Congenital stationary night blindness in mice - a tale of two Cacna1f mutants.

BACKGROUND: Mutations in CACNA1F, which encodes the Ca(v)1.4 subunit of a voltage-gated L-type calcium channel, cause X-linked incomplete congenital stationary night blindness (CSNB2), a condition of defective retinal neurotransmission which results in night blindness, reduced visual acuity, and diminished ERG b-wave. We have characterized two putative murine CSNB2 models: an engineered null-mutant, with a stop codon (G305X); and a spontaneous mutant with an ETn insertion in intron 2 of Cacna1f (nob2). METHODS: Cacna1f ( G305X ): Adults were characterized by visual function (photopic optokinetic response, OKR); gene expression (microarray) and by cell death (TUNEL) and synaptic development (TEM). Cacna1f ( nob2 ): Adults were characterized by properties of Cacna1f mRNA (cloning and sequencing) and expressed protein (immunoblotting, electrophysiology, filamin [cytoskeletal protein] binding), and OKR. RESULTS: The null mutation in Cacna1f ( G305X ) mice caused loss of cone cell ribbons, failure of OPL synaptogenesis, ERG b-wave and absence of OKR. In Cacna1f ( nob2 ) mice alternative ETn splicing produced ~90% Cacna1f mRNA having a stop codon, but ~10% mRNA encoding a complete polypeptide. Cacna1f ( nob2 ) mice had normal OKR, and alternatively-spliced complete protein had WT channel properties, but alternative ETn splicing abolished N-terminal protein binding to filamin. CONCLUSIONS: Ca(v)1.4 plays a key role in photoreceptor synaptogenesis and synaptic function in mouse retina. Cacna1f ( G305X ) is a true knockout model for human CSNB2, with prominent defects in cone and rod function. Cacna1f ( nob2 ) is an incomplete knockout model for CSNB2, because alternative splicing in an ETn element leads to some full-length Ca(v)1.4 protein, and some cones surviving to drive photopic visual responses.

Temperature dependence of Cav1.4 calcium channel gating.

The CACNA1F gene encodes the pore-forming subunit of the L-type Cav1.4 voltage-gated calcium channel (VGCC) and plays a central role in tonic vesicular release at photoreceptor ribbon synapses. The main objective of this study was to examine the effects of temperature on human Cav1.4 cDNA clone VGCCs. With 20 mM Ba2+ as charge carrier, increasing the temperature from 23 degrees C to 37 degrees C increases whole-cell conductance, shifts the voltage-dependence of activation to more hyperpolarized voltages, and accelerates the degree of recovery from inactivation over a given time, but does not significantly alter the half-inactivation potential (Vh). The window current for Cav1.4 was also shifted to more hyperpolarized voltages, observable from approximately -35 mV to +20 mV at 37 degrees C in 20 mM Ba2+. Several comparable results were observed when characterizing Cav1.2 at temperatures ranging from 23 degrees C to 37 degrees C. However, one difference between Cav1.4 and Cav1.2 was the temperature dependence of voltage-dependent inactivation kinetics. Increasing temperature from 23 degrees C to 37 degrees C accelerates Cav1.4 inactivation kinetics approximately 50-fold, whereas Cav1.2 only accelerates approximately 10-fold over the same temperature range. The time constant of inactivation (tauh) temperature coefficient (Q10) was 18.8 for Cav1.4 over a temperature range of 23 degrees to 33 degrees C (corresponding to an activation energy Ea=221 kJ/mol), compared with Cav1.2 with a Q10 of 3 (Ea=90 kJ/mol) recorded under identical conditions. In addition, Cav1.4 was also tested using 2 mM Ca2+ as a charge carrier and similar changes in current-voltage Boltzmann parameters and gating kinetics were observed. Hence, despite the accelerated inactivation kinetics of Cav1.4 channels observed at near physiological temperatures the window current is preserved and could allow for tonic glutamate release from photoreceptors in the retina during dark adapted conditions.

Functional analysis of congenital stationary night blindness type-2 CACNA1F mutations F742C, G1007R, and R1049W.

