Shepherd VA and Beilby MJ

Journal of Membrane Biology, 1999, 170: 229 - 242

Abstract

An extracellular mucilage composed of sulphated and carboxylated polysaccharides modifies the response to hypotonic challenge in the charophyte alga Lamprothamnium papulosum. Marine cells with substantial extracellular mucilage layers respond to hypotonic challenge by depolarising, without dramatically increasing conductance and without cessation of cytoplasmic streaming, and with I/V profiles either linear, or typical of the K⁺ state (slow regulating cells). Young cells, lacking mucilage, also respond by depolarising, but increase their conductance and transiently stop cytoplasmic streaming (fast regulating cells). We report that the extracellular mucilage is degraded by heparinase enzyme at pH 7.5 and 25 ^oC. Heparinased mucilage lost its capacity for staining with Toluidine Blue or Alcian Blue at pH 1, indicating that the structure of the sulphated mucilage had been disrupted. Heparinased slow-regulating cells had similar resting membrane potential differences to untreated cells (6 treated cells: -90.6 mV, SE (standard error): 12.3 mV; 9 untreated cells; -93.8 mV, SE: 9.6 mV) but greater resting conductance, G_s (5 treated cells: 57 Sm^-2, SE: 14.8 Sm^-2 ; 9 controls: 17.2  Sm^-2, SE: 2.1 Sm^-2 ). On exposure to hypotonic solution, heparinased slow-regulating cells depolarised by up to 160 mV, usually to positive potentials, and cytoplasmic streaming ceased. The cell conductance increased ~four-fold at the reversal potential, G_rev (to 214  Sm^-2, SE: 99.2  Sm^-2 , 4 cells). By contrast, untreated cells depolarised by less than 100 mV, and G_rev increased only 1.3-fold (to 17.2 Sm^-2, SE: 2.1  Sm^-2, 6 cells). The heparinased cells resumed streaming after several minutes despite having positive membrane potentials, and after 60 minutes G_rev remained very high (139.9  Sm^-2, SE: 71.6  Sm^-2, 4 treated cells) with the cells retaining zero or positive membrane potentials. The cells did not enter the K⁺ state. In contrast, untreated cells recovered negative membrane potentials, G_rev declined to less than Gs (12.4  Sm^-2, SE: 3.1  Sm^-2, 4 untreated cells) and the cells entered the K⁺ state. The effect was reversible and washout of heparinase overnight resulted in reversion to normal slow-regulating behaviour. Fast-regulating cells with thin, weakly sulphated mucilage were less affected by heparinase, with similar patterns of hypotonic response to untreated equivalent cells. This suggests that the thick, sulphated polysaccharide mucilage is normally integral to the slow-regulating response. The interactions between the mucilage and ion-channels in mediating the response to hypotonic challenge is discussed.

Keywords : Lamprothamium papulosum, hypotonic effect, heparinase, marine algae, turgor regulation, sulphated polysaccharide, mucilage, electrophysiology, stretch-activated channels