Plasma membrane-endoplasmic reticulum coupling probed with genetically-encoded voltage sensors

Area of Science:

• Cellular biology
• Neuroscience
• Biophysics

Background:

• The endoplasmic reticulum (ER) is a vital organelle in eukaryotic cells, maintaining a membrane potential crucial for cellular functions.
• Studying ER membrane potential is challenging, limiting our understanding of its role in cellular processes.
• Voltage gradients across the ER membrane influence ion flux and cellular signaling.

Purpose of the Study:

• To develop and validate novel genetically-encoded optical probes for measuring ER membrane potential.
• To investigate the electrical coupling between the plasma membrane and the ER.
• To explore the functional implications of ER membrane potential dynamics in cellular signaling.

Main Methods:

• Development of hybrid voltage sensor (hVoS) derived probes targeted to the ER using specific protein motifs (Sec61β, cytochrome P450, cytochrome b5).
• Utilized patch-clamp fluorometry to record ER membrane potential changes in response to plasma membrane voltage steps.
• Employed equivalent circuit analysis to estimate the resistance of plasma membrane-ER contacts.

Main Results:

• Successfully generated and targeted optical probes to the ER, enabling visualization of ER membrane potential.
• Demonstrated that plasma membrane voltage changes elicit rapid, synchronous voltage changes at the ER membrane.
• Identified distinct probe responses suggesting localization to different ER compartments, with Sec61β-hVoS indicating proximity to voltage-gated ion channels.
• Estimated the resistance of direct electrical contacts between the plasma membrane and ER.

Conclusions:

• The developed optical probes provide powerful tools for real-time imaging of ER membrane potential.
• Direct electrical coupling between the plasma membrane and ER facilitates rapid voltage signal transmission.
• This mechanism is crucial for regulating ER functions, particularly in excitable cells.