Congenital stationary night blindess-2 (incomplete congenital stationary night blindness (iCSNB) or CSNB-2) is a nonprogressive, X-linked retinal disease which can lead to clinical symptoms such as myopia, hyperopia, nystagmus, strabismus, decreased visual acuity, and impaired scotopic vision. These clinical manifestations are linked to mutations found in the CACNA1F gene which encodes for the Ca(v)1.4 voltage-gated calcium channel. To better understand the physiological effects of these mutations, three missense mutants, F742C, G1007R and R1049W, previously shown to be mutated in patients with CSNB-2, were transiently expressed in human embryonic kidney (HEK) tsA-201 cells and characterized using whole-cell patch clamp. The G1007R mutation is located in transmembrane segment 5 (S5) of domain III and R1049W is located in the extracellular linker between S5 and the P-loop of domain III. Both mutants produced full length proteins that targeted to the membrane but did not support ionic currents. In 20 mM Ba(2+), F742C (S6 domain II) produced a approximately 21 mV hyperpolarizing shift in half activation potential (V(a[1/2])) and a approximately 23 mV hyperpolarizing shift in half inactivation potential (V(h[1/2])). Additionally, F742C displayed slower inactivation kinetics and a smaller whole cell conductance (G(max)). In physiological 2 mM Ca(2+), F742C produced a approximately 19 mV hyperpolarizing shift in V(a[1/2]). These findings suggest that the pathology of CSNB-2 in patients with these missense mutations in the Ca(v)1.4 calcium channel is the result in either a gain of function (F742C) or a loss of function (G1007R, R1049W).

Effects of extracellular pH on neuronal calcium channel activation.

Previous studies have shown that extracellular pH (pHo) alters gating and permeation properties of cardiac L- and T-type channels. However, a comprehensive study investigating the effects of pHo on all other voltage-gated calcium channels is lacking. Here, we report the effects of pHo on activation parameters slope factor (S), half-activation potential (Va), reversal potential (Erev), and maximum slope conductance (Gmax) of the nine known neuronal voltage-gated calcium channels transiently expressed in tsA-201 cells. In all cases, acidification of the extracellular bathing solution results in a depolarizing shift in the activation curve and reduction in peak current amplitudes. Relative to a physiological pHo of 7.25, statistically significant depolarizing shifts in Va were observed for all channels at pHo 7.00 except Cav1.3 and 3.2, which showed significant shifts at pHo 6.75 and below. All channels displayed significant reductions in Gmax relative to pHo 7.25 at pHo 7.00 except Cav1.2, 2.1, and 3.1 which required acidification to pHo 6.75. Upon acidification Cav3 channels displayed the largest changes in Vas and exhibited the largest reduction in Gmax compared with other channel subtypes. Taken together, these results suggest that significant modulation of calcium channel currents can occur with changes in pHo. Acidification of the external solution did not produce significant shifts in observed Erevs or blockade of outward currents for any of the nine channel subtypes. Finally, we tested a simple Woodhull-type model of current block by assuming blockade of the pore by a single proton. In all cases, the amount of blockade observed could not be explained in these simple terms, suggesting that proton modulation is more complicated, involving more than one site or gating modification as has been previously described for cardiac L- and T-type channels.

Temperature dependence of T-type calcium channel gating.

T-type calcium channel isoforms are expressed in a multitude of tissues and have a key role in a variety of physiological processes. To fully appreciate the physiological role of distinct channel isoforms it is essential to determine their kinetic properties under physiologically relevant conditions. We therefore characterized the gating behavior of expressed rat voltage-dependent calcium channels (Ca(v)) 3.1, Ca(v)3.2, and Ca(v)3.3, as well as human Ca(v)3.3 at 21 degrees C and 37 degrees C in saline that approximates physiological conditions. Exposure to 37 degrees C caused significant increases in the rates of activation, inactivation, and recovery from inactivation, increased the current amplitudes, and induced a hyperpolarizing shift of half-activation for Ca(v)3.1 and Ca(v)3.2. At 37 degrees C the half-inactivation showed a hyperpolarizing shift for Ca(v)3.1 and Ca(v)3.2 and human Ca(v)3.3, but not rat Ca(v)3.3. The observed changes in the kinetics were significant but not identical for the three isoforms, showing that the ability of T-type channels to conduct calcium varies with both channel isoform and temperature.

The Drosophila cacts2 mutation reduces presynaptic Ca2+ entry and defines an important element in Cav2.1 channel inactivation.

