Tracking Voltage-dependent Conformational Changes in Skeletal Muscle Sodium Channel during Activation

Abstract

The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.

Na channel inactivation from open and closed states

“…Fig. 4 is a revision of the model in the introduction to incorporate the Csi data, the fluorescence data (11,12), scorpion toxin studies (14,15), and evidence from mutations (13,16). The four domains of an Na channel are indicated by circles, with shading to indicate activation of their S4 segments.…”

“…Some features in the structure and behavior of the domains that must be taken into account are detailed in the Table 1, where the last two rows are based on the fluorescence data of Bezanilla and colleagues (11,12).…”

Mapping the Interaction Site for a β-Scorpion Toxin in the Pore Module of Domain III of Voltage-gated Na+ Channels

“…Fluorescent labeling studies show that the four voltage sensors in the different domains are functionally specialized. The voltage sensors in domains I and II activate most rapidly and drive the rapid activation of Na ϩ conductance, whereas the voltage sensor in domain IV activates slowly with a time course similar to fast inactivation (28). Detailed mapping of the amino acid residues in the ISS2-S6, IVS1-S2, and IVS3-S4 segments that are required for high-affinity binding of ␣-scorpion toxins has led to the conclusion that these three extracellular loops form the receptor site for ␣-scorpion toxins with a similar conformation as we have proposed here for the receptor site for ␤-scorpion toxins (29).…”

A Geometric Comparison of Single Chain Multi-State Models of Ion Channel Gating

“…The rate functions are simple exponentials (the most most commonly employed functional form for voltage-dependent rates (Zagotta et al, 1994b;Chanda and Bezanilla, 2002;Kargol et al, 2002;Piper et al, 2003)) of the form…”

“…Typically, the states are ordered in a partial sequence with each state connected to usually two, sometimes three, or at most four others. The rate functions are often restricted by dictating that all or a set of the forward rates be identical or integer multiples of each other, and the reverse rates similarly (Zagotta et al, 1994b;Roux et al, 1998;Bezanilla, 2000;Bähring et al, 2001;Chanda and Bezanilla, 2002;Piper et al, 2003). Aggregation of similarly behaving states can also be performed to yield some simplification (Kienker, 1989;Kijima and Kijima, 1997).…”