Optical probe responses on sarcoplasmic reticulum. Oxacarbocyanines

Area of Science:

• Biochemistry
• Membrane Transport
• Spectroscopy

Background:

• Sarcoplasmic reticulum (SR) Ca2+ transport is crucial for muscle contraction.
• Oxacarbocyanine dyes are used as optical probes for membrane potential and ion binding.
• Understanding dye responses is key to accurately measuring SR function.

Purpose of the Study:

• To analyze the absorbance and fluorescence changes of oxacarbocyanine dyes during ATP-induced Ca2+ transport in rabbit SR.
• To elucidate the specific binding interactions of Ca2+ and ATP with the SR membrane.
• To determine how ionic conditions affect dye responses during Ca2+ uptake.

Main Methods:

• Spectroscopic analysis (absorbance and fluorescence) of oxacarbocyanine dyes (Di-O-C5(3)).
• Experiments conducted in varying ionic media (KCl, MgCl2, sucrose).
• Measurement of apparent dissociation constants for Ca2+ and ATP binding.

Main Results:

• Oxacarbocyanine fluorescence decreases with Ca2+ or ATP binding at the active site (apparent Kd of 0.2 µM for Ca2+, 5 µM for ATP).
• ATP binding effects are observable without divalent cations.
• Millimolar Ca2+ concentrations and ATP-induced uptake cause changes related to low-affinity Ca2+ binding sites, influenced by K+ and Mg2+ competition.

Conclusions:

• Oxacarbocyanine dyes respond to both high-affinity active site binding and low-affinity non-specific binding of Ca2+ and ATP.
• Ionic strength and composition significantly impact dye optical signals.
• These findings are critical for accurate interpretation of membrane potential during SR Ca2+ transport studies.