, Kathryn Abele, Wei K. Zhang, Lawrence Geyman, Bochao Lin, Yong Yu, Eftychios Pnevmatikakis, Jian Yang. Biophys. J. 104(2)s: 462a. Full-Text PDF Open Archive