Amiloride block of the mechanosensitive cation channel in Xenopus oocytes

Abstract

1. Patch clamp recording techniques have been used to investigate the block by amiloride of the mechanosensitive cation-selective channel in frog (Xenopus laevis) oocytes. 2. Cell-attached and outside-out patch recording configurations were employed to study the differences in block produced when amiloride was present at either the extracellular (external) or intracellular (internal) membrane face. 3. External amiloride causes a highly voltage-dependent 'flickery' block of single mechanosensitive channel currents in which inward mechanosensitive current recorded at negative potentials is reduced in amplitude but outward mechanosensitive current recorded at positive potentials is almost unaffected. 4. At -100 mV the apparent dissociation constant (Kd) for external amiloride block is 0.5 mM. The extracellular concentration dependence of amiloride block yields a Hill coefficient equal to 2, inconsistent with a single site blocking stoichiometry. 5. The shapes of current-voltage relationships measured in different external amiloride concentrations also indicate deviations from a simple channel plug model in which a single blocking cation is driven into the channel by the membrane potential. 6. Internal amiloride causes a voltage-independent 'flickery' block of mechanosensitive channel currents which equally reduces both inward and outward mechanosensitive currents. 7. The present data indicate that a minimum of two amiloride binding sites are necessary to predict external amiloride block. A model involving a voltage-dependent conformational change with subsequent voltage-independent co-operative binding of two amiloride molecules is found to explain the data. 8. The relevance of the present actions of amiloride on mechanosensitive channels is discussed in relation to reports of amiloride-inhibitable cation flux pathways involved in a number of basic physiological functions including mechanosensitivity of sensory cells, volume regulation and fertilization.