X-linked lissencephaly and "double cortex" are allelic human disorders mapping to Xq22.3-Xq23 associated with arrest of migrating cerebral cortical neurons. We identified a novel 10 kb brain-specific cDNA interrupted by a balanced translocation in an XLIS patient that encodes a novel 40 kDa predicted protein named Doublecortin. Four double cortex/X-linked lissencephaly families and three sporadic double cortex patients show independent doublecortin mutations, at least one of them a de novo mutation. Doublecortin contains a consensus Abl phosphorylation site and other sites of potential phosphorylation. Although Doublecortin does not contain a kinase domain, it is homologous to the amino terminus of a predicted kinase protein, indicating a likely role in signal transduction. Doublecortin, along with the newly characterized mDab1, may define an Abl-dependent pathway regulating neuronal migration.

KCNQ (K V 7) potassium channels underlie subthreshold M-currents that stabilize the neuronal resting potential and prevent repetitive firing of action potentials. Here, antibodies against four different KCNQ2 and KCNQ3 polypeptide epitopes show these subunits concentrated at the axonal initial segment (AIS) and node of Ranvier. AIS concentration of KCNQ2 and KCNQ3, like that of voltage-gated sodium (Na V ) channels, is abolished in ankyrin-G knock-out mice. A short motif, common to KCNQ2 and KCNQ3, mediates both in vivo ankyrin-G interaction and retention of the subunits at the AIS. This KCNQ2/KCNQ3 motif is nearly identical to the sequence on Na V ␣ subunits that serves these functions. All identified Na V and KCNQ genes of worms, insects, and molluscs lack the ankyrin-G binding motif. In contrast, vertebrate orthologs of Na V ␣ subunits, KCNQ2, and KCNQ3 (including from bony fish, birds, and mammals) all possess the motif. Thus, concerted ankyrin-G interaction with KCNQ and Na V channels appears to have arisen through convergent molecular evolution, after the division between invertebrate and vertebrate lineages, but before the appearance of the last common jawed vertebrate ancestor. This includes the historical period when myelin also evolved.

Mutations in the gene encoding the K ϩ channel KCNQ2 cause neonatal epilepsy and myokymia, indicating that KCNQ2 regulates the excitability of CNS neurons and motor axons, respectively. We show here that KCNQ2 channels are functional components of axon initial segments and nodes of Ranvier, colocalizing with ankyrin-G and voltage-dependent Na ϩ channels throughout the CNS and PNS. Retigabine, which opens KCNQ channels, diminishes axonal excitability. Linopirdine, which blocks KCNQ channels, prolongs the repolarization of the action potential in neonatal nerves. The clustering of KCNQ2 at nodes and initial segments lags that of ankyrin-G during development, and both ankyrin-G and KCNQ2 can be coimmunoprecipitated in the brain. KCNQ3 is also a component of some initial segments and nodes in the brain. The diminished activity of mutant KCNQ2 channels accounts for neonatal epilepsy and myokymia; the cellular locus of these effects may be axonal initial segments and nodes.

Members of the Kv7 family (Kv7.2-Kv7.5) generate a subthreshold K ؉ current, the M؊ current. This regulates the excitability of many peripheral and central neurons. Recent evidence shows that Kv7.2 and Kv7.3 subunits are targeted to the axon initial segment of hippocampal neurons by association with ankyrin G. Further, spontaneous mutations in these subunits that impair axonal targeting cause human neonatal epilepsy. However, the precise functional significance of their axonal location is unknown. Using electrophysiological techniques together with a peptide that selectively disrupts axonal Kv7 targeting (ankyrin G-binding peptide, or ABP) and other pharmacological tools, we show that axonal Kv7 channels are critically and uniquely required for determining the inherent spontaneous firing of hippocampal CA1 pyramids, independently of alterations in synaptic activity. This action was primarily because of modulation of action potential threshold and resting membrane potential (RMP), amplified by control of intrinsic axosomatic membrane properties. Computer simulations verified these data when the axonal Kv7 density was three to five times that at the soma. The increased firing caused by axosomatic Kv7 channel block backpropagated into distal dendrites affecting their activity, despite these structures having fewer functional Kv7 channels. These results indicate that axonal Kv7 channels, by controlling axonal RMP and action potential threshold, are fundamental for regulating the inherent firing properties of CA1 hippocampal neurons.axon initial segment ͉ CA1 pyramidal neurons ͉ M-current ͉ KCNQ channels N euronal Kv7 (KCNQ) channels form a noninactivating K ϩ current (also known as the MϪ current); this turns on at subthreshold potentials and regulates the excitability of a variety of peripheral and central neurons (1-3). Recent immunohistochemical evidence has shown that the principal subunits forming native M channels, Kv7.2 and Kv7.3 (3,4), are concentrated at the axon initial segment (AIS) and nodes of Ranvier of central and peripheral principal neurons (5-9), where they colocalize with Na ϩ channels. Like Na ϩ channels, they contain an ankyrin G-binding motif that targets them to the AIS (5, 8). They are also expressed at lower densities at the soma and possibly dendrites and synaptic terminals (4,6,7,10,11).Spontaneous mutations in Kv7 subunits cause epilepsy in humans (2) and mice (12). The hippocampus is strongly implicated in epilepsy (13) and accordingly, previous somatic recordings from these neurons have indicated that the Kv7 current is involved in determining several aspects of neuronal excitability, including the resting membrane potential (RMP), spike frequency adaptation, and burst suppression (e.g., refs. 14-16). However, the specific contribution made by Kv7 channels in the AIS to these or other manifestations of excitability has not been determined. This is important, because some human epileptogenic mutations impair axonal Kv7 subunit expression (7).We have used selective pharmacological and mol...

