A markedly disrupted skeletal network with abnormally distributed intramembrane particles in complete protein 4.1-deficient red blood cells (allele 4.1 Madrid): implications regarding a critical role of protein 4.1 in maintenance of the integrity of the red blood cell membrane.

Electron microscopic (EM) studies were performed to clarify the interactions of membrane proteins in the red blood cell membrane structure in situ of a homozygous patient with total deficiency of protein 4.1 who carried a point mutation of the downstream translation initiation codon (AUG --> AGG) of the protein 4.1 gene [the 4.1 (-) Madrid; Dalla Venezia et al, J Clin Invest 90:1713, 1992]. Immunologically, as expected, protein 4.1 was completely missing in the red blood cell membrane structure in situ. A markedly disrupted skeletal network was observed by EM using the quick-freeze deep-etching method and the surface replica method, although the number of spectrin molecules was only minimally reduced (395 +/- 63/microm2; normal, 504 +/- 36/microm2). The number of basic units in the skeletal network was strikingly reduced (131 +/- 21/microm2; normal, 548 +/- 39/microm2), with decreased small-sized units (17 +/- 4/microm2; normal, 384 +/- 52/microm2) and increased large-sized units (64% +/- 14%; normal, 5% +/- 1%). Concomitantly, immuno-EM disclosed striking clustering of spectrin molecules with aggregated ankyrin molecules in the red blood cell membrane structure in situ. Although no quantitative abnormalities in the number and size distribution of the intramembrane particles were observed, there was a disappearance of regular distribution, with many clusters of various sizes, probably reflecting the distorted skeletal network. Therefore, protein 4.1 suggests by EM to play a crucial role in maintenance of the normal integrity of the membrane structure in situ not only of the skeletal network but also of the integral proteins.