Dendrotoxin (DTX) homologues are powerful blockers of K + channels that contain certain subfamily Kv1 (1.1±1.6) a-and b-subunits, in (a) 4 (b) 4 stoichiometry. DTX k inhibits potently Kv1.1-containing channels only, whereas aDTX is less discriminating, but exhibits highest affinity for Kv1.2. Herein, the nature of interactions of DTX k with native K + channels composed of Kv1.1 and 1.2 (plus other) subunits were examined, using 15 site-directed mutants in which amino acids were altered in the 3 10 -helix, b-turn, a-helix and random-coil regions. The mutants' antagonism of high-affinity [ 125 I]DTX k binding to Kv1.1-possessing channels in rat brain membranes and blockade of the Kv1.1 current expressed in oocytes were quantified. Also, the levels of inhibition of [ 125 I]aDTX binding to brain membranes by the DTX k mutants were used to measure their high-and low-affinity interactions, respectively, with neuronal Kv1.2-containing channels that possess Kv1.1 as a major or minor constituent. Displacement of toxin binding to either of these subtypes was not altered by single substitution with alanine of three basic residues in the random-coil region, or R52 or R53 in the a-helix; accordingly, representative mutants (K17A, R53A) blocked the Kv1.1 current with the same potency as the natural toxin. In contrast, competition of the binding of the radiolabelled aDTX or DTX k was dramatically reduced by alanine substitution of K26 or W25 in the b-turn whereas changing nearby residues caused negligible alterations. Consistently, W25A and K26A exhibited diminished functional blockade of the Kv1.1 homo-oligomer. The 3 10 -helical N-terminal region of DTX k was found to be responsible for recognition of Kv1.1 channels because mutation of K3A led to <1246-fold reduction in the inhibitory potency for [ 125 I]DTX k binding and a large decrease in its ability to block the Kv1.1 current; the effect of this substitution on the affinity of DTX k for Kv1.2-possessing oligomers was much less dramatic (<16-fold).

We have examined the effects of co-expression of Kvbeta1.1 and Kvbeta2.1 subunits on the gating of rat brain Kv1.4 channels, expressed in Xenopus oocytes. Expression of Kv1.4 subunits alone produced a rapidly inactivating "A" type current, which activated at potentials beyond -60 mV in a solution containing high levels of rubidium. Current activation curves obtained from tail current measurements were fitted with a Boltzmann function, with V1/2 = -47 mV and k = 10 mV. Neither the Kvbeta1.1 nor Kvbeta2.1 subunits altered the voltage dependence of activation. Both subunits accelerated the activation time constant of Kv1.4, without affecting its voltage dependence. Surprisingly, the Kvbeta2.1 subunit, which lacks an N-terminal inactivation domain, was almost as effective as the Kvbeta1.1 subunit in speeding up Kv1.4. Steady-state inactivation of Kv1.4 was unchanged upon co-expression with either Kvbeta1.1 or Kvbeta2.1 subunits. Kv1.4 recovered from inactivation with two time constants; apart from an approximately 50% lengthening of the slow time constant with a high Kvbeta2.1 injection ratio, neither time constant was altered by either the Kvbeta1.1 or Kvbeta2.1 subunits, suggesting little interaction with recovery from C-type inactivation. Clearly, beta subunits have the potential to modify the gating of Kv1.4 channels in the brain more subtly than has been suggested previously.

Voltage-gated K(+) channels (Kv1) are important in glia, being required for cell proliferation. Herein, reactive astrocytes from a rat cerebellar lesion were shown to contain Kv1.1, -1.2, -1.3, -1.4, and -1.6 alpha plus beta1.1 subunits, as well as an unusual beta2.1 constituent; the latter was also found in a glioblastoma C6 cell line, together with Kv1.1, -1.3, and -1.6 and beta1.1 subunits. Reverse transcriptase-polymerase chain reaction revealed a novel product of the beta2 gene in C6 cells and reactive astrocytes, but not in cultured astrocytes or rat normal brain. Its cloning identified a variant, Kvbeta2.1A, alternatively spliced between I24 and Y39. Despite this 14 residue deletion, Kvbeta2.1A assembled cotranslationally with Kv1.1 or -1.2 and, when coexpressed with Kv1. 4 in oocytes, increased the inactivation rate of this K(+) current. Whereas the full-length beta2.1 gave a large increase in the amplitude of the Kv1.1 current in oocytes, the effect of beta2.1A varied from a modest elevation of the current to a slight suppression in some cases. In summary, this is the first report of the existence of an alternatively spliced product of the Kvbeta2.1 gene in C6 cells and reactive astrocytes, and supports the involvement of its core region (residues 39 onward) in assembly with alpha subunits while excluding a contribution of the adjacent 14 residues to accelerating the inactivation of Kv1.4.