Mutations in a plasma membrane Ca2+-ATPase gene cause deafness in deafwaddler mice.

Hearing loss is the most common sensory deficit in humans. Because the auditory systems of mice and humans are conserved, studies on mouse models have predicted several human deafness genes and identified new genes involved in hearing. The deafwaddler (dfw) mouse mutant is deaf and displays vestibular/motor imbalance. Here we report that the gene encoding a plasma membrane Ca2+-ATPase type 2 pump (Atp2b2, also known as Pmca2) is mutated in dfw. An A-->G nucleotide transition in dfw DNA causes a glycine-to-serine substitution at a highly conserved amino-acid position, whereas in a second allele, dfw2J, a 2-base-pair deletion causes a frameshift that predicts a truncated protein. In the cochlea, the protein Atp2b2 is localized to stereocilia and the basolateral wall of hair cells in wild-type mice, but is not detected in dfw2J mice. This indicates that mutation of Atp2b2 may cause deafness and imbalance by affecting sensory transduction in stereocilia as well as neurotransmitter release from the basolateral membrane. These mutations affecting Atp2b2 in dfw and dfw2J are the first to be found in a mammalian plasma membrane calcium pump and define a new class of deafness genes that directly affect hair-cell physiology.

Mutant beta-spectrin 4 causes auditory and motor neuropathies in quivering mice.

The autosomal recessive mouse mutation quivering (qv), which arose spontaneously in 1953, produces progressive ataxia with hind limb paralysis, deafness and tremor. Six additional spontaneous alleles, qvJ, qv2J, qv3J, qv4J, qvlnd and qvlnd2J, have been identified. Ear twitch responses (Preyer's reflex) to sound are absent in homozygous qv/qv mice, although cochlear morphology seems normal and cochlear potentials recorded at the round window are no different from those of control mice. However, responses from brainstem auditory nuclei show abnormal transmission of auditory information, indicating that, in contrast to the many known mutations causing deafness originating in the cochlea, deafness in qv is central in origin. Here we report that quivering mice carry loss-of-function mutations in the mouse beta-spectrin 4 gene (Spnb4) that cause alterations in ion channel localization in myelinated nerves; this provides a rationale for the auditory and motor neuropathies of these mice.

Cloning of genomic and complementary DNA from Shaker, a putative potassium channel gene from Drosophila.

On the basis of electrophysiological analysis of Shaker mutants, the Shaker locus of Drosophila melanogaster has been proposed to encode a structural component of a voltage-dependent potassium channel, the A channel. Unlike sodium channels, acetylcholine receptors, and calcium channels, K+ channels have not been purified biochemically. To facilitate biochemical studies of a K+ channel, genomic DNA from the Shaker locus has been cloned. Rearrangements in five Shaker mutants have been mapped to a 60-kilobase segment of the genome. Four complementary DNA clones have been analyzed. These clones indicate that the Shaker gene contains multiple exons distributed over at least 65 kilobases of genomic DNA in the region where the mutations mapped. Furthermore, the gene may produce several classes of alternatively spliced transcripts. Two of the complementary DNA clones have been sequenced and their sequences support the hypothesis that Shaker encodes a component of a K+ channel.

Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila.

Potassium currents are crucial for the repolarization of electrically excitable membranes, a role that makes potassium channels a target for physiological modifications that alter synaptic efficacy. The Shaker locus of Drosophila is thought to encode a K+ channel. The sequence of two complementary DNA clones from the Shaker locus is reported here. The sequence predicts an integral membrane protein of 70,200 daltons containing seven potential membrane-spanning sequences. In addition, the predicted protein is homologous to the vertebrate sodium channel in a region previously proposed to be involved in the voltage-dependent activation of the Na+ channel. These results support the hypothesis that Shaker encodes a structural component of a voltage-dependent K+ channel and suggest a conserved mechanism for voltage activation.

A family of three mouse potassium channel genes with intronless coding regions.

To understand the molecular mechanisms responsible for generating physiologically diverse potassium channels in mammalian cells, mouse genomic clones have been isolated with a potassium channel complementary DNA, MBK1, that is homologous to the Drosophila potassium channel gene, Shaker. A family of three closely related potassium channel genes (MK1, MK2, and MK3) that are encoded at distinct genomic loci has been isolated. Sequence analysis reveals that the coding region of each of these three genes exists as a single uninterrupted exon in the mouse genome. This organization precludes the generation of multiple forms of the protein by alternative RNA splicing, a mechanism known to characterize the Drosophila potassium channel genes Shaker and Shab. Thus, mammals may use a different strategy for generating diverse K+ channels by encoding related genes at multiple distinct genomic loci, each of which produces only a single protein.

