Intracellular calcium buffering capacity in isolated squid axons

Area of Science:

• Neuroscience
• Cell Physiology
• Biochemistry

Background:

• Intracellular calcium regulation is crucial for neuronal function.
• Understanding calcium buffering mechanisms in axons is essential for comprehending nerve impulse transmission and cellular homeostasis.
• The role of mitochondria and other intracellular buffers in managing calcium loads requires further elucidation.

Purpose of the Study:

• To quantify changes in ionized calcium within squid axons under varying calcium loading conditions.
• To differentiate the buffering capacity of mitochondrial versus non-mitochondrial systems in response to calcium influx.
• To characterize the properties and capacity of the FCCP-insensitive calcium buffering system.

Main Methods:

• Utilized multiwavelength differential absorption spectroscopy with the Ca binding dye Arsenazo III to measure ionized calcium changes in isolated squid axons.
• Calibrated absorbance changes in situ using in vitro solutions mimicking axoplasm composition with CaEGTA buffers.
• Induced calcium loads through microinjection, stimulation in high calcium seawater, and soaking in low calcium saline solutions.
• Employed carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) to inhibit mitochondrial proton gradients and assess its impact on calcium buffering.

Main Results:

• Ionized calcium in squid axoplasm increased by approximately 0.6 nM per µM of calcium load across a range of 50–2,500 µmol/kg.
• Inhibition of mitochondrial function with FCCP revealed that 93–95% of the imposed calcium load was buffered by an FCCP-insensitive system.
• This FCCP-insensitive buffer system demonstrated a high capacity, remaining unsaturated even at the largest imposed calcium loads, suggesting a capacity of at least several millimolar.
• While only 6% of endogenous calcium is stored in FCCP-sensitive (mitochondrial) buffers, this system accommodates up to 30% of an imposed exogenous calcium load.

Conclusions:

• Squid axons possess a substantial intracellular calcium buffering system that is largely independent of mitochondrial function.
• This non-mitochondrial buffer system plays a dominant role in managing large, imposed exogenous calcium loads.
• The findings highlight the significant capacity of non-mitochondrial mechanisms in maintaining calcium homeostasis within axons.