Hyperpolarization-activated HCN channels are modulated by direct binding of cyclic nucleotides. For HCN2 channels, cAMP shifts the voltage dependence for activation, with relatively little change in the maximal conductance. By contrast, in spHCN channels, cAMP relieves a rapid inactivation process and produces a large increase in maximum conductance. Our results suggest that these two effects of cAMP represent the same underlying process. We also find that spHCN inactivation occurs not by closure of a specialized inactivation gate, as for other voltage-dependent channels, but by reclosure of the same intracellular gate opened upon activation. Effectively, the activation gate exhibits a "desensitization to voltage," perhaps by slippage of the coupling between the voltage sensors and the gate. Differences in the initial coupling efficiency could allow cAMP to produce either the inactivation or the shift phenotype by strengthening effective coupling: a shift would naturally occur if coupling is already strong in the absence of cAMP.

In a myasthenic syndrome associated with fatigable generalized weakness and recurrent attacks of respiratory and bulbar paralysis since birth, nerve stimulation at physiologic rates rapidly decremented the compound muscle action potential. Intercostal muscle studies revealed no abnormality of the resting membrane potential, evoked quantal release, synaptic potentials, acetylcholine receptor channel kinetics, or endplate ultrastructure, but endplate potentials depolarizing the resting potential to ؊40 mV failed to excite action potentials. Pursuing this clue, we sequenced SCN4A encoding the skeletal muscle sodium channel (Na v1.4) and detected two heteroallelic mutations involving conserved residues not present in 400 normal alleles: S246L in the S4͞S5 cytoplasmic linker in domain I, and V1442E in the S3͞S4 extracellular linker in domain IV. The genetically engineered V1442E-Na channel expressed in HEK cells shows marked enhancement of fast inactivation close to the resting potential, and enhanced use-dependent inactivation on high-frequency stimulation; S246L is likely a benign polymorphism. The V1442E mutation in SCN4A defines a novel disease mechanism and a novel phenotype with myasthenic features.

Voltage-gated sodium channels are essential for the propagation of action potentials in nociceptive neurons.

Jingzhaotoxin-I (JZTX-I), a 33-residue polypeptide, is derived from the Chinese tarantula Chilobrachys jingzhao venom based on its ability to evidently increase the strength and the rate of vertebrate heartbeats. The toxin has three disulfide bonds with the linkage of I-IV, II-V, and III-VI that is a typical pattern found in inhibitor cystine knot molecules. Its cDNA determined by rapid amplification of 3-and 5-cDNA ends encoded a 62-residue precursor with a small proregion of eight residues. Whole-cell configuration indicated that JZTX-I was a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. Although JZTX-I also exhibits the interaction with channel isoforms expressing in mammalian and insect sensory neurons, its affinity for tetrodotoxin-resistant subtype in mammalian cardiac myocytes (IC 50 ‫؍‬ 31.6 nM) is ϳ30-fold higher than that for tetrodotoxin-sensitive subtypes in latter tissues. Not affecting outward delayrectified potassium channels expressed in Xenopus laevis oocytes and tetrodotoxin-resistant sodium channels in mammal sensory neurons, JZTX-I hopefully represents a potent ligand to discriminate cardiac sodium channels from neuronal tetrodotoxin-resistant isoforms. Furthermore, different from any reported spider toxins, the toxin neither modifies the current-voltage relationships nor shifts the steady-state inactivation of sodium channels. Therefore, JZTX-I defines a new subclass of spider sodium channel toxins. JZTX-I is an ␣-like toxin first reported from spider venoms. The result provides an important witness for a convergent functional evolution between spider and other animal venoms.

1 We have used the whole-cell patch-clamp technique to study the e ects of 4-sulphoniccalixarenes and some other poly-sulphonic acid agents, such as suramin and basilen blue, on volume-regulated anion channel (VRAC) currents in cultured endothelial cells (CPAE cells). 2 The 4-sulphonic-calixarenes induced a fast inhibition at positive potentials but were ine ective at negative potentials. At small positive potentials, 4-sulphonic-calix[4]arene was a more e ective inhibitor than 4-sulphonic-calix[6]arene and -calix[8]arene, which became more e ective at more positive potentials. 3 Also suramin and basilen blue induced a voltage dependent current inhibition, reaching a maximum around +40 mV and declining at more positive potentials. 4 The voltage dependence of inhibition was modelled by assuming that these negatively charged molecules bind to a site inside VRAC that senses a fraction d of the applied electrical ®eld, ranging beween 0.16 to 0.32. 4-Sulphonic-calix[4]arene, suramin and basilen blue bind and occlude VRAC at moderate potentials, but permeate the channel at more positive potentials. 4-Sulphonic-calix[6]arene and -calix[8]arene however do not permeate the channel. From the structural information of the calixarenes, we estimate a lower and upper limit of 11*12 and 17*12 A Ê 2 respectively for the crosssectional area of the pore.

