Near-membrane [Ca2+] transients resolved using the Ca2+ indicator FFP18

Area of Science:

• Cellular Biology
• Biophysics
• Neuroscience

Background:

• Calcium ions (Ca2+) regulate critical cellular functions like contraction and neurotransmission.
• A discrepancy exists between Ca2+ levels required for in vitro membrane processes and those measured in intact cells.
• Standard Ca2+ indicators average signals, masking localized near-membrane Ca2+ dynamics.

Purpose of the Study:

• To investigate localized Ca2+ dynamics at the cell membrane.
• To introduce and validate a novel Ca2+ indicator, FFP18, for near-membrane measurements.
• To resolve the paradox of Ca2+-sensitive membrane processes activation.

Main Methods:

• Development of FFP18, a Ca2+ indicator with low affinity and high water solubility for selective near-membrane localization.
• Confocal microscopy to assess FFP18's intracellular distribution, confirming >65% plasma membrane association.
• High-speed digital imaging of Ca2+ transients using FFP18 and fura-2 during membrane depolarization.

Main Results:

• FFP18 selectively localized to the plasma membrane.
• Near-membrane Ca2+ rose rapidly (within 20 ms) to micromolar levels, peaking at 50-100 ms.
• Cytoplasmic Ca2+ (measured by fura-2) increased slowly, reaching only hundreds of nanomolar during the same period.
• FFP18 data revealed significantly faster and higher Ca2+ transients near the membrane compared to bulk cytoplasm.

Conclusions:

• Localized, rapid Ca2+ transients occur directly beneath the plasma membrane.
• These near-membrane Ca2+ spikes provide a mechanism for activating numerous Ca2+-sensitive membrane processes.
• FFP18 is a valuable tool for studying localized Ca2+ signaling at the cell surface.