Light at the End of the Channel

The propagation of action potential spikes along neuronal membranes is a feature of the nervous system so fundamental and so well known as to seem commonplace. But the molecular foundation of this familiar biological phenomenon—the voltage-dependent conformational changes leading to opening of Na+, K+, and Ca2+ channels—presents puzzles in protein behavior unencountered elsewhere in macromolecular chemistry. How is the membrane electric field sensed by these proteins? By what sort of allosteric gymnastics do the proteins respond to changes in transmembrane voltage as they switch among closed and open conformations? These kinds of questions have provided most of the driving force for research in ion channel biophysics since Hodgkin and Huxley 1952xHodgkin, A.L. and Huxley, A.F. J. Physiol. (Lond.). 1952; 117: 500–544CrossrefSee all ReferencesHodgkin and Huxley 1952 created the field a half-century ago. It is only in the past few years, however, that we have begun to derive convincing pictures of the physical movements that occur upon voltage gating of channels. These recent advances have all emerged from juxtaposing the usual electrophysiological measurements with clever chemical interventions. Thus, Horn’s group (Yang and Horn 1995xYang, N. and Horn, R. Neuron. 1995; 15: 213–218Abstract | Full Text PDF | PubMed | Scopus (280)See all ReferencesYang and Horn 1995) introduced a molecular cherry-picking method to show that the charge-laden “S4” transmembrane sequence of Na+ channels literally moves outward across the membrane upon depolarization and thereby initiates the events leading to channel opening. By a related chemical-modification strategy, experiments from Yellen’s lab (Liu et al. 1997xLiu, Y., Holmgren, M., Jurman, M.E., and Yellen, G. Neuron. 1997; 19: 175–184Abstract | Full Text | Full Text PDF | PubMed | Scopus (356)See all ReferencesLiu et al. 1997) located the end point of the K+ channel gating pathway, the pore-occluding “gate” itself. And recently, Isacoff and colleagues added another weapon to the arsenal by fluorescently labeling K+ channels in a site-directed manner, in order to follow optically the gating-induced changes in the local environment of the introduced chromophore (Mannuzzu et al. 1996xMannuzzu, L.M., Moronne, M.M., and Isacoff, E.Y. Science. 1996; 271: 213–216Crossref | PubMedSee all ReferencesMannuzzu et al. 1996).In this issue of Neuron, Cha and coworkers (1999) exploit this latter fluorescence method to answer a central question that has been hovering over the field from its very beginnings: are the voltage-driven conformational changes equivalent in the four equivalent channel “subunits,” and do these changes occur independently? Or, alternatively, do the different subunits play different roles in the gating process? All “S4-type” ion channels are built as tetrameric complexes, with four similar or identical subunits surrounding the pore. In voltage-gated K+ channels, these subunits are actually separate polypeptides, but Na+ and Ca2+ channels are built from four “honorary subunits”: homologous domains lying in tandem in a single polypeptide chain. The strict homotetrameric character of K+ channels makes it next to impossible to distinguish individual K+ channel subunits during gating. The present experiments avoid this ambiguity by focusing on Na+ channels, which can be straightforwardly labeled with fluorescent probes at specific sites confined to single domains (but often with molecular biological headaches). The authors attach fluorescent probes individually to equivalent S4-associated residues in domains I, II, III, and IV and observe the optical signals associated with each probe as the channels gate. These site-specific fluorescence signals report the goings-on in individual domains, in contrast to conventional electrical signals, to which all four subunits necessarily contribute. The results show that the four domains of Na+ channels are functionally nonequivalent: they have distinct gating personalities.The specific focus of this study is a classical topic in Na+ channels: the coupling of voltage-dependent activation to channel inactivation, a separate gating process that ensures swift, reliable spike repolarization. Many S4-type channels spontaneously inactivate upon sustained depolarization, and it is now widely accepted that inactivation is directly coupled to channel opening. In its simplest form, this idea demands that only the open conformation of the channel is capable of inactivating. Conversely, inactivated channels cannot directly close upon repolarization, but must first emerge out of the inactivated state into the open state. This effect was first observed in groundbreaking measurements of charge movement (“gating currents”) by Armstrong and Bezanilla 1977xArmstrong, C.M. and Bezanilla, F. J. Gen. Physiol. 1977; 70: 567–590Crossref | PubMedSee all ReferencesArmstrong and Bezanilla 1977 in Na+ channels. Depolarization elicits prompt on gating currents, as the S4 segments, poised for the activating voltage change, move outward as the first step toward channel opening. However, once the channel has opened and then inactivated a few milliseconds later, the return of gating charge upon repolarization is slow: it takes several milliseconds for the channel to escape from inactivation. The process by which this off gating current is slowed is called “charge immobilization,” and in Cha et al. 1999xCha, A., Ruben, P.C., George, A.L. Jr., Fujimoto, E., and Bezanilla, F. Neuron. 1999; 22: 73–87Abstract | Full Text | Full Text PDF | PubMed | Scopus (127)See all ReferencesCha et al. 1999 this phenomenon is followed on each of the separate S4 domains.Cha and coworkers place fluorescent probes at equivalent positions near the external end of each S4 segment. The fluorescence signals (reporting the local conformational change near the specific residue) are then followed simultaneously with gating current (reporting the movement of S4 charge averaged over all four domains). The results are striking. In domains I and II, the fluorescence change is rapid in both the on and off directions. A different result is obtained with domains III and IV, however. Here, the optical signals follow more complicated kinetics, as if the chromophore’s environment changes on both the fast timescale of activation and the slower timescale of inactivation. Moreover, if the depolarizing pulse is maintained long enough for inactivation to set in, the fluorescence signal is immobilized, as is 60%–70% of the gating charge. This effect is not seen for S4 residues in domains I or II, which is a clear functional distinction between the four nominally equivalent channel sequences. This result is not entirely unexpected, since the cytoplasmic linker between domains III and IV, and the S4 residue in domain IV itself, are known to be mutagenic hot spots for Na+ channel inactivation (7xWest, J.W., Patton, D.E., Scheuer, T., Wang, Y., Goldin, A.L., and Catterall, W.A. Proc. Natl. Acad. Sci. USA. 1992; 89: 10910–10914Crossref | PubMedSee all References, 3xChahine, M., George, A.L. Jr., Zhou, M., Ji, S., Sun, W., Barchi, R.L., and Horn, R. Neuron. 1994; 12: 281–294Abstract | Full Text PDF | PubMed | Scopus (235)See all References). Though the detailed mechanism of Na+ channel inactivation is unknown, it is satisfying that the fluorescence immobilization experiments point to domains III and IV as specifically linked to inactivation.So a surprisingly simple picture emerges from these experiments. In domains I and II, the voltage-sensing S4 segments move back and forth freely in response to voltage changes, oblivious to whether the channel protein is open or inactivated. In the two other domains, once the S4 segments have moved outward upon depolarization, they become immobilized by the conformational change leading to the inactivated state. This picture, far more detailed than could have been achieved by electrophysiological measurements alone, is the first mechanistic fruit of the new site-specific optical techniques applied to ion channels.