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

The endoplasmic reticulum (ER) forms an elaborate contiguous network extending through the cytoplasm of eukaryotic cells. The ER is surrounded by a membrane that separates its lumen from the cytoplasm. The ER membrane harbors channels and pumps capable of controlling ion flux and creating a voltage gradient. Because the ER membrane potential is difficult to study experimentally, little is known about how voltage influences its many vital functions. Here, we introduce optical probes of ER membrane potential derived from the hybrid voltage sensor (hVoS) family of genetically encoded voltage sensors. Probes were targeted to the ER using motifs from three ER proteins, Sec61β, cytochrome P450, and cytochrome b₅ type A. As shown recently with other types of ER voltage sensors, patch-clamp fluorometry recording with our new probes demonstrated that voltage steps applied to the plasma membrane elicit a voltage change at the ER membrane. These probes exhibited subtle differences in their responses suggesting they target different ER compartments. The steeper voltage dependence of Sec61β-hVoS (mCerulean3-Sec61β) signals suggested that this probe targets an ER compartment rich in voltage-gated ion channels. The ER voltage change is slow, but its onset is virtually synchronous with the plasma membrane voltage step. This suggests a direct electrical coupling into the ER lumen through plasma membrane-ER contacts. Analysis with the aid of an equivalent circuit provided an estimate of the resistance of these contacts. The rapid, direct transmission of voltage changes from the plasma membrane to the ER provides a mechanism for regulating ER function that could be especially important in excitable cells. The sensors introduced here provide researchers with powerful tools for imaging ER voltage and assessing its impact on cellular function.