Hyperpolarization-activated cation (HCN) channels are believed to be involved in the generation of cardiac pacemaker depolarizations as well as in the control of neuronal excitability and plasticity. The contributions of the four individual HCN channel isoforms (HCN1±4) to these diverse functions are not known. Here we show that HCN2-de®cient mice exhibit spontaneous absence seizures. The thalamocortical relay neurons of these mice displayed a near complete loss of the HCN current, resulting in a pronounced hyperpolarizing shift of the resting membrane potential, an altered response to depolarizing inputs and an increased susceptibility for oscillations. HCN2-null mice also displayed cardiac sinus dysrhythmia, a reduction of the sinoatrial HCN current and a shift of the maximum diastolic potential to hyperpolarized values. Mice with cardiomyocytespeci®c deletion of HCN2 displayed the same dysrhythmia as mice lacking HCN2 globally, indicating that the dysrhythmia is indeed caused by sinoatrial dysfunction. Our results de®ne the physiological role of the HCN2 subunit as a major determinant of membrane resting potential that is required for regular cardiac and neuronal rhythmicity.

The mechanisms by which proteins gain entry into membranes is a fundamental problem in biology. Here, we present the first crystal structure of a thiol-activated cytolysin, perfringolysin O, a member of a large family of toxins that kill eukaryotic cells by punching holes in their membranes. The molecule adopts an unusually elongated shape rich in beta sheet. We have used electron microscopy data to construct a detailed model of the membrane channel form of the toxin. The structures reveal a novel mechanism for membrane insertion. Surprisingly, the toxin receptor, cholesterol, appears to play multiple roles: targeting, promotion of oligomerization, triggering a membrane insertion competent form, and stabilizing the membrane pore.

Malfunctions of potassium channels are increasingly implicated as causes of neurological disorders. However, the functional roles of the large-conductance voltage-and Ca 2؉ -activated K ؉ channel (BK channel), a unique calcium, and voltage-activated potassium channel type have remained elusive. Here we report that mice lacking BK channels (BK ؊/؊ ) show cerebellar dysfunction in the form of abnormal conditioned eye-blink reflex, abnormal locomotion and pronounced deficiency in motor coordination, which are likely consequences of cerebellar learning deficiency. At the cellular level, the BK ؊/؊ mice showed a dramatic reduction in spontaneous activity of the BK ؊/؊ cerebellar Purkinje neurons, which generate the sole output of the cerebellar cortex and, in addition, enhanced short-term depression at the only output synapses of the cerebellar cortex, in the deep cerebellar nuclei. The impairing cellular effects caused by the lack of postsynaptic BK channels were found to be due to depolarization-induced inactivation of the action potential mechanism. These results identify previously unknown roles of potassium channels in mammalian cerebellar function and motor control. In addition, they provide a previously undescribed animal model of cerebellar ataxia. P otassium channels are the largest and most diverse class of ion channels underlying electrical signaling in the brain (1). By causing highly regulated, time-dependent, and localized polarization of the cell membrane, the opening of K ϩ channels mediates feedback control of excitability in a variety of cell types and conditions (1). Consequently, K ϩ channel dysfunctions can cause a range of neurological disorders (2-6), and drugs that target K ϩ channels hold promise for a variety of clinical applications (7).Among the wide range of voltage-and calcium-gated K ϩ channel types, one stands out as unique: the large-conductance voltage-and Ca 2ϩ -activated K ϩ channel (BK channel, also termed Slo or Maxi-K) differs from all other K ϩ channels in that it can be activated by both intracellular Ca 2ϩ ions and membrane depolarization (8). These channels are widely expressed in central and peripheral neurons, as well as in other tissues (9), and are regarded as a promising drug target (10). However, the functions of the BK channels in vivo have not previously been directly tested in any vertebrate species. We therefore decided to examine the functions of these channels by inactivating the gene encoding the pore-forming channel protein. MethodsA complete description of the methods is given in Supporting Methods, which is published as supporting information on the PNAS web site.Generation of BK Channel ␣ Subunit-Deficient Mice. In the targeting vector (Fig. 5, which is published as supporting information on the PNAS web site), the pore exon was flanked by a single loxP site and a floxed neo͞tk cassette. Correctly targeted embryonic stem cells were injected into C57BL͞6 blastocysts and resulting chimeric mice mated with C57BL͞6. Homozygous BK-deficient mice (F 2 generation) ...

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