Voltage-gated Ca2+ channels in nerve terminals open in response to action potentials and admit Ca2+, the trigger for neurotransmitter release. The cacophony gene encodes the primary presynaptic voltage-gated Ca2+ channel in Drosophila motor-nerve terminals. The cac(ts2) mutant allele of cacophony is associated with paralysis and reduced neurotransmission at non-permissive temperatures but the basis for the neurotransmission deficit has not been established. The cac(ts2) mutation occurs in the cytoplasmic carboxyl tail of the alpha1-subunit, not within the pore-forming trans-membrane domains, making it difficult to predict the mutation's impact. We applied a Ca2+-imaging technique at motor-nerve terminals of mutant larvae to test the hypothesis that the neurotransmission deficit is a result of impaired Ca2+ entry. Presynaptic Ca2+ signals evoked by single and multiple action potentials showed a temperature-dependent reduction. The amplitude of the reduction was sufficient to account for the neurotransmission deficit, indicating that the site of the cac(ts2) mutation plays a role in Ca2+ channel activity. As the mutation occurs in a motif conserved in mammalian high-voltage-activated Ca2+ channels, we used a heterologous expression system to probe the effect of this mutation on channel function. The mutation was introduced into rat Ca(v)2.1 channels expressed in human embryonic kidney cells. Patch-clamp analysis of mutant channels at the physiological temperature of 37 degrees C showed much faster inactivation rates than for wild-type channels, demonstrating that the integrity of this motif is critical for normal Ca(v)2.1 channel inactivation.

NMDA receptors mediate calcium accumulation in myelin during chemical ischaemia.

Central nervous system myelin is a specialized structure produced by oligodendrocytes that ensheaths axons, allowing rapid and efficient saltatory conduction of action potentials. Many disorders promote damage to and eventual loss of the myelin sheath, which often results in significant neurological morbidity. However, little is known about the fundamental mechanisms that initiate myelin damage, with the assumption being that its fate follows that of the parent oligodendrocyte. Here we show that NMDA (N-methyl-d-aspartate) glutamate receptors mediate Ca2+ accumulation in central myelin in response to chemical ischaemia in vitro. Using two-photon microscopy, we imaged fluorescence of the Ca2+ indicator X-rhod-1 loaded into oligodendrocytes and the cytoplasmic compartment of the myelin sheath in adult rat optic nerves. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor antagonist NBQX completely blocked the ischaemic Ca2+ increase in oligodendroglial cell bodies, but only modestly reduced the Ca2+ increase in myelin. In contrast, the Ca2+ increase in myelin was abolished by broad-spectrum NMDA receptor antagonists (MK-801, 7-chlorokynurenic acid, d-AP5), but not by more selective blockers of NR2A and NR2B subunit-containing receptors (NVP-AAM077 and ifenprodil). In vitro ischaemia causes ultrastructural damage to both axon cylinders and myelin. NMDA receptor antagonism greatly reduced the damage to myelin. NR1, NR2 and NR3 subunits were detected in myelin by immunohistochemistry and immunoprecipitation, indicating that all necessary subunits are present for the formation of functional NMDA receptors. Our data show that the mature myelin sheath can respond independently to injurious stimuli. Given that axons are known to release glutamate, our finding that the Ca2+ increase was mediated in large part by activation of myelinic NMDA receptors suggests a new mechanism of axo-myelinic signalling. Such a mechanism may represent a potentially important therapeutic target in disorders in which demyelination is a prominent feature, such as multiple sclerosis, neurotrauma, infections (for example, HIV encephalomyelopathy) and aspects of ischaemic brain injury.

Mutation analysis of the sodium/hydrogen exchanger gene (NHE5) in familial paroxysmal kinesigenic dyskinesia.

Familial Paroxysmal Kinesigenic Dyskinesia (PKD) is an autosomal dominant condition characterized by attacks of dystonia or chorea triggered by sudden movements. Recently two separate loci for PKD, Episodic Kinesigenic Dyskinesia 1 (EKD1) and Episodic Kinesigenic Dyskinesia 2 (EKD2), have been mapped to chromosome 16 but the causative genes have not been identified. The Na(+)/H(+) exchanger gene (NHE5) involved in regulating intracellular pH lies in the EKD2 region. The coding region of the NHE5 gene in familial PKD was sequenced. We did not identify any mutations in the exons, intron/exon boundaries or the 5' and 3'UTR. This excludes mutations in the coding region of the NHE5 gene as a cause for familial PKD, but does not rule out a possible role of sequence variants in introns or regulatory regions.

Molecular and functional characterization of a family of rat brain T-type calcium channels.

