Brain spectrin (fodrin) interacts with phospholipids as revealed by intrinsic fluorescence quenching and monolayer experiments

Abstract

We demonstrate that phospholipid vesicles affect the intrinsic fluorescence of isolated brain spectrin. In the present studies we tested the effects of vesicles prepared from phosphatidylcholine (PtdCho) alone, in addition to vesicles containing PtdCho mixed with other phospholipids [phosphatidylethanolamine (PtdEtn) and phosphatidylserine] as well as from total lipid mixture extracted from brain membrane. The largest effect was observed with PtdEtn/PtdCho (3:2 molar ratio) vesicles; the effect was markedly smaller when vesicles were prepared from egg yolk PtdCho alone. Brain spectrin injected into a subphase induced a substantial increase in the surface pressure of monolayers prepared from phospholipids. Results obtained with this technique indicated that the largest effect is again observed with monolayers prepared from a PtdEtn/PtdCho mixture. The greatest effect was observed when the monolayer contained 50-60% PtdEtn in a PtdEtn/PtdCho mixture. This interaction occurred at salt and pH optima close to physiological conditions (0.15 M NaCl, pH7.5). Experiments with isolated spectrin subunits indicated that the effect of the beta subunit on the monolayer surface pressure resembled that measured with the whole molecule. Similarly to erythrocyte spectrin-membrane interactions, brain spectrin interactions with PtdEtn/PtdCho monolayer were competitively inhibited by isolated erythrocyte ankyrin. This also suggests that the major phospholipid-binding site is located in the beta subunit and indicates the possible physiological significance of this interaction.

Spectrin and phospholipids — the current picture of their fascinating interplay

“…Moreover, the inner monolayer of the erythrocyte membrane contains more PE than PS [87,88]. Such specificity was also seen in the case of non-erythroid spectrin, as reported by Diakowski et al [89], who showed that purified brain spectrin induced an increase in surface pressure in lipid monolayers composed of PE/PC, PS/PC and PC. The strongest effect of spectrin was observed on monolayers containing 50-60% of PE.…”

“…However, ankyrin inhibited only one of the two possible lipid-binding sites. A similar level of ankyrin-mediated inhibition of lipid interactions was observed for nonerythroid (brain) spectrin [89]. Moreover, it appeared that β-spectrin was inhibited much more strongly than α-spectrin [89,125].…”

“…A similar level of ankyrin-mediated inhibition of lipid interactions was observed for nonerythroid (brain) spectrin [89]. Moreover, it appeared that β-spectrin was inhibited much more strongly than α-spectrin [89,125]. The ankyrin-binding domain of erythroid spectrin, first described by Kennedy et al in 1991 [126], was found within the region-spanning 14 th and 15 th repeats of erythroid β-spectrin (Fig.…”

“…This is nicely reflected by the fact that the affinity of spectrin to PE/PC monolayers considerably increases with a decrease in the initial surface pressure, and the protein is able to efficiently penetrate "loose" (approx. 9 mN/m, far from the 30-35 mN/m assigned for natural membranes) PE/PC monolayers [89,90]. Moreover, both native spectrin [136] and the isolated ankyrin-sensitive PE/PC lipid-binding domain of beta spectrin [103] induced an increase in lipid hydrocarbon chain mobility of either PE/PC vesicles or natural membranes.…”

Identification of quantitative trait loci that modify the severity of hereditary spherocytosis in wan, a new mouse model of band-3 deficiency

“…For example, spectrin is known to interact with phosphatidylserine (PS) at multiple sites, and this interaction is mediated by the ␤-subunit. 45,46 Hence, ␤-spectrin polymorphisms could enhance spectrin-PS interactions. Alternatively, polymorphic ␤-spectrin alleles could bind other, currently unknown, integral membrane proteins with differing affinities.…”