Calcium influx in internally dialyzed squid giant axons

Area of Science:

• Neuroscience
• Cell Physiology
• Biophysics

Background:

• Understanding calcium (Ca) influx mechanisms is crucial for neuronal function.
• Previous methods for measuring Ca influx in axons had limitations.
• The role of intracellular ions and energy substrates in Ca transport requires further elucidation.

Purpose of the Study:

• To develop and validate a novel method for quantifying Ca influx in internally dialyzed squid axons.
• To investigate the dependence of Ca influx on intracellular calcium ([Ca++]i), sodium ([Na+]i), and adenosine triphosphate (ATP).
• To characterize the components of Ca influx sensitive and insensitive to these factors and membrane potential.

Main Methods:

• Utilized internally dialyzed squid axons with controlled exposure to radioactive external medium.
• Employed ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) for calcium buffering.
• Included mitochondrial inhibitors (cyanide, oligomycin, FCCP) to prevent Ca uptake.

Main Results:

• Established a baseline Ca influx of 0.14 pmol.cm-2.s-1 under standard conditions ([ATP]=2 mM, [Ca++]i=0.06 microM, [Na+]i=70 mM).
• Demonstrated that ATP removal reduced Ca influx, while Na+ removal abolished Na+-dependent influx.
• Observed that Ca influx increased sigmoidally with [Ca++]i up to 1 microM in the presence of ATP and Na+.
• Identified that 50-60% of total Ca influx was insensitive to Na+, Ca++, and ATP, but sensitive to membrane potential and partially inhibited by Co++.

Conclusions:

• The developed method reliably measures Ca influx in squid axons.
• Both Na+ and ATP play significant roles in regulating Ca influx, likely through distinct pathways.
• A substantial portion of Ca influx is independent of Na+, ATP, and intracellular Ca concentration, suggesting alternative regulatory mechanisms.