Cytoplasmic calcium buffers in intact human red cells

Area of Science:

• Cellular Physiology
• Biochemistry
• Ion Transport

Background:

• Precise understanding of cytoplasmic calcium (Ca2+) buffering in human red blood cells is crucial for elucidating Ca2+-dependent cellular functions.
• Existing methods for measuring intracellular Ca2+ concentrations ([Ca2+]i) have limitations in covering the full range from nanomolar to millimolar.

Purpose of the Study:

• To precisely quantify the cytoplasmic Ca2+ buffering behavior in intact human red blood cells across a wide range of [Ca2+]i.
• To identify and characterize the components responsible for Ca2+ buffering within the red blood cell cytoplasm.

Main Methods:

• Human red blood cells with inhibited Ca2+ pumps (via ATP depletion or vanadate) were used.
• Intracellular Ca2+ buffering was analyzed using 45Ca2+ equilibrium distribution measurements.
• Measurements were performed across varying external Ca2+ concentrations ([Ca2+]o) under controlled cell volume and pH.

Main Results:

• The primary Ca2+ buffer, alpha, demonstrated a consistent value (0.34 ± 0.01) across donors, indicating high capacity and low affinity.
• An additional Ca2+ buffering complex with low capacity (~80 µmol (340 g Hb)−1) and intermediate affinity (K(D,app) ~4-50 µM) was detected.
• Putative Ca2+ buffers with submicromolar dissociation constants were below the detection limit (<2 µmol (340 g Hb)−1).

Conclusions:

• The cytoplasmic Ca2+ buffering in human red blood cells is dominated by a consistent low-affinity buffer (alpha).
• A novel intermediate-affinity Ca2+ buffer exists, contributing significantly at higher intracellular Ca2+ levels.
• These findings refine our understanding of calcium homeostasis and its regulation in red blood cells.