Patch-clamp study of ion-channels in membrane vesicles prepared from the parasitic helminths Ascaris suum and Schistosoma mansoni

Abstract

Ascaris lumbricoides and Schistosoma mansoni are major parasites of man in the developing world. The possible development of drug resistant strains means that continuing and improved treatments require the selection of novel target sites. Membrane ion-channels have proved to be suitable targets for many therapeutic agents. The purpose of this thesis was to further characterise distinct ion-channels present in parasite membranes. The properties of the ion-channels found in membranes of the two parasite species were assessed using standard single-channel recording techniques.

Membrane vesicles prepared from the bag region of the somatic muscle cells of the parasitic nematode Ascaris suum (a close relative of A. lumbricoides) contain highconductance, voltage-sensitive, Ca-dependent chloride channels. The effect of altered pH on these channels was investigated using the patch-clamp technique and isolated inside-out membrane patches. Changes in pH had little effect on channel conductances and reversal potentials. Under control conditions (symmetrical pH 7.2) the channel had the highest probability of opening at ~-35mV (the resting, membrane potential of the cell). At positive membrane potentials the probability of opening decreased. The Boltzmann equation was used to describe the relationship between membrane potential and probability of channel opening, and to calculate the effective gating charge. Reduction of external pH produced an increase in the probability of channel opening at hyperpolarised membrane potentials. An increase in internal pH caused a voltage independent increase in the probability of channel opening and made the effective gating charge less negative. The effect of reducing internal pH was marked: the channel then opened most frequently at positive membrane potentials and the probability of opening at -35mV was greatly reduced.

The decrease in internal pH changed the polarity of the effective gating charge.

A simple model was constructed to describe the effects of pH on channel gating. Treatment of S. mansoni in low pH media results in the formation of vesicles from the tegument. Parasites pre-treated with 5-N-(octadecanoyl)aminofluorescein (AF18) and examined using fluorescence microscopy showed at least some of the vesicles were outer tegument in origin. Scanning electron microscopy studies show the vesicles possess a smooth surface suitable for the application of the patch-clamp technique.

Transmission electron microscopy studies indicate that some vesicles posses the unique heptalaminate membranes found in the outer tegument.

Patchclamp studies have shown the presence of a large-conductance, non-selective cation, channel. Other channel types with different ionic selectivities and conductances were observed.

The properties of the observed ion-channel types are discussed; comment is made on these ion-channels as possible target sites.