Repulsion between tetraethylammonium ions in cloned voltage-gated potassium channels.

Tetraethylammonium ion (TEA+) blocks voltage-gated K+ channels by acting at two sites located at opposite ends of the aqueous pore. This allowed us to test two predictions made by models of ion permeation, namely that K+ channels can be simultaneously occupied by multiple ions and that the ions repel each other. We show that externally applied TEA+ antagonize block by internal TEA+ and vice versa. The antagonism is less than predicted for competitive binding, hence TEA+ may occupy both sites simultaneously. External TEA+ and internal TEA+ reduce each others affinity 4- to 5-fold. In addition, K+ antagonizes block by TEA+ at the opposite side of the membrane, and external TEA+ antagonizes is block by internal Ba2+. The antagonism between ions applied at opposite sides of the membrane may be common to all cations binding to K+ channels.

Hypomyelination alters K+ channel expression in mouse mutants shiverer and Trembler.

Voltage-gated K+ channels are localized to juxtaparanodal regions of myelinated axons. To begin to understand the role of normal compact myelin in this localization, we examined mKv1.1 and mKv1.2 expression in the dysmyelinating mouse mutants shiverer and Trembler. In neonatal wild-type and shiverer mice, the focal localization of both proteins in axon fiber tracts is similar, suggesting that cues other than mature myelin can direct initial K+ channel localization in shiverer mutants. In contrast, K+ channel localization is altered in hypomyelinated axonal fiber tracts of adult mutants, suggesting that abnormal myelination leads to channel redistribution. In shiverer adult, K+ channel expression is up-regulated in both axons and glia, as revealed by immunocytochemistry, RNase protection, and in situ hybridization studies. This up-regulation of K+ channels in hypomyelinated axon tracts may reflect a compensatory reorganization of ionic currents, allowing impulse conduction to occur in these dysmyelinating mouse mutants.

Deletion of the K(V)1.1 potassium channel causes epilepsy in mice.

Mice lacking the voltage-gated potassium channel alpha subunit, K(V)1.1, display frequent spontaneous seizures throughout adult life. In hippocampal slices from homozygous K(V)1.1 null animals, intrinsic passive properties of CA3 pyramidal cells are normal. However, antidromic action potentials are recruited at lower thresholds in K(V)1.1 null slices. Furthermore, in a subset of slices, mossy fiber stimulation triggers synaptically mediated long-latency epileptiform burst discharges. These data indicate that loss of K(V)1.1 from its normal localization in axons and terminals of the CA3 region results in increased excitability in the CA3 recurrent axon collateral system, perhaps contributing to the limbic and tonic-clonic components of the observed epileptic phenotype. Axonal action potential conduction was altered as well in the sciatic nerve--a deficit potentially related to the pathophysiology of episodic ataxia/myokymia, a disease associated with missense mutations of the human K(V)1.1 gene.

The plasma membrane calcium ATPase and disease.

The plasma membrane calcium ATPase (PMCA) uses energy to pump calcium (Ca2+) ions out of the cytosol into the extracellular milieu, usually against a strong chemical gradient. This energy expenditure is necessary to maintain a relatively low intracellular net Ca2+ load. Mammals have four genes (ATP2B1-ATP2B4), encoding the proteins PMCA1 through PMCA4. Transcripts from each of these genes are alternatively spliced to generate several variant proteins that are in turn post-translationally modified in a variety of ways. Expressed ubiquitously and with some level of functional redundancy in most vital tissues, only one of the four genes--Atp2b2--has been causally linked through naturally occuring mutations to disease in mammals: specifically to deafness and ataxia in spontaneous mouse mutants. In humans, a missense amino acid substitution in PMCA2 modifies the severity of hearing loss. Targeted null mutations of the Atp2b1 and Atp2b4 genes in mouse are embryonic lethal and cause a sperm motility defect, respectively. These phenotypes point to complex human diseases like hearing loss, cardiac function and infertility. Changes in PMCA expression are associated with other diseases including cataract formation, carciniogenesis, diabetes, and cardiac hypertension and hypertrophy. Severity of these diseases may be affected by subtle changes in expression of the PMCA isoforms expressed in those tissues.

