Title Molecular motions of the outer ring of charge of the sodiumchannel : Do they couple to slow inactivation ?

In contrast to fast inactivation, the molecular basis of sodium (Na) channel slow inactivation is poorly understood. It has been suggested that structural rearrangements in the outer pore mediate slow inactivation of Na channels similar to C-type inactivation in potassium (K) channels. We probed the role of the outer ring of charge in inactivation gating by paired cysteine mutagenesis in the rat skeletal muscle Na channel (rNav1.4). The outer charged ring residues were substituted with cysteine, paired with cysteine mutants at other positions in the external pore, and coexpressed with rat brain 1 in Xenopus oocytes. Dithiolthreitol (DTT) markedly increased the current in E403C E758C double mutant, indicating the spontaneous formation of a disulfide bond and proximity of the carbons of these residues of no more than 7 Å. The redox catalyst Cu(II) (1,10-phenanthroline) 3 (Cu(phe) 3 ) reduced the peak current of double mutants (E403C E758C, E403C D1241C, E403C D1532C, and D1241C D1532C) at a rate proportional to the stimulation frequency. Voltage protocols that favored occupancy of slow inactivation states completely prevented Cu(phe) 3 modification of outer charged ring paired mutants E403C E758C, E403C D1241C, and E403C D1532C. In contrast, voltage protocols that favored slow inactivation did not prevent Cu(phe) 3 modification of other double mutants such as E403C W756C, E403C W1239C, and E403C W1531C. Our data suggest that slow inactivation of the Na channel is associated with a structural rearrangement of the outer ring of charge. key words: rNav1.4 channel • cysteine mutagenesis • disulfide bond • electrophysiology I N T R O D U C T I O N The voltage-gated Na channel mediates the rapid upstroke of action potential and is responsible for excitation and conduction in nerve and muscle. Upon depolarization, the channel activates a process that involves outward movement of all four charged S4 segments (Stuhmer et al., 1989; Hirschberg et al., 1995; Yang and Horn, 1995; Yang et al., 1996; Kontis et al., 1997). Na channel opening is transient, with inactivation rapidly ensuing. Na channels undergo several different kinetically distinct forms of inactivation. The time constants of recovery from inactivated states range from milliseconds (fast inactivation) to hundreds of milliseconds (intermediate inactivation) and seconds (slow inactivation) (Simoncini and Stuhmer, 1987; Ruff et al., 1988; Nuss et al., 1996; Kambouris et al., 1998). In Shaker K channels, N-type inactivation is mediated by an intracellular region at the NH 2 terminus acting as “ball” occluding the pore (Hoshi et al., 1990). The analogous region in the Na channel is the cytoplasmic III-IV linker, which serves as a tethered pore blocker binding to an inactivation gate receptor in the intracellular mouth of the channel (Armstrong, 1981; Stuhmer et al., 1989; Vassilev et al., 1989; Patton et al., 1992; West et al., 1992). Unlike fast inactivation, the molecular basis of slow inactivation in Na channels is poorly understood. However, slow inactivation plays a critical role in membrane excitability and firing properties by governing the availability of Na channels in neurons and muscle (Ruff et al., 1988; Fleidervish et al., 1996). The physiological significance of slow inactivation in Na channels has been established by studies demonstrating that abnormalities of slow inactivation are associated with a variety of human diseases, including hyperkalemic periodic paralysis (Cummins and Sigworth, 1996; Hayward et al., 1999; Bendahhou et al., 2002), Brugada syndrome (Veldkamp et al., 2000), and epilepsy (Alekov et al., 2001; Spampanato et al., 2001). In Shaker K channels, C-type inactivation, which can be slow, is a mechanism of inactivation that involves a conformational rearrangement of the outer pore. C-type inactivation persists when N-type inactivation is deleted (Hoshi et al., 1991), is inhibited by extracellular tetraethylammonium (Choi et al., 1991) and is sensitive to extracellular [K ] and mutations in the outer pore (Lopez-Barneo et al., 1993). The outer ring of charge (E403, E758, D1241, and D1532 in domains I-IV, respectively) is located three Address correspondence to Gordon F. Tomaselli, Department of Medicine, Johns Hopkins University School of Medicine, 720 Rutland Ave./Ross 844, Baltimore, MD 21205. Fax: (410) 955-7953; email: gtomasel@jhmi.edu Abbreviations used in this paper: DTT, dithiolthreitol; MTS, methane-