The recent development of techniques for recording currents through single ionic channels has led to the identification of a K+-specific channel that is activated by cytoplasmic Ca2+. The channel has complex properties, being activated by depolarizing voltages and having a voltage-sensitivity that is modulated by cytoplasmic Ca2+ levels. The conduction behaviour of the channel is also unusual, its high ionic selectivity being displayed simultaneously with a very high unitary conductance. Very little is known about the biochemistry of this channel, largely due to the lack of a suitable ligand for use as a biochemical probe for the channel. We describe here a protein inhibitor of single Ca2+-activated K+ channels of mammalian skeletal muscle. This inhibitor, a minor component of the venom of the Israeli scorpion, Leiurus quinquestriatus, reversibly blocks the large Ca2+-activated K+ channel in a simple biomolecular reaction. We have partially purified the active component, a basic protein of relative molecular mass (Mr) approximately 7,000.

Voltage-sensitive sodium channels and calcium channels are homologous proteins with distinctly different selectivity for permeation of inorganic cations. This difference in function is specified by amino acid residues located within P-region segments that link presumed transmembrane elements S5 and S6 in each of four repetitive Domains I, II, III, and IV. By analyzing the selective permeability of Na+, K+, and Ca2+ in various mutants of the mu 1 rat muscle sodium channel, the results in this paper support the concept that a conserved motif of four residues contributed by each of the Domains I-IV, termed the DEKA locus in sodium channels and the EEEE locus in calcium channels, determines the ionic selectivity of these channels. Furthermore, the results indicate that the Lys residue in Domain III of the sodium channel is the critical determinant that specifies both the impermeability of Ca2+ and the selective permeability of Na+ over K+. We propose that the alkylammonium ion of the Lys(III) residue acts as an endogenous cation within the ion binding site/selectivity filter of the sodium channel to tune the kinetics and affinity of inorganic cation binding within the pore in a manner analogous to ion-ion interactions that occur in the process of multi-ion channel conduction.

The gating kinetics of a Cat'-activated K' channel from adult rat muscle plasma membrane are studied in artificial planar bilayers . Analysis of single-channel fluctuations distinguishes two Ca 2 '-and voltage-dependent processes : (a) short-lived channel closure (<1 ms) events appearing in a bursting pattern ; (b) opening and closing events ranging from one to several hundred milliseconds in duration . The latter process is studied independently of the first and is denoted as the primary gating mode .

Peptide toxins with high affinity, divergent pharmacological functions, and isoform-specific selectivity are powerful tools for investigating the structure-function relationships of voltagegated sodium channels (VGSCs). Although a number of interesting inhibitors have been reported from tarantula venoms, little is known about the mechanism for their interaction with VGSCs. We show that huwentoxin-IV (HWTX-IV), a 35-residue peptide from tarantula Ornithoctonus huwena venom, preferentially inhibits neuronal VGSC subtypes rNav1.2, rNav1.3, and hNav1.7 compared with muscle subtypes rNav1.4 and hNav1.5. Of the five VGSCs examined, hNav1.7 was most sensitive to HWTX-IV (IC 50 ϳ 26 nM). Following application of 1 M HWTX-IV, hNav1.7 currents could only be elicited with extreme depolarizations (>؉100 mV). Recovery of hNav1.7 channels from HWTX-IV inhibition could be induced by extreme depolarizations or moderate depolarizations lasting several minutes. Site-directed mutagenesis analysis indicated that the toxin docked at neurotoxin receptor site 4 located at the extracellular S3-S4 linker of domain II. Mutations E818Q and D816N in hNav1.7 decreased toxin affinity for hNav1.7 by ϳ300-fold, whereas the reverse mutations in rNav1.4 (N655D/ Q657E) and the corresponding mutations in hNav1.5 (R812D/ S814E) greatly increased the sensitivity of the muscle VGSCs to HWTX-IV. Our data identify a novel mechanism for sodium channel inhibition by tarantula toxins involving binding to neurotoxin receptor site 4. In contrast to scorpion ␤-toxins that trap the IIS4 voltage sensor in an outward configuration, we propose that HWTX-IV traps the voltage sensor of domain II in the inward, closed configuration.

Mutational and biophysical analysis suggests that an intracellular COOH-terminal domain of the large conductance Ca 2؉ -activated K ؉ channel (BK channel) contains Ca 2؉ -binding site(s) that are allosterically coupled to channel opening. However the structural basis of Ca 2؉ binding to BK channels is unknown. To pursue this question, we overexpressed the COOH-terminal 280 residues of the Drosophila slowpoke BK channel (Dslo-C280) as a FLAG-and His 6-tagged protein in Escherichia coli. We purified Dslo-C280 in soluble form and used a 45 Ca 2؉ -overlay protein blot assay to detect Ca 2؉ binding. Dslo-C280 exhibits specific binding of 45 Ca 2؉ in comparison with various control proteins and known EF-hand Ca 2؉ -binding proteins. A mutation (D5N5) of Dslo-C280, in which five consecutive Asp residues of the ''Ca-bowl'' motif are changed to Asn, reduces 45 Ca 2؉ -binding activity by 56%. By electrophysiological assay, the corresponding D5N5 mutant of the Drosophila BK channel expressed in HEK293 cells exhibits lower Ca 2؉ sensitivity for activation and a shift of Ϸ؉80 mV in the midpoint voltage for activation. This effect is associated with a decrease in the Hill coefficient (N) for activation by Ca 2؉ and a reduction in apparent Ca 2؉ affinity, suggesting the loss of one Ca 2؉ -binding site per monomer. These results demonstrate a functional correlation between Ca 2؉ binding to a specific region of the BK protein and Ca 2؉ -dependent activation, thus providing a biochemical approach to study this process.