Molecular basis of altered excitability in Shaker mutants of Drosophila melanogaster.

Mutations in the Shaker (Sh) locus of Drosophila melanogaster have differing effects on action potential duration and repolarization in neurons as well as on A-type K+ channels (I(A)) in muscle. The molecular basis of three exemplary Sh alleles (Sh(KS133), Sh(E62) and Sh5) has been identified. They are point mutation in the Sh transcription unit expressing aberrant voltage-gated A-type K+ channels. Replicas of each mutation have been introduced by in vitro mutagenesis into Sh cDNA. The expression of in vitro transcribed mutant Sh cRNA in Xenopus laevis oocytes reproduced the specific phenotypic traits of each Sh allele. The lack of I(A) in Sh(KS133) is due to a missense mutation within a sequence motif occurring in all hitherto characterized voltage-gated K+ channel forming proteins. The reduction of I(A) in Sh(E62) is due to a mutation in an AG acceptor site. The intervening sequence between exon 19 and 20 is not spliced in Sh(E62) RNA. As a consequence Sh(E62) flies do not contain the full complement of Sh K+ forming proteins. Finally, the Sh5 mutation leads to an altered voltage dependence of K+ channel activation and inactivation as well as to an accelerated rate of recovery from inactivation. This is due to a missense mutation altering the amino acid sequence of the proposed transmembrane segment S5 of the Sh K+ channels. Segment S5 is located adjacently to the presumed voltage sensor of voltage-gated ion channels. The results explain the altered properties of excitable cells in Sh mutants and provide a general model for the possible role of A-type K+ channels in modulation action potential profiles

Phallolysin. A mushroom toxin, forms proton and voltage gated membrane channels.

Phallolysin, a water soluble protein of Mr 34,000 produced by the poisonous mushroom Amanita phalloides, causes lysis of various mammalian cell types. Lysis is thought to be initiated by the formation of ion permeable membrane channels. We therefore studied the interaction of phallolysin with solvent-free planar lipid bilayers. In the presence of low phallolysin concentrations (10-100 nM) single channel current fluctuations were observed. Unit channel conductances are 44 pS in 500 mM NaCl and 77 pS in 1 M NaCl. Although the channel does not significantly discriminate between alkali cations, its permeability to Cl- is lower (PK+/PCl- = 4/1). Gating kinetics display a pronounced bursting behavior and a dependence on membrane voltage, cis side pH-value, and on membrane lipid composition. An equivalence relation between membrane voltage and proton concentration was found, i.e. a pH change of one unit is equivalent to a corresponding voltage change of 130 mV. Dependence on the amount of negatively charged lipids is explained by changes of the actual pH due to surface charge effects