In-depth structure-function studies of voltage-gated Na+ channels and peptide toxins are continuously increasing our understanding of their interaction. In this study, an effective yeast expression system was used to study the role of 14 N- and C-terminal residues from the alpha-like toxin BmK M1 from the Chinese scorpion Buthus martensii Karsch. With the use of site-directed mutagenesis, all of these residues were individually substituted by one or more amino acids, resulting in a total of 19 mutants. These were then subjected to a bioassay on mice, an elaborate electrophysiological characterization on three cloned voltage-gated Na+ channels (Nav1.2, Nav1.5, and para), and a circular dichroism analysis. Our results reveal large mutant-dependent differences that emphasize important and specific roles for the studied residues. By mutating single amino acids, we were able to redirect the alpha-like characteristics of BmK M1 (active on both mammals and insects) to either much higher mammal specificity or, in a few cases, total insect specificity. This study therefore represents a thorough mapping and elucidation of three epitopes that underlie the molecular basis of the mammalian and insecticidal potency of the scorpion alpha-like toxin, BmK M1 on voltage-gated Na+ channels.

1 Fluoxetine (Prozac) is widely used as an antidepressant drug and is assumed to be a selective 5-hydroxytryptamine (5-HT) reuptake inhibitor (SSRI). Claims that its bene®cial psychotropic e ects extend beyond those in treatment of depression have drawn clinical and popular attention to this compound, raising the question of whether there is anything exceptional about the supposed selective actions. 2 We have used the voltage clamp technique to study the e ect of¯uoxetine on a neuronal, voltagedependent potassium (K + ) channel (RCK1; Kv1.1), expressed in Xenopus laevis oocytes. This channel subunit is abundantly expressed in the central nervous system and K + channels containing this subunit are involved in the repolarization process of many types of neurones. 3 Blockade of the K + currents by¯uoxetine was found to be use-and dose-dependent. Wash-out of this compound could not be achieved. Fluoxetine did not a ect the ion selectivity of this K + channel, as the reversal potential was unaltered. 4 Slowing of both activation and deactivation kinetics of the channel by¯uoxetine was observed, including tail current crossover upon repolarization. 5 Hodgkin-Huxley type of models and more generalized Markov chain models were used to ®t the kinetics of the data. Based upon a Markov kinetic scheme, our data can be interpreted to mean that blockade of¯uoxetine consists of two components: a voltage-independent occurring in the last closed, but available state of the channel, and a voltage-dependent occurring in the open state. 6 This study describes the ®rst biophysical working model for the mechanism of action of¯uoxetine on a neuronal, voltage-dependent K + channel, RCK1. Although this channel is not very potently blocked by¯uoxetine when expressed in oocytes, this study may help us to understand some of the clinical symptoms seen with elevated serum concentrations of this SSRI.

1 We have used the whole-cell patch clamp technique to study the e ect of¯uoxetine, a commonly used antidepressant drug, on the volume-regulated anion channel (VRAC) in calf pulmonary artery endothelial (CPAE) cells. We also examined its e ects on other Cl 7 channels, i.e. the Ca 2+ -activated Cl 7 current (I Cl,Ca ) and the cystic ®brosis transmembrane conductance regulator (CFTR) to assess the speci®city of this compound for VRAC. 2 At pH 7.4¯uoxetine induced a fast and reversible block of the volume-sensitive chloride current (I Cl,swell ), with a K i value of 6.0+0.5 mM (n=6-9). The blocking e ciency increased with increasing extracellular pH (K i =0.32+0.01 mM at pH 8.8, n=3-9), indicating that the blockade is mediated by the uncharged form of¯uoxetine. 3 Fluoxetine inhibited Ca 2+ -activated Cl 7 currents, I Cl,Ca , activated by loading CPAE cells via the patch pipette with 1000 nM free Ca 2+ (K i =10.7+1.6 mM at pH 7.4, n=3-5). The CFTR channel, transiently transfected in CPAE cells, was also inhibited with a K i value of 26.9+9.4 mM at pH 7.4 (n=3). 4 This study describes for the ®rst time the e ects of¯uoxetine on anion channels. Our data reveal a potent block of VRAC at¯uoxetine concentrations close to plasma concentrations. The results suggest a hydrophobic interaction with high a nity between uncharged¯uoxetine and volumeactivated chloride channels. Ca 2+ -activated Cl 7 currents and CFTR are also blocked by¯uoxetine, revealing a novel characteristic of the drug as a chloride channel modulator.