N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake

Area of Science:

• Cell biology
• Molecular pharmacology
• Immunology

Background:

• The P2X7 receptor, a purinergic receptor, plays a role in cellular signaling through channel and pore formation.
• Fluorescent imaging is crucial for studying receptor expression and localization.
• Fusion proteins require functional validation due to potential alterations.

Purpose of the Study:

• To investigate the functional impact of N-terminal fluorescent protein fusions on the human P2X7 receptor.
• To evaluate surface expression, channel activity, and pore formation of human P2X7 receptor fusion constructs.

Main Methods:

• Transfection of cells with human P2X7 receptor fusion constructs (e.g., eGFP, 3HA).
• Assessment of surface receptor expression.
• Measurement of calcium influx to determine channel opening.
• YO-PRO-1 dye uptake assay to evaluate pore formation upon BzATP stimulation.

Main Results:

• N-terminal eGFP fusion to the human P2X7 receptor impaired channel opening and pore formation, despite normal surface expression.
• N-terminal 3HA tagging also disrupted pore formation, indicating sensitivity to even small N-terminal modifications.
• These findings contrast with observations for the rat P2X7 receptor.

Conclusions:

• The N-terminus of the human P2X7 receptor contains critical signaling motifs essential for its function.
• N-terminal tagging, even with small tags like eGFP or 3HA, can abolish human P2X7 receptor pore formation.
• Functional human P2X7 receptor fusion proteins cannot be constructed by simple N-terminal tagging.