Voltage-gated calcium channels represent a heterogenous family of calcium-selective channels that can be distinguished by their molecular, electrophysiological, and pharmacological characteristics. We report here the molecular cloning and functional expression of three members of the low voltage-activated calcium channel family from rat brain (alpha(1G), alpha(1H), and alpha(1I)). Northern blot and reverse transcriptase-polymerase chain reaction analyses show alpha(1G), alpha(1H), and alpha(1I) to be expressed throughout the newborn and juvenile rat brain. In contrast, while alpha(1G) and alpha(1H) mRNA are expressed in all regions in adult rat brain, alpha(1I) mRNA expression is restricted to the striatum. Expression of alpha(1G), alpha(1H), and alpha(1I) subunits in HEK293 cells resulted in calcium currents with typical T-type channel characteristics: low voltage activation, negative steady-state inactivation, strongly voltage-dependent activation and inactivation, and slow deactivation. In addition, the direct electrophysiological comparison of alpha(1G), alpha(1H), and alpha(1I) under identical recording conditions also identified unique characteristics including activation and inactivation kinetics and permeability to divalent cations. Simulation of alpha(1G), alpha(1H), and alpha(1I) T-type channels in a thalamic neuron model cell produced unique firing patterns (burst versus tonic) typical of different brain nuclei and suggests that the three channel types make distinct contributions to neuronal physiology.

The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily.

The pituitary adenylate cyclase-activating polypeptide (PACAP)/ glucagon superfamily includes nine hormones in humans that are related by structure, distribution (especially the brain and gut), function (often by activation of cAMP), and receptors (a subset of seven-transmembrane receptors). The nine hormones include glucagon, glucagon-like peptide-1 (GLP-1), GLP-2, glucose-dependent insulinotropic polypeptide (GIP), GH-releasing hormone (GRF), peptide histidine-methionine (PHM), PACAP, secretin, and vasoactive intestinal polypeptide (VIP). The origin of the ancestral superfamily members is at least as old as the invertebrates; the most ancient and tightly conserved members are PACAP and glucagon. Evidence to date suggests the superfamily began with a gene or exon duplication and then continued to diverge with some gene duplications in vertebrates. The function of PACAP is considered in detail because it is newly (1989) discovered; it is tightly conserved (96% over 700 million years); and it is probably the ancestral molecule. The diverse functions of PACAP include regulation of proliferation, differentiation, and apoptosis in some cell populations. In addition, PACAP regulates metabolism and the cardiovascular, endocrine, and immune systems, although the physiological event(s) that coordinates PACAP responses remains to be identified.

P/Q-type calcium channels mediate the activity-dependent feedback of syntaxin-1A.

Spatial and temporal changes in intracellular calcium concentrations are critical for controlling gene expression in neurons. In many neurons, activity-dependent calcium influx through L-type channels stimulates transcription that depends on the transcription factor CREB by activating a calmodulin-dependent pathway. Here we show that selective influx of calcium through P/Q-type channels is responsible for activating expression of syntaxin-1A, a presynaptic protein that mediates vesicle docking, fusion and neurotransmitter release. The initial P/Q-type calcium signal is amplified by release of calcium from intracellular stores and acts through phosphorylation that is dependent on the calmodulin-dependent kinase CaM K II/IV, protein kinase A and mitogen-activated protein kinase kinase. Initiation of syntaxin-1A expression is rapid and short-lived, with syntaxin-1A ultimately interacting with the P/Q-type calcium channel to decrease channel availability. Our results define an activity-dependent feedback pathway that may regulate synaptic efficacy and function in the nervous system.

Ancient divergence of insulin and insulin-like growth factor.

Studies on the evolutionary pathway of the insulin gene family suggest that insulin and insulin-like growth factor (IGF) became distinct molecules only after the vertebrates arose. A single molecule with identity to both insulin and IGF was reported in amphioxus. To study the origin of insulin, we selected tunicates because their ancestors are thought to be a nodal point in the evolution of vertebrates. This is the first report of separate insulin and IGF molecules from invertebrates. Two cDNAs were isolated from the tunicate Chelyosoma productum: One cDNA encodes a distinct preproinsulin with B, C, and A domains, whereas the other encodes tunicate preproIGF, including all five domains in their proper sequence. Both mRNAs are expressed in the nervous system, digestive tract, heart, and possibly the gonad but not in branchial basket or tunic. Hence, insulin and igf genes have similar expression patterns. In situ methods confirm the polymerase chain reaction evidence that tunicate insulin and igf mRNAs are expressed in cortical cells of the neural ganglion. We conclude that insulin and IGF have maintained separate gene lineages in both vertebrate and protochordate evolution and, thus, a distinct evolutionary history of more than 600 million years.

