Increased Cation Conductance in Human Erythrocytes Artificially Aged by Glycation

• Published in: The Journal of membrane biology Vol. 235; no. 3; pp. 177 - 189
• Main Authors: Kucherenko, Yuliya V, Bhavsar, Shefalee K, Grischenko, Valentin I, Fischer, Uwe R, Huber, Stephan M, Lang, Florian
• Format: Journal Article
• Language: English
• Published: New York New York : Springer-Verlag 01.06.2010; Springer-Verlag; Springer Nature B.V
• Subjects: Biochemistry; Biomedical and Life Sciences; Calcium - metabolism; calcium channels; Calcium Channels, L-Type - drug effects; Calcium Channels, L-Type - metabolism; Cell Size - drug effects; Cellular biology; Electric Conductivity; Erythrocyte Aging - drug effects; Erythrocytes; Glucose - pharmacology; Glycated Hemoglobin A - metabolism; glycation; Glycosylation; Hemoglobin; Human Physiology; Humans; Life Sciences; Membranes; Patch-Clamp Techniques; Peptides; Phosphatidylserines - pharmacology; TRPC; TRPC Cation Channels - drug effects; TRPC Cation Channels - metabolism; α1C peptide; Hemoglobin; Calcium channels; α1C peptide; TRPC; Glycation
• ISSN: 0022-2631; 1432-1424
• DOI: 10.1007/s00232-010-9265-2

Excessive glucose concentrations foster glycation and thus premature aging of erythrocytes. The present study explored whether glycation-induced erythrocyte aging is paralleled by features of suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface and cell shrinkage. Both are triggered by increases of cytosolic Ca²⁺ concentration ([Ca²⁺]i), which may result from activation of Ca²⁺ permeable cation channels. Glycation was accomplished by exposure to high glucose concentrations (40 and 100 mM), phosphatidylserine exposure estimated from annexin binding, cell shrinkage from decrease of forward scatter, and [Ca²⁺]i from Fluo3-fluorescence in analysis via fluorescence-activated cell sorter. Cation channel activity was determined by means of whole-cell patch clamp. Glycation of total membrane proteins, immunoprecipitated TRPC3/6/7, and immunoprecipitated L-type Ca²⁺ channel proteins was estimated by Western blot testing with polyclonal antibodies used against advanced glycation end products. A 30-48-h exposure of the cells to 40 or 100 mM glucose in Ringer solution (at 37°C) significantly increased glycation of membrane proteins, hemoglobin (HbA₁c), TRPC3/6/7, and L-type Ca²⁺ channel proteins, enhanced amiloride-sensitive, voltage-independent cation conductance, [Ca²⁺]i, and phosphatidylserine exposure, and led to significant cell shrinkage. Ca²⁺ removal and addition of Ca²⁺ chelator EGTA prevented the glycation-induced phosphatidylserine exposure and cell shrinkage after glycation. Glycation-induced erythrocyte aging leads to eryptosis, an effect requiring Ca²⁺ entry from extracellular space.