Mutations in the potassium channel subunit KCNQ2 lead to benign familial neonatal convulsions, a dominantly inherited form of generalized epilepsy. In heterologous cells, KCNQ2 expression yields voltage-gated potassium channels that activate slowly (tau, approximately 0.1 sec) at subthreshold membrane potentials. KCNQ2 associates with KCNQ3, a homolog, to form heteromeric channels responsible for the M current (I(M)) in superior cervical ganglion (SCG) neurons. Muscarinic acetylcholine and peptidergic receptors inhibit SCG I(M), causing slow EPSPs and enhancing excitability. Here, we use KCNQ2N antibodies, directed against a conserved N-terminal portion of the KCNQ2 polypeptide, to localize KCNQ2-containing channels throughout mouse brain. We show that KCNQ2N immunoreactivity, although widespread, is particularly concentrated at key sites for control of rhythmic neuronal activity and synchronization. In the basal ganglia, we find KCNQ2N immunoreactivity on somata of dopaminergic and parvalbumin (PV)-positive (presumed GABAergic) cells of the substantia nigra, cholinergic large aspiny neurons of the striatum, and GABAergic and cholinergic neurons of the globus pallidus. In the septum, GABAergic, purinergic, and cholinergic neurons that contribute to the septohippocampal and septohabenular pathways exhibit somatic KCNQ2 labeling. In the thalamus, GABAergic nucleus reticularis neurons that regulate thalamocortical oscillations show strong labeling. In the hippocampus, many PV-positive and additional PV-negative interneurons exhibit strong somatic staining, but labeling of pyramidal and dentate granule somata is weak. There is strong neuropil staining in many regions. In some instances, notably the hippocampal mossy fibers, evidence indicates this neuropil staining is presynaptic.

Mutations that reduce the function of KCNQ2 channels cause neuronal hyperexcitability, manifested as epileptic seizures and myokymia. These channels are present in nodes of Ranvier in rat brain and nerve and have been proposed to mediate the slow nodal potassium current I(Ks). We have used immunocytochemistry, electrophysiology and pharmacology to test this hypothesis and to determine the contribution of KCNQ channels to nerve excitability in the rat. When myelinated nerve fibres of the sciatic nerve were examined by immunofluorescence microscopy using antibodies against KCNQ2 and KCNQ3, all nodes showed strong immunoreactivity for KCNQ2. The nodes of about half the small and intermediate sized fibres showed labelling for both KCNQ2 and KCNQ3, but nodes of large fibres were labelled by KCNQ2 antibodies only. In voltage-clamp experiments using large myelinated fibres, the selective KCNQ channel blockers XE991 (IC50 = 2.2 microm) and linopirdine (IC50 = 5.5 microm) completely inhibited I(Ks), as did TEA (IC50 = 0.22 mm). The KCNQ channel opener retigabine (10 microm) shifted the activation curve to more negative membrane potentials by -24 mV, thereby increasing I(Ks). In isotonic KCl 50% of I(Ks) was activated at -62 mV. The activation curve shifted to more positive potentials as [K+]o was reduced, so that the pharmacological and biophysical properties of I(Ks) were consistent with those of heterologously expressed homomeric KCNQ2 channels. The ability of XE991 to selectively block I(Ks) was further exploited to study I(Ks) function in vivo. In anaesthetized rats, the excitability of tail motor axons was indicated by the stimulus current required to elicit a 40% of maximal compound muscle action potential. XE991 (2.5 mg kg(-1) i.p.) eliminated all nerve excitability functions previously attributed to I(Ks): accommodation to 100 ms subthreshold depolarizing currents, the post-depolarization undershoot in excitability, and the late subexcitability after a single impulse or short trains of impulses. Due to reduced spike-frequency adaptation after XE991 treatment, 100 ms suprathreshold current injections generated long trains of action potentials. We conclude that the nodal I(Ks) current is mediated by KCNQ channels, which in large fibres of rat sciatic nerve appear to be KCNQ2 homomers.

