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1. Patch clamp techniques were used to measure the ionic dependence of amiloride block of single mechanosensitive (MS) channels in frog (Xenopus laevis) oocytes. 2. The primary aim was to determine whether the difference in potency of amiloride block of MS channels in frog oocytes (IC50 = 0.5 mM) and chick auditory hair cells (IC50 = 50 microM) was due to the different ionic recording solutions. 3. Amiloride block of the oocyte MS channel does not vary significantly with complete substitution of external Na+ (i.e. 100 mM) with K+ in Ca(2+)-free recording solution (in both Na+ and K+ the IC50 = 0.5 mM). 4. A physiological concentration (1.8 mM) of external Ca2+ blocks the oocyte MS channel and reduces the potency of amiloride block (IC50 = 1.1 mM) without altering the voltage-dependence or the HIll coefficient (n = 1.8) of amiloride block. The reduction in potency can be explained by surface charge screening by Ca2+ which reduces the effective amiloride surface concentration. 5. The present results indicate that factors other than ionic recording conditions must underlie the difference in potency of amiloride block of MS channels in oocytes and auditory hair cells.

1. Patch clamp recording techniques have been used to compare the block caused by amiloride and some of its structural analogues of the mechanosensitive (MS) cation selective channel in frog (Xenopus laevis) oocytes. 2. Like amiloride, the amiloride analogues dimethylamiloride (DMA), benzamil and bromohexamethyleneamiloride (BrHMA) block the MS channel in a highly voltage-dependent manner. 3. All analogues tested were more potent blockers than amiloride with IC50's of 500 microM (amiloride), 370 microM (DMA), 95 microM (benzamil) and 34 microM (BrHMA). 4. Hill plots gave Hill coefficients of 2 (amiloride), 1.8 (DMA), 1 (benzamil) and 1.2 (BrHMA) indicating that the binding of two ligand molecules may be necessary for the block caused by amiloride, DMA and possibly BrHMA whereas only a single ligand molecule may be required for the block by benzamil. 5. The potential use of BrHMA as a light-activated, covalent label of the MS channel protein is discussed. 6. The amiloride analogue 'fingerprinting' of the blocking site on the MS channel indicates it is structurally different from previously described amiloride-sensitive ion transport pathways but may be related to the amiloride binding site on outer hair cells of the ear.