Atp2b2, encoding plasma membrane Ca2+-ATPase type 2, (PMCA2) exhibits tissue-specific first exon usage in hair cells, neurons, and mammary glands of mice.

Atp2b2 encodes the plasma membrane Ca(2+)-ATPase type 2 (PMCA2) expressed in various tissues, including stereocilia of cochlear and vestibular hair cells, cerebellar Purkinje cells, and lactating mammary epithelia. Mutations of the gene lead to deafness, ataxia, and reduced Ca(2+) levels in milk. Heterozygous mutants also have abnormal hearing, suggesting that precise regulation of Atp2b2 is required for normal function. In this study, we describe Atp2b2 5'-untranslated region genomic structure and transcript usage in mice. Using 5'-rapid amplification of cDNA ends, we observed four transcripts: types alpha, beta, mu and delta, each splicing into a common ATG-containing exon. Types alpha and beta correspond to previously published mammalian cDNA sequences. Types mu and delta constitute novel 5'-untranslated region sequences, and were observed at high levels only in lactating mammary gland. Using real-time reverse transcriptase polymerase chain reaction, we quantified relative transcript usage across several tissues. We show that alpha and beta are abundant throughout the CNS, as well as the cochlea. When we microdissected the cochlea into hair cell and spiral ganglion containing fractions, we found that cochlear hair cell expression is mediated through the type alpha transcript. In situ hybridization studies in cerebellum using exon-specific probes revealed that alpha dominates in Purkinje neurons, while beta is enriched in cerebellar granule neurons. We compared 5'-untranslated region sequence across multiple species, and found high conservation around the first exons for alpha and beta in mammals, but not other species. The regions around the mu and delta first exons are highly conserved between rat and mouse, but less so with other species. Our results show that expression of Atp2b2 is highly regulated, using four different transcriptional start regions, two of which are differentially expressed in neuronal tissue. This suggests that unique regulatory mechanisms are used to control Atp2b2 expression in different types of cells.

BOLD-fMRI of PTZ-induced seizures in rats.

PURPOSE: To develop a non-invasive method for exploring seizure initiation and propagation in the brain of intact experimental animals. METHODS: We have developed and applied a model-independent statistical method--Hierarchical Cluster Analysis (HCA)--for analyzing BOLD-fMRI data following administration of pentylenetetrazol (PTZ) to intact rats. HCA clusters voxels into groups that share similar time courses and magnitudes of signal change, without any assumptions about when and/or where the seizure begins. RESULTS: Epileptiform spiking activity was monitored by EEG (outside the magnet) following intravenous PTZ (IV-PTZ; n=4) or intraperitoneal PTZ administration (IP-PTZ; n=5). Onset of cortical spiking first occurred at 29+/-16 s (IV-PTZ) and 147+/-29 s (IP-PTZ) following drug delivery. HCA of fMRI data following IV-PTZ (n=4) demonstrated a single dominant cluster, involving the majority of the brain and first activating at 27+/-23s. In contrast, IP-PTZ produced multiple, relatively small, clusters with heterogeneous time courses that varied markedly across animals (n=5); activation of the first cluster (involving cortex) occurred at 130+/-59 s. With both routes of PTZ administration, the timing of the fMRI signal increase correlated with onset of EEG spiking. CONCLUSIONS: These experiments demonstrate that fMRI activity associated with seizure activity can be analyzed with a model-independent statistical method. HCA indicated that seizure initiation in the IV- and IP-PTZ models involves multiple regions of sensitivity that vary with route of drug administration and that show significant variability across animal subjects. Even given this heterogeneity, fMRI shows clear differences that are not apparent with typical EEG monitoring procedures, in the activation patterns between IV and IP-PTZ models. These results suggest that fMRI can be used to assess different models and patterns of seizure activation.

Immunological evidence for a relationship between the dendrotoxin-binding protein and the mammalian homologue of the Drosophila Shaker K+ channel.

Polyclonal antibodies were raised against two synthetic peptides from different parts of the predicted amino acid sequence of the mouse homologue (MBK1) of the Drosophila Shaker K+ channel. The antibodies recognized the toxin-binding subunit of the dendrotoxin-binding proteins from rat and bovine brain. The results suggest that the dendrotoxin-binding protein is related to the expression products of the mammalian homologue of the Shaker gene.

Prostaglandin E2 inhibits the potassium current in sensory neurons from hyperalgesic Kv1.1 knockout mice.

