Mutations, molecules, and myotonia

The linkages between a protein 's primary sequence, three-dimensional structure, and the detailed manifestations of its function are well appreciated. Exploring and understanding these linkages have proven remarkably complex, however. While some functions may be localized exclusively to one protein region, others may be distributed or even diffusely coded in the structure. The kinetics of the tetrodotoxin-sensitive Na + channel and its voltage-gated siblings provide many examples of such complex and distributed coupling. The channel consists of four domains, each a repeat of the fundamental unit that is the primary motif of the voltage-dependent K + channel. In each domain, six membrane-spanning regions (S1-$6) give the channel its essential form, with charged groups embedded within these regions, presumably imparting the channel 's characteristic sensitivity to membrane potential. Two sets of hydropbilic loops are important for function: One set of loops (the $5-$6 linkers in each domain) on the extracellular side line the pore region; another such loop, between domains III and IV on the cytoplasmic side, is critical for fast inactivation. Clearly, the various elements of this channel 's gating and permeability are linked to its detailed structure; alteration of charge in the $4 segments changes activation (Stiihmer et al., 1989), and alteration of hydrophobicity in the cytoplasmic linker between domains III and IV slows inactivation (West et al., 1992). Neither observation, however, is unambiguous regarding mechanism. Not all $4 charges give the same functional alteration in channel activation, and noncharged residues in these and nearby regions also affect the voltage dependence of gating. This observation is indeed expected if the underlying conformational changes involve sliding or rotational motions of domains, which would be sensitive to a wide variety of interactions at their interfaces. Inactivation properties, too, are determined by a broad variety of structures, which may be physically and functionally removed from the III-IV linker itself, as exemplified by a set of naturally occurring Na + channel mutations. Familial skeletal muscle disorders such as hyperkalemic periodic paralysis and paramyotonia congenita have been linked to alterations in the Na + channel gene (Fontaine et al., 1990; see Rfidel et al., 1993, for review) and subsequently localized to diverse sites including the cytoplasmic end of the $6 segments of domains II and IV, the $5 segments in domains II and III, as well as to changes in the extracellular ends of domain IV's $3 and $4 segments (see Fig. 1 of Hayward et al., 1996, in this issue). How could such diverse changes give rise to altered inactivation, and concomitant disease? This question is examined in this (Hayward et al., 1996) and the February (]i et al., 1996) issues of The Journal of General Physiology. One hypothesis to explain such distributed participation in inactivation is that this reaction is analogous to a pharmacological blocking reaction, as originally proposed by Armstrong (1970). While Na + channels may not have a ball at the end of a chain, binding of the IIIIV linker to the inner channel vestibule may serve the same function. Such binding would involve a specific reaction that would ultimately depend, like a jigsaw piece fitting into its designated spot, on the detailed structures of both the linker and its binding site. Mutations, both proximal and distant, that altered ei ther structure would impact on the channels ' inactivation kinetics, while spontaneous rearrangements of their tertiary or quaternary structure might cause transient and reversible changes in reaction rates (so-called "mode" shifts). Moreover, inactivation is in turn linked to movement of one or more activation gates, so mutations or modes in these gates or in regions that affect their kinetics would indirectly influence inactivation properties. Building a more complete understanding in the face of such complex linkages is one of the major challenges one faces in these studies. Several strategies have now emerged to aid our understanding of s t ructurefunction links. One approach, p ioneered by Horn and others (e.g., Yang et al., 1996), probes cysteine availability in $4 and other channel regions as a fimction of membrane potential. Such studies provide new, intimate views of the inner workings of the target channel by showing how certain portions of the $4 shift their accessibility f rom the cytoplasmic to the extracellular side of the channel with depolarization. Another important approach seeks to pair specific mutations in a manner that allows them to compensate