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Two types of high voltage-activated calcium channels in SH-SY5Y human neuroblastoma cells
1Department of Pharmacology, Northwestern University Medical School, Chicago, IL 60611.
Abstract:
Voltage-activated calcium channel currents were recorded from differentiated human neuroblastoma cells. SK-N-SH-SY5Y (SH-SY5Y) line, using patch-clamp techniques. Experimental solutions were designed to suppress sodium and potassium channel currents, and barium ions were used as the charge carrier. Two distinct types of calcium channel currents (N- and L-like) were identified based on their time-dependent inactivation, pharmacology and single-channel conductances. N- and L-like calcium channel currents were evoked by step depolarizing pulses to potentials more positive than -40 mV from a holding potential of -100 mV. The N-like component showed time-dependent inactivation during maintained depolarization with a time constant of tau f approximately 100 ms, whereas the L-like currents showed very slow inactivation with a time constant of tau s approximately 1,000 ms. Steady-state inactivation of currents evoked from a holding potential of -100 mV had two distinct components. One component involved the reduction of the transient current and had a half-maximal current at approximately -66 mV, whereas the other component involved the reduction of the steady-state current in the range of -35 to 0 mV with a half-maximal current at approximately -17 mV. Bay K 8644 (5 microM), had two distinct actions, one was the increase (50%) of the current associated with a depolarizing pulse to +10 mV. The second action was the increase in the peak amplitude of the tail current and the slowing of the deactivation kinetics. Omega-conotoxin at 1 microM irreversibly reduced the N-like current, sparing a component that was still sensitive to 5 microM Bay K 8644. The single-channel currents recorded with the cell-attached configuration of the patch clamp revealed two distinct conductances: a large approximately 28 pS and a small approximately 16 pS, corresponding to the L- and N-like channels, respectively. Bay K 8644 at 5 microM increased the mean open time of L-like single channel currents without changing single-channel conductance.
Insights
This study identified two distinct voltage-activated calcium channel currents (N- and L-like) in human neuroblastoma cells using patch-clamp techniques. These channels exhibit different inactivation rates and conductances, providing insights into neuronal excitability.
Area of Science:
- Neuroscience
- Cell Biology
- Pharmacology
Background:
- Voltage-activated calcium channels play crucial roles in neuronal function.
- Human neuroblastoma cell lines are valuable models for studying neuronal ion channels.
Purpose of the Study:
- To characterize distinct types of voltage-activated calcium channel currents in differentiated human neuroblastoma cells (SH-SY5Y).
- To investigate the electrophysiological properties, pharmacology, and single-channel behavior of these identified calcium channel currents.
Main Methods:
- Patch-clamp electrophysiology was employed to record whole-cell and single-channel currents.
- Experimental solutions were optimized to isolate calcium currents, using barium as the charge carrier.
- Pharmacological agents (Bay K 8644 and omega-conotoxin) were used to differentiate channel subtypes.
Main Results:
- Two distinct calcium channel currents, termed N-like and L-like, were identified based on inactivation kinetics and voltage dependence.
- N-like currents showed rapid inactivation (τf ≈ 100 ms), while L-like currents exhibited slow inactivation (τs ≈ 1,000 ms).
- Pharmacological profiling revealed differential sensitivity to Bay K 8644 and omega-conotoxin, with distinct single-channel conductances (≈16 pS for N-like, ≈28 pS for L-like).
Conclusions:
- Differentiated SH-SY5Y neuroblastoma cells express at least two distinct types of voltage-activated calcium channels.
- These channels possess unique electrophysiological and pharmacological properties, contributing to the complex modulation of neuronal excitability.
- The findings provide a basis for further investigation into the specific roles of these calcium channel subtypes in neuronal signaling and disease.