Prostaglandin E(2) (PGE(2)) enhances the sensitivity of sensory neurons to various forms of noxious stimulation. This occurs, in part, by the suppression of a delayed rectifier-like potassium current in these neurons. However, the molecular identity of this current remains unclear. Recent studies demonstrated that a mutant mouse lacking a delayed rectifier potassium channel gene, Kv1.1, displayed lowered thresholds to thermal stimulation in behavioral assays of pain perception, i.e. the Kcna1-null mice were hyperalgesic. Here we examined whether PGE(2) can alter the sensitivity of Kcna1-null mice to noxious stimulation and examine the capability of PGE(2) to inhibit the potassium current in these knockout mice. Behavioral assays were used to assess the effect of PGE(2) on either thermal hyperalgesia or mechanical sensitivities. In addition, the whole-cell patch-clamp technique was used to study the effects of PGE(2) on the total potassium current recorded from isolated mouse sensory neurons. Even with a reduced threshold to thermal stimulation, PGE(2) could still sensitize the response of Kcna1-null mice to thermal and mechanical stimulation by amounts that were similar to that in wild type mice. The activation properties of the potassium current were similar for both the wild type and the Kcna1-null mice, whereas the inactivation properties were different in cells exhibiting large amounts of steady-state inactivation (>50%) measured at +20 mV. PGE(2) suppressed the total potassium current in both groups of mice by 40-50% without altering the voltage dependence of activation. In addition, PGE(2) produced similar amounts of suppression in both groups of mice when currents were examined with the steady-state inactivation protocol. Based on these results, it is unlikely that Kv1.1 is the molecular identity of the potassium channel(s) modulated by PGE(2) to sensitize nociceptive sensory neurons. Also, the enhanced thermal sensitivity as observed in the Kcna1-null mice might be due to more central neurons of the pain sensing pathway.

Evidence of altered inhibition in layer V pyramidal neurons from neocortex of Kcna1-null mice.

Mice lacking the potassium channel subunit KCNA1 exhibit a severe epileptic phenotype beginning at an early postnatal age. The precise cellular physiological substrates for these seizures are unclear, as is the site of origin. Since KCNA1 mRNA in normal mice is expressed in the neocortex, we asked whether neurons in the neocortex of three to four week-old Kcna1-null mutants exhibit evidence of hyperexcitability. Layer V pyramidal neurons were directly visualized in brain slices with infrared differential-interference contrast microscopy and evaluated with cellular electrophysiological techniques. There were no significant differences in intrinsic membrane properties and action potential shape between Kcna1-null and wild-type mice, consistent with previous findings in hippocampal slice recordings. However, the frequency of spontaneous post-synaptic currents was significantly higher in Kcna1-null compared to wild-type mice. The frequency of spontaneous inhibitory post-synaptic currents and miniature (action-potential-independent) inhibitory post-synaptic currents was also significantly higher in Kcna1-null compared to wild-type mice. However, the frequency of spontaneous and miniature excitatory post-synaptic currents was not different in these two groups of animals. Comparison of the amplitude and kinetics of miniature inhibitory and excitatory post-synaptic currents revealed differences in amplitude, rise time and half-width between Kcna1-null and wild-type mice. Our data indicate that the inhibitory drive onto layer V pyramidal neurons is increased in Kcna1 knockout mice, either directly through an increased spontaneous release of GABA from presynaptic terminals contacting layer V pyramidal neurons, or an enhanced excitatory synaptic input to inhibitory interneurons.

Hyperalgesia in mice lacking the Kv1.1 potassium channel gene.

Hyperalgesia and morphine induced antinociception were measured in mice lacking the gene for the Shaker-like voltage-gated potassium channel Kv1.1 alpha subunit. The effects of varying gene dosage were studied by comparing homozygous null (-/-) versus heterozygous (+/-) and wildtype (+/+) littermates. Hyperalgesia was measured using the paw flick assay, hot plate assay and formalin induced hind paw licking. It was observed that null mutant animals had significantly shorter latencies to response in the paw flick (36%) and hot plate (27%) assays while their licking times after hind paw injection of formalin was increased in both the first (74%) and second (65%) phases of the response compared to wildtype controls. Morphine induced antinociception in Kv1.1 null mutant animals was blunted. These studies indicate that Kv1.1 plays an important role in nociceptive and antinociceptive signaling pathways.

Differential expression of voltage-gated potassium channel genes in auditory nuclei of the mouse brainstem.

