We cloned two L L subunits of large-conductance calcium-activated potassium (BK) channels, hKCNMB3 (BKL L1) and hKCNMB4 (BKL L4). Profiling mRNA expression showed that hKCNMB3 expression is enriched in testis and hKCNMB4 expression is very prominent in brain. We coexpressed BK channel K K (BKK K) and BKL L4 subunits in vitro in CHO cells. We compared BKK K/L L4 mediated currents with those of smooth muscle BKK K/L L1 channels. BKL L4 slowed activation kinetics more significantly, led to a steeper apparent calcium sensitivity, and shifted the voltage range of BK current activation to more negative potentials than BKL L1. BKK K/L L4 channels were not blocked by 100 nM charybdotoxin or iberiotoxin, and were activated by 17L L-estradiol.z 2000 Federation of European Biochemical Societies.

We examined whether the N-terminus of Kv4.2 A-type channels (4.2NT) possesses an autoinhibitory N-terminal peptide domain, which, similar to the one of Shaker, mediates inactivation of the open state. We found that chimeric Kv2.1(4.2NT) channels, where the cytoplasmic Kv2.1 N-terminus had been replaced by corresponding Kv4.2 domains, inactivated relatively fast, with a mean time constant of 120 ms as compared to 3.4 s in Kv2.1 wild-type. Notably, Kv2.1(4.2NT) showed features typically observed for Shaker N-type inactivation: fast inactivation of Kv2.1(4.2NT) channels was slowed by intracellular tetraethylammonium and removed by N-terminal truncation (Delta40). Kv2.1(4.2NT) channels reopened during recovery from inactivation, and recovery was accelerated in high external K+. Moreover, the application of synthetic N-terminal Kv4.2 and ShB peptides to inside-out patches containing slowly inactivating Kv2.1 channels mimicked N-type inactivation. Kv4.2 channels, after fractional inactivation, mediated tail currents with biphasic decay, indicative of passage through the open state during recovery from inactivation. Biphasic tail current kinetics were less prominent in Kv4.2/KChIP2.1 channel complexes and virtually absent in Kv4.2Delta40 channels. N-type inactivation features of Kv4.2 open-state inactivation, which may be suppressed by KChIP association, were also revealed by the finding that application of Kv4.2 N-terminal peptide accelerated the decay kinetics of both Kv4.2Delta40 and Kv4.2/KChIP2.1 patch currents. However, double mutant cycle analysis of N-terminal inactivating and pore domains indicated differences in the energetics and structural determinants between Kv4.2 and Shaker N-type inactivation.

Voltage-gated ion channels generate cellular excitability, cause diseases when mutated, and act as drug targets in hyperexcitability diseases, such as epilepsy, cardiac arrhythmia and pain. Unfortunately, many patients do not satisfactorily respond to the present-day drugs. We found that the naturally occurring resin acid dehydroabietic acid (DHAA) is a potent opener of a voltage-gated K channel and thereby a potential suppressor of cellular excitability. DHAA acts via a non-traditional mechanism, by electrostatically activating the voltage-sensor domain, rather than directly targeting the ion-conducting pore domain. By systematic iterative modifications of DHAA we synthesized 71 derivatives and found 32 compounds more potent than DHAA. The most potent compound, Compound 77, is 240 times more efficient than DHAA in opening a K channel. This and other potent compounds reduced excitability in dorsal root ganglion neurons, suggesting that resin-acid derivatives can become the first members of a new family of drugs with the potential for treatment of hyperexcitability diseases.

Small conductance calcium-activated potassium channels (SK, K Ca ) are a family of voltageindependent K + channels with a distinct physiology and pharmacology. The bee venom toxin apamin inhibits exclusively the three cloned SK channel subtypes (SK1, SK2 and SK3) with different affinity, highest for SK2, lowest for SK1 and intermediate for SK3 channels. The high selectivity of apamin made it a valuable tool to study the molecular makeup and function of native SK channels. Three amino acids located in the outer vestibule of the pore are of particular importance for the different apamin sensitivities of SK channels. Chimeric SK1 channels, enabling the homomeric expression of the rat SK1 (rSK1) subunit and containing the core domain (S1-S6) of rSK1, are apamin insensitive. By contrast, channels formed by the human orthologue hSK1 are sensitive to apamin. This finding hinted to the involvement of regions beyond the pore as determinants of apamin sensitivity, since hSK1 and rSK1 have an identical amino acid sequence in the pore region. Here we investigated which parts of the channels outside the pore region are important for apamin sensitivity by constructing chimeras between apamin insensitive and sensitive SK channel subunits and by introducing point mutations. We demonstrate that a single amino acid situated in the extracellular loop between the transmembrane segments S3 and S4 has a major impact on apamin sensitivity. Our findings enabled us to convert the hSK1 channel into a channel that was as sensitive for apamin as SK2, the SK channel with the highest sensitivity. Ca 2+ -activated K + channels (K Ca ) 1 are activated by rises in intracellular Ca 2+ . The K Ca potassium channel family comprises of at least three subfamilies, K Ca 1-3 (1). Channels containing the K Ca 1.1 α-subunit (BK channels) have large single channel conductance and are maximally activated by micromolar concentrations of intracellular free calcium and concurrent depolarization (2). Their kinetic and pharmacological properties are modified upon assembly with membrane standing ß-subunits (3). The K Ca 2 subfamily of small-conductance Ca 2+ -activated K + channels, also known as SK channels, has three closely related members SK1 (K Ca 2.1), SK2 (K Ca 2.2) and SK3 (K Ca 2.3), which are characterized by a small single channel conductance. The IK channel (K Ca 3.1) shows an intermediate single channel conductance. Both SK and IK channels are voltage independent and activated by submicromolar concentrations of intracellular free Ca 2+ . The gating of SK and IK channels is induced upon Ca 2+ binding to calmodulin, which is constitutively bound to each channel subunit. Ca 2+ * This work was supported by a Wellcome Prize studentship (T.F.) and a Wellcome Trust Senior Research fellowship (M.S.).Address correspondence to: Martin Stocker, Laboratory of Molecular Pharmacology, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK, UKPMC Funders Group Author Manuscript UKPMC Funders Group Author Manuscriptbindin...

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