Voltage-gated Na+ channels play a fundamental role in the excitability of nerve and muscle cells. Defects in fast Na+ channel inactivation can cause hereditary muscle diseases with hyper- or hypoexcitability of the sarcolemma. To explore the kinetics and gating mechanisms of noninactivating muscle Na+ channels on a molecular level, we analyzed single channel currents from wild-type and five mutant Na+ channels. The mutations were localized in different protein regions which have been previously shown to be important for fast inactivation (D3-D4-linker, D3/S4-S5, D4/S4-S5, D4/S6) and exhibited distinct grades of defective fast inactivation with varying levels of persistent Na+ currents caused by late channel reopenings. Different gating schemes were fitted to the data using hidden Markov models with a correction for time interval omission and compared statistically. For all investigated channels including the wild-type, two open states were necessary to describe our data. Whereas one inactivated state was sufficient to fit the single channel behavior of wild-type channels, modeling the mutants with impaired fast inactivation revealed evidence for several inactivated states. We propose a single gating scheme with two open and three inactivated states to describe the behavior of all five examined mutants. This scheme provides a biological interpretation of the collected data, based on previous investigations in voltage-gated Na+ and K+ channels.

Voltage-gated Na+ channels are the basis for the initiation and conduction of action potentials in excitable cells. The channel's main α-subunit, containing the selectivity filter and the gating machinery of the channel, consists of four domains (D1-D4), each of which contains six transmembrane segments (S1-S6). The S4 segments contain positively charged amino acids constituting the so-called voltage sensors that can move relative to the rest of the protein upon voltage changes. Movement of the voltage sensors out of their resting position upon membrane depolarization leads to an opening of the gate, a process called activation. Conventionally, its reversal upon repolarization, when the channel is transferred back from the open to the closed state, is termed deactivation. During a maintained depolarization, wild-type (WT) Na+ channels, after a brief initial opening, are transferred to another nonconducting state within milliseconds, due to closure of a second gate, and remain closed. This process is called fast inactivation and the conformational state correspondingly, fast inactivated state. A part of the protein located between domains D3 and D4 on the intracellular side of the membrane is responsible for fast inactivation. It is believed that the inactivation gate consists of three-to-four hydrophobic amino acids (isoleucine-phenylalanine-methionine-(threonine), i.e., IFM(T); denoted IFM, thereafter) that might bind to a receptor site within the inner mouth of the pore, blocking the permeation pathway. Previous work revealed the cytoplasmic loops connecting the S4 and S5 segments and the intracellular parts of the S6 segments in D3 and D4 as primary candidates for forming an IFM binding site (1,2).

To deepen the understanding of the molecular mechanism of fast inactivation we performed single channel recordings for channels containing mutations in the regions critical for the fast inactivation process. Analysis of the macroscopic whole-cell currents showed an altered gating of the mutant channels, in particular a disrupted inactivation with a slowing of the current relaxation, the presence of more or less prominent noninactivating, persistent Na+ currents in a range of 3%-55% of the initial peak current and a 10-20 mV positive shift of steady-state inactivation ((3,4), A. Alekov and H. Lerche, unpublished.)

In contrast to macroscopic currents, single ion channel data contain information on the dwell times in different protein conformations and the correlations between them. Thus, they can give further insight into the channel kinetics. Most commonly, single ion channel currents are described by hidden Markov models. In these models, the switching between the different protein configurations, called states, is described by a Markov chain. The number of these states and the allowed transitions between them is comprised graphically in the gating scheme.

One approach to analyze the data has been introduced by Horn and Lange (5). The authors fitted given gating schemes to the idealized, noise-free time series. Since the inevitable anti-aliasing filter limits the time resolution of the recording system and since the idealization of the current records relies on heavy filtering, brief openings and closings are missed. Several methods to cope with these missed events have been proposed, which are mostly approximative (6-9). An exact solution to the problem of time interval omission has been given by Hawkes et al. (10), who derived recursion formulas for the resulting apparent open and apparent closed time distributions.