Rapid energy-efficient signaling along vertebrate axons is achieved through intricate subcellular arrangements of voltage-gated ion channels and myelination. One recently appreciated example is the tight colocalization of K v 7 potassium channels and voltage-gated sodium (Na v ) channels in the axonal initial segment and nodes of Ranvier. The local biophysical properties of these K v 7 channels and the functional impact of colocalization with Na v channels remain poorly understood. Here, we quantitatively examined K v 7 channels in myelinated axons of rat neocortical pyramidal neurons using high-resolution confocal imaging and patch-clamp recording. K v 7.2 and 7.3 immunoreactivity steeply increased within the distal two-thirds of the axon initial segment and was mirrored by the conductance density estimates, which increased from ϳ12 (proximal) to 150 pS m Ϫ2 (distal). The axonal initial segment and nodal M-currents were similar in voltage dependence and kinetics, carried by K v 7.2/7.3 heterotetramers, 4% activated at the resting membrane potential and rapidly activated with single-exponential time constants (ϳ15 ms at 28 mV). Experiments and computational modeling showed that while somatodendritic K v 7 channels are strongly activated by the backpropagating action potential to attenuate the afterdepolarization and repetitive firing, axonal K v 7 channels are minimally recruited by the forward-propagating action potential. Instead, in nodal domains K v 7.2/7.3 channels were found to increase Na v channel availability and action potential amplitude by stabilizing the resting membrane potential. Thus, K v 7 clustering near axonal Na v channels serves specific and context-dependent roles, both restraining initiation and enhancing conduction of the action potential.

Acetylcholine excites many central and autonomic neurons through inhibition of M-channels, slowly activating, noninactivating voltage-gated potassium channels. We here provide information regarding the in vivo distribution and biochemical characteristics of human brain KCNQ2 and KCNQ3, two channel subunits that form M-channels when expressed in vitro, and, when mutated, cause the dominantly inherited epileptic syndrome, benign neonatal familial convulsions. KCNQ2 and KCNQ3 proteins are colocalized in a somatodendritic pattern on pyramidal and polymorphic neurons in the human cortex and hippocampus. Immunoreactivity for KCNQ2, but not KCNQ3, is also prominent in some terminal fields, suggesting a presynaptic role for a distinct subgroup of M-channels in the regulation of action potential propagation and neurotransmitter release. KCNQ2 and KCNQ3 can be coimmunoprecipitated from brain lysates. Further, KCNQ2 and KCNQ3 are coassociated with tubulin and protein kinase A within a Triton X-100-insoluble protein complex. This complex is not associated with low-density membrane rafts or with N-methyl-Daspartate receptors, PSD-95 scaffolding proteins, or other potassium channels tested. Our studies thus provide a view of a signaling complex that may be important for cognitive function as well as epilepsy. Analysis of this complex may shed light on the unknown transduction pathway linking muscarinic acetylcholine receptor activation to M-channel inhibition. M utations in ion channel genes have been shown to cause a large number of forms of epilepsy and other episodic disorders in humans and inbred mice (1-3). Independent efforts led by Leppert (4-6) and Steinlein (7,8) to identify genes responsible for benign neonatal familial convulsions (BNFC), a dominantly inherited epilepsy associated with frequent seizures in the first weeks of life and a 10-16% risk of seizure recurrence after infancy, resulted in the cloning of two potassium channel subunits, KCNQ2 and KCNQ3. KCNQ2 and KCNQ3 have been characterized electrophysiologically with heterologous expression systems (8-12). Coexpression of KCNQ2 and KCNQ3 in Xenopus oocytes resulted in currents that were much larger than those observed when either subunit was expressed alone, leading to the suggestion that heteromultimeric KCNQ2͞KCNQ3 channels were expressed in vivo (9, 10). In oocytes or HEK cells, currents mediated by these heterologously expressed KCNQ2͞ KCNQ3 channels were enhanced by activation of the cAMPdependent protein kinase (PKA); this effect was abolished by site-directed mutations that eliminated a single PKA consensus site near the amino terminus of the KCNQ2 polypeptide (10). Mutations causing BNFC were found to cause only slight reductions in current compared with wild-type controls, suggesting that small differences in the activity of these KCNQ channels in vivo might be sufficient to cause epilepsy (10). When KCNQ2 and KCNQ3 cDNAs were coexpressed in oocytes, currents were observed that exhibited kinetic and voltagedependent properties similar to M-currents...