1. Individual Na+ channels from isolated guinea‐pig ventricular heart cells were studied using the patch‐clamp technique. To localize the selectivity region of the channels we investigated their blockade by a permanently charged quaternary ammonium ion (QX‐314, 2‐(triethylamino)‐N‐(2,6‐dimethylphenyl)acetamide, 0‐5 mM) that was applied to the cytoplasmic side of the channel. 2. Resolution of individual blocking events was enhanced by covalent removal of fast inactivation following brief internal exposure to the enzyme papain. The improved resolution reveals the existence of two distinct modalities of blockade: reduction of unitary current, and millisecond interruptions of current. 3. Both modes of internal block could be potentiated by lowering external Na+ concentration. This finding argues that the two corresponding sites of interaction are both located within the channel pore. 4. Analysis of the voltage dependence of block placed both binding sites deep within the pore, at 70% of the electric field from the cytoplasmic entrance. Combined with recent studies localizing block by external Cd2+, the present results argue that the selectivity region of Na+ channels is quite narrow (spanning about 10% of the electric field), and located near the external side of the channel. 5. The manner in which the two blocking processes interact, along with the physical proximity of their binding sites, leads us to propose that the block configuration responsible for the reduction in unitary current serves as a transition intermediate that catalyses formation of the discrete‐block complex.
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