In this study, we investigated the mechanism underlying the production of inwardly rectifying subconductance states induced in large conductance Ca(2+)-activated K+ channels (maxi K(Ca) channels) by the small, homologous proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin-I (DTX). Low-resolution bilayer recordings of BPTI-induced substates display excess noise that is well described by a beta-distribution characteristic of a filtered, two-state process. High-resolution patch recordings of maxi K(Ca) channels from vascular smooth muscle cells confirm that the BPTI-induced substate is actually comprised of rapid, voltage-dependent transitions between the open state and a nearly closed state. Patch recordings of DTX-induced substates also exhibit excess noise consistent with a similar two-state fluctuation process that occurs at rates faster than those measured for the BPTI-induced substate. The results indicate that these examples of ligand-induced substates originate by a fluctuating barrier mechanism that is similar to one class of models proposed by Dani, J.A., and J.A. Fox (1991. J. Theor. Biol. 153: 401-423) to explain subconductance behavior of ion channels. To assess the general impact of such rapid fluctuations on the practical measurement of unitary currents by amplitude histograms, we simulated single-channel records for a linear, three-state scheme of C (closed)-O(open)-S(substate). This simulation defines a range of transition rates relative to filter frequency where rapid fluctuations can lead to serious underestimation of actual unitary current levels. On the basis of these experiments and simulations, we conclude that fluctuating barrier processes and open channel noise may play an important physiological role in the modulation of ion permeation.

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