Voltage-gated potassium (Kv) channels may play an important role in the encoding of auditory information. Towards understanding the roles of Shaker and Shaw-like channels in this process, we examine here the expression of Kv1.1, Kv1.2, Kv3.1, and Kv3.3 in the central auditory nuclei of the mouse using quantitative in situ hybridization techniques. We establish rank order for each channel's expression in each region, finding that the medial nucleus of the trapezoid body shows the highest signal for each of the four channel genes. In other auditory nuclei differential expression is found among and between members of both Shaker and Shaw subfamilies. Of particular interest is the stark contrast between high level expression of Kv1.1 and very low level expression of Kv3.1 in the octopus cell area of the cochlear nucleus and in the lateral superior olivary nucleus. These unique expression patterns suggest that Kv channel gene expression is regulated to allow brainstem auditory neurons to transmit temporally patterned signals with high fidelity. In instances where specific cell types can be tentatively identified, we discuss the possible contribution made by these channel genes to the physiological properties of those neurons.

Effects of PMCA2 mutation on DPOAE amplitudes and latencies in deafwaddler mice.

The deafwaddler (dfw) mouse mutant is caused by a spontaneous mutation in the gene that encodes a plasma membrane Ca(2+) ATPase (type 2), PMCA2 (Street et al., 1998. Nat. Genet. 19, 390-394), which is expressed in cochlear and vestibular hair cells. Distortion product otoacoustic emission (DPOAE) amplitudes and latencies were examined in control mice, deafwaddler mutants, and controls treated with the drug furosemide. Furosemide causes a transient reduction of DPOAEs (Mills et al., 1993. J. Acoust. Soc. Am. 94, 2108-2122). We wanted to determine whether DPOAEs obtained in furosemide-treated mice were similar or different from results obtained in +/dfw mice. DPOAE amplitude and phase were measured as a function of f(2)/f(1) ratio. These data were converted into waveforms using inverse fast Fourier transform, and their average latency was used to estimate DPOAE group delay. Homozygous deafwaddlers did not produce DPOAEs. Heterozygous deafwaddlers (+/dfw) had increased DPOAE thresholds and reduced amplitudes at high frequencies, compared to controls. To the extent that DPOAEs depend on functional outer hair cells (OHCs), abnormal DPOAEs in +/dfw mice suggest that PMCA2 is important for OHC function at high frequencies. Similar to the effects of furosemide, the mutation reduced DPOAEs for low-level stimuli; in contrast to furosemide, the mutation altered DPOAEs elicited by high levels.

Voltage-gated K+ channels of the mammalian brain.

Research on voltage-gated K+ channels of the mammalian brain has seen a flood of new data in the last 2 years. A genetic approach, based on the Shaker mutation of Drosophila, led to cDNA clones for mammalian voltage-gated K+ channels. K+ channel proteins were detected independently and purified with the help of channel specific toxins. From these studies the structure of two families of mammalian K+ channels emerged. One family is defined molecularly by the sequence homology of its members, the other by binding sites for the snake toxin dendrotoxin. The two families have several members in common. The voltage-gated K+ channels of mammalian brain are oligomers of glycosilated peptides of 65-95 kDa. The primary structure of these subunits is characterized by six to eight potential transmembrane regions, including the S4 region, the voltage-sensor of the channels. Associated with at least some K+ channels are 38- and 42-kDa peptides of unknown function. The channels give rise to non- or slow-inactivating K+ currents that are regulated through phosphorylation by both cAMP-dependent and an endogenous kinase.

Physical mapping of potassium channel gene clusters on mouse chromosomes three and six.

Mammalian voltage-gated K channel genes have been divided into four subfamilies (Shaker, Shab, Shal, and Shaw) based on their sequence identity and similarity to related genes in Drosophila. Genetic mapping of the voltage-gated K channel genes has shown that similar multigene clusters exist on mouse Chr 3 and 6 and suggests that the clusters may have arisen through chromosomal duplication. In this report, YAC-based physical maps of the clustered mouse Shaker-like K channel genes have been constructed using restriction endonuclease and yeast chromosome fragmentation approaches. These data define the physical spacing as 5'-Kcna3-(60 kb)-Kcna2-(90 kb)-Kcna8-3' on Chr 3, and as 5'-Kcna6-(80 kb)-Kcna1-(110 kb)-Kcna5-3' on Chr 6, with all genes oriented in a head-to-tail manner within their respective clusters. These detailed physical maps of both K channel gene clusters provide additional support for the idea of an ancient genome tetraploidization event.