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Use of FRET-Sensor 'Mermaid' to Detect Subtle Changes in Membrane Potential of Primary Mouse PASMCs
Ruth C Dartsch1, Simone Kraut1, Tim Mayer2
1Cardiopulmonary Institute (CPI), Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Justus-Liebig-University, 35392 Giessen, Germany.
Abstract:
Subtle changes in the membrane potential of pulmonary arterial smooth muscle cells (PASMCs) are pivotal for controlling pulmonary vascular tone, e.g., for initiating Hypoxic Pulmonary Vasoconstriction, a vital mechanism of the pulmonary circulation. In our study, we evaluated the ability of the fluorescence resonance energy transfer (FRET)-based voltage-sensor Mermaid to detect such subtle changes in membrane potential. Mouse PASMCs were isolated and transduced with Mermaid-encoding lentiviral vectors before the acceptor/donor emission ratio was assessed via live cell FRET-imaging. Mermaid's sensitivity was tested by applying specific potassium chloride (KCl) concentrations. These KCl concentrations were previously validated by patch clamp recordings to induce depolarization with predefined amplitudes that physiologically occur in PASMCs. Mermaid's emission ratio dose-dependently increased upon depolarization with KCl. However, Mermaid formed unspecific intracellular aggregates, which limited the usefulness of this voltage sensor. When analyzing the membrane rim only to circumvent these unspecific signals, Mermaid was not suitable to resolve subtle changes in the membrane potential of ≤10 mV. In summary, we found Mermaid to be a suitable alternative for reliably detecting qualitative membrane voltage changes of more than 10 mV in primary mouse PASMCs. However, one should be aware of the limitations associated with this voltage sensor.
Insights
The voltage sensor Mermaid can detect significant (over 10 mV) membrane potential changes in pulmonary arterial smooth muscle cells (PASMCs). However, it struggles with subtle voltage shifts and forms intracellular aggregates, limiting its use for precise measurements.
Area of Science:
- Physiology
- Cell Biology
- Biophysics
Background:
- Pulmonary arterial smooth muscle cells (PASMCs) control vascular tone through membrane potential changes.
- Hypoxic Pulmonary Vasoconstriction relies on these subtle electrical signaling events.
- Accurate voltage sensing is crucial for understanding pulmonary circulation regulation.
Purpose of the Study:
- To evaluate the FRET-based voltage sensor Mermaid for detecting subtle membrane potential changes in mouse PASMCs.
- To assess Mermaid's sensitivity and specificity in response to physiologically relevant stimuli.
- To determine the limitations of Mermaid for studying pulmonary vascular tone regulation.
Main Methods:
- Isolation and lentiviral transduction of mouse PASMCs with Mermaid.
- Live-cell FRET imaging to assess Mermaid's emission ratio.
- Application of potassium chloride (KCl) to induce controlled membrane depolarization.
- Validation of KCl-induced depolarization using patch clamp recordings.
Main Results:
- Mermaid's emission ratio increased dose-dependently with KCl-induced depolarization.
- Unspecific intracellular Mermaid aggregates were observed, limiting sensor utility.
- Mermaid could not resolve subtle membrane potential changes of ≤10 mV, even when analyzing the membrane rim.
- The sensor reliably detected qualitative voltage changes >10 mV.
Conclusions:
- Mermaid is a viable tool for detecting significant (>10 mV) membrane potential changes in PASMCs.
- Limitations include intracellular aggregation and reduced sensitivity to subtle voltage fluctuations.
- Careful consideration of Mermaid's limitations is necessary for accurate interpretation of results in pulmonary circulation research.
