Voltage-gated potassium channels (Kv) of the Shaker-related superfamily are assembled from membrane-integrated alpha subunits and auxiliary beta subunits. The beta subunits may increase Kv channel surface expression and/or confer A-type behavior to noninactivating Kv channels in heterologous expression systems. The interaction of Kv alpha and Kv beta subunits depends on the presence or absence of several domains including the amino-terminal N-type inactivating and NIP domains and the Kv alpha and Kv beta binding domains. Loss of function of Kv beta 1.1 subunits leads to a reduction of A-type Kv channel activity in hippocampal and striatal neurons of knock-out mice. This reduction may be correlated with altered cognition and motor control in the knock-out mice.

Voltage-gated ion channels contain a positively charged transmembrane segment termed S4. Recent evidence suggests that depolarisation of the membrane potential causes this segment to undergo conformational changes that, in turn, lead to the opening of the channel pore. In order to define these conformational changes in structural terms, we have introduced single cysteine substitutions into the S4 segment of the prototypical Shaker K+ channel at various positions and expressed the mutants in Xenopus oocytes. The cells were depolarised to induce K+ currents and the effect of application of 100 microM parachloromercuribenzenesulphonate (PCMBS) on these currents was examined by the two-electrode voltage-clamp technique. PCMBS inhibited K+ currents elicited by mutants L358C, L361C, V363C and L366C, but not those by V367C and S376C. Since PCMBS is a membrane-impermeable cysteine-modifying reagent, the data suggest that depolarisation must have caused the S4 segment to move out of the lipid bilayer into the extracellular phase rendering the residues at positions 358, 361, 363 and 366 susceptible to PCMBS attack. The lack of effect of PCMBS on V367C suggests that the exposure of S4 terminates at L366. Detailed analysis of L361C mutant revealed that the S4 movement can occur even below the resting potential of the cell, at which potential voltage-gated K+ channels are normally in a non-conducting closed state.

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SUMMARY1. Immunoglobulin G (IgG) prepared from the plasma ofpatients with a presynaptic disorder of neuromuscular transmission (Lambert-Eaton myasthenic syndrome, l.e.m.s.), or from normal pooled control human plasma, was injected into mice (10 mg daily) for up to 99 days.2. Micro-electrodes were used to record end-plate potentials from the diaphragm muscle bathed in normal Krebs solution containing tubocurarine (1-04-6 /LM).3. At 0.5 Hz nerve stimulation frequency, the quantal content was significantly reduced (P < 0-01-P < 0-001) in mice treated with six l.e.m.s. patients' IgG each compared with paired controls. The pooled quantal content was 55 + 3 (n = 110 end-plates) for all test animals and 131+9 (n = 47) for all controls (P < 0-001).4. During short trains at 20 or 40 Hz nerve stimulation, control muscles showed marked depression, while test muscles showed either facilitation or less marked depression. Quantal content throughout these trains remained lower than in controls.5. The results indicate that IgG antibody from l.e.m.s. patients can induce a similar physiological disorder in injected mice, and they support the view that this antibody interferes with evoked release of transmitter in l.e.m.s. by binding to nerve terminal determinants.

The human and rat forms of the Kv2.1 channel have identical amino acids over the membrane-spanning regions and differ only in the N-and C-terminal intracellular regions. Rat Kv2.1 activates much faster than human Kv2.1. Here we have studied the role of the N-and C-terminal residues that determine this difference in activation kinetics between the two channels. For this, we constructed mutants and chimeras between the two channels, expressed them in oocytes, and recorded currents by two-electrode voltage clamping. In the N-terminal region, mutation Q67E in the rat channel displayed a slowing of activation relative to rat wild type, whereas mutation D75E in the human channel showed faster activation than human wild type. In the C-terminal region, we found that some residues within the region of amino acids 740 -853 ("CTA" domain) were also involved in determining activation kinetics. The electrophysiological data also suggested interactions between the N and C termini. Such an interaction was confirmed directly by using a glutathione S-transferase (GST) fusion protein with the N terminus of Kv2.1, which we showed to bind to the C terminus of Kv2.1. Taken together, these data suggest that exposed residues in the T1 domain of the N terminus, as well as the CTA domain in the C terminus, are important in determining channel activation kinetics and that these N-and C-terminal regions interact.