The murine Bis1 seizure gene and the Kcnab2 gene encoding the beta2-subunit of the K+ channel are different.

We tested the hypothesis that Bis1, a gene involved in seizure regulation in mice which has been localized to the distal part of chromosome 4, was the same as the gene Kcnab2, encoding the beta2-subunit for voltage-dependent K+ channels. Two facts suggested this hypothesis: Kcnab2 is located in the 3.1-cM confidence interval containing Bis1 and many studies have shown an involvement of K+ channels in the genesis of seizures. DNA sequence analysis of the coding sequence for Kcnab2 from JE/Le mice revealed no structural alterations which might affect Kcnab2 function. However, several nucleotide changes were observed.

Characterization of three episodic ataxia mutations in the human Kv1.1 potassium channel.

Episodic ataxia (EA) is a rare inherited neurological disorder due to mutation in the voltage-dependent Kv1.1 potassium channel. In nine unrelated families, a different missense point mutation at highly conserved positions has been reported. We have previously characterized six of the EA mutants. In this study, three recently identified mutations were introduced into the human Kv1.1 cDNA and expressed in Xenopus oocytes. Compared to wild type, T226A and T226M reduced the current amplitude by > 95%, shifted the voltage dependence by 15 mV, and slowed activation and deactivation kinetics. Currents from G311S were approximately 25% of wild type, less steeply voltage-dependent and had more pronounced C-type inactivation. These altered gating properties will reduce the delayed-rectifier potassium current which may underlie the symptoms of EA.

Episodic ataxia mutations in Kv1.1 alter potassium channel function by dominant negative effects or haploinsufficiency.

Subunits of the voltage-gated potassium channel Kv1.1 containing mutations responsible for episodic ataxia (EA), a human inherited neurological disease, were expressed in Xenopus oocytes. Five EA subunits formed functional homomeric channels with lower current amplitudes and altered gating properties compared with wild type. Two EA mutations located in the first cytoplasmic loop, R239S and F249I, yielded minimal or no detectable current, and Western blot analysis showed reduced protein levels. Coinjection of equal amounts of EA and wild-type mRNAs, mimicking the heterozygous condition, resulted in current amplitudes and gating properties that were intermediate between wild-type and EA homomeric channels, suggesting that heteromeric channels are formed with a mixed stoichiometry of EA and wild-type subunits. To examine the relative contribution of EA subunits in forming heteromeric EA and wild-type channels, each EA subunit was made insensitive to TEA, TEA-tagged, and coexpressed with wild-type subunits. TEA-tagged R239S and F249I induced the smallest shift in TEA sensitivity compared with homomeric wild-type channels, whereas the other TEA-tagged EA subunits yielded TEA sensitivities similar to coexpression of wild-type and TEA-tagged wild-type subunits. Taken together, these results show that the different mutations in Kv1.1 affect channel function and indicate that both dominant negative effects and haplotype insufficiency may result in the symptoms of EA.

Forskolin's structural analogue 1,9-dideoxyforskolin has Ca2+ channel blocker-like action in rat cerebellar granule cells.

Forskolin, routinely used as a specific activator of the cAMP pathway, is also a blocker of various ionic channels in a cAMP-independent way. We investigated, in rat cerebellar granule cells in culture, the effects of forskolin and its structural analogue 1,9-dideoxyforskolin on Ca2+ entry. Changes in cytosolic free Ca2+ concentration ([Ca]i) were monitored using fura-2 microfluorimetry. The increase in [Ca]i observed in response to membrane depolarization by 30 mM KCI was reduced by 20% in the presence of 100 microM forskolin, and by 71% with the same concentration of 1,9-dideoxyforskolin. A dose-response curve for 1,9-dideoxyforskolin gave an estimated IC50 of 54 microM. Additional experiments using the patch-clamp technique showed that 100 microM 1,9-dideoxyforskolin inhibit voltage-activated Ca2+ currents by 63%, although forskolin had no significant effect in the same conditions. This blocking effect of 1,9-dideoxyforskolin is not specific of a given Ca2+ channel type.

Forskolin blocks the transient K current of rat cerebellar granule neurons.

The action of forskolin (FSK) on voltage-activated K currents was investigated in cerebellar granule cells. FSK reversibly inhibited both A-type (IA) and non-inactivating K currents. IA blockade was more precisely characterized. This effect of FSK was shown to be voltage- and concentration-dependent with an IC50 of 19 microM at +50 mV. 1,9-Dideoxyforskolin (1,9-ddxFSK), a derivative of FSK that does not activate adenylate cyclase, specifically blocked IA, while cAMP-increasing agents had no direct effect on the K currents. The possibility that the non-cAMP mediated effect of FSK occurs through a channel-blocking mechanism and its eventual implications for neuronal excitability are discussed.