Expression and alternative processing of a chicken gene encoding both growth hormone-releasing hormone and pituitary adenylate cyclase-activating polypeptide.

The chicken growth hormone-releasing hormone (GRF) gene was isolated, sequenced, and characterized. In addition, three different mRNAs were isolated from juvenile and adult brain. The first cDNA encoded for a GRF(1-46), the second cDNA encoded for a GRF(1-43) due to a sliding intron boundary, and the third skipped exon four and encoded only GRF(33-46). We also determined that juvenile chicken mRNA encoding GRF is expressed in the brain and gonads, but not in the pituitary, heart, liver, kidney, crop, small intestine, large intestine, eye, and muscle. This gene is also interesting in terms of evolution because another neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), is encoded within the same gene (grf/pacap) in chicken, but on a separate gene (pacap) in mammals. We showed previously that these two neuropeptides were encoded in the same cDNA in fish, but the present evidence in chicken suggests a gene duplication in stem mammals.

Sequence and expression of cDNA for pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH)-like peptide in catfish.

Growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase activating polypeptide (PACAP) are two neuropeptides that are associated with the release of pituitary growth hormone. Here a cDNA of 2501 base pairs encoding both a PACAP and a GHRH-like peptide was isolated from a brain cDNA library made from Thai catfish (Clarias macrocephalus). The organization is unlike that of the mammalian gene where PACAP and PACAP-related peptide (PRP) are encoded in one gene, and the GHRH peptide is on a separate gene. Northern analysis of catfish brain mRNA indicated that PACAP/GHRH-like mRNA has three sizes; bands of 6000, 2500, and 1000 bases suggest alternative splicing of the gene. Reverse transcriptase/PCR assay detected PACAP/GHRH-like mRNA in tissues from the brain, testis, ovary, and stomach, but not from the pancreas, pituitary, muscle, and liver. Our hypothesis that the two mammalian genes encoding GHRH or PACAP originated from a gene duplication between fish and tetrapods is supported by the present findings of similar mRNA organization and pattern of expression for the one fish gene and two mammalian genes.

Catfish express two forms of insulin-like growth factor-I (IGF-I) in the brain. Ubiquitous IGF-I and brain-specific IGF-I.

Insulin-like growth factor-I (IGF-I) is expressed not only in liver, but also in brain and other tissues. This ubiquitous IGF-I has a complex pattern of expression due to multiple transcription start sites, polyadenylation sites and exon skipping. We have isolated a cDNA encoding a brain-specific IGF-I from a catfish brain cDNA library. Also, a fragment encoding ubiquitous IGF-I was amplified from brain and liver mRNA and the deduced protein shown to be distinct (66% sequence identity) from brain-specific IGF-I. Consistent with other IGF-I prepropeptides, the brain-specific IGF-I has a 43-residue signal peptide followed by B, C, A, D, and E domains. Retained in the catfish brain-specific IGF-I peptide are residues predicted to be involved with the correct tertiary folding, disulfide linkages, and receptor binding. Northern blot analysis of poly(A+)-rich mRNA from brain indicated a single 1600-base pair transcript; a band was not detected from mRNA of liver, stomach, pancreas, pituitary, blood, herring brain, or brain poly(A-) RNA. A sensitive reverse transcriptase/polymerase chain reaction assay also showed that brain-specific IGF-I mRNA was expressed solely in the Thai catfish brain but not liver, stomach, pancreas, pituitary, ovary, and African catfish brain.

Primary structure of neuropeptide Y from brains of the American alligator (Alligator mississippiensis).

The purification of NPY from brains of the American alligator (Alligator mississippiensis) was achieved using reverse-phase high performance liquid chromatography (HPLC). The amino acid sequence was determined using automated Edman degradation as Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu- Asp-Ala-Pro-Ala-Glu-Asp-Met-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile- Asn-Leu - Ile-Thr-Arg-Gln-Arg-Tyr. Alligator NPY is the first non-mammalian vertebrate to have 100% sequence identity to human NPY. The conservation of alligator NPY suggests that serine in position 7 of chicken NPY evolved after the birds and reptiles diverged from a common Archosaurian ancestor. Furthermore, the sequence identity between alligator and human NPY suggests this sequence is the same as the ancestral amniote NPY.

Primary structure of two forms of gonadotropin-releasing hormone from brains of the American alligator (Alligator mississippiensis).

Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brains of the American alligator, Alligator mississippiensis, using reverse-phase high-pressure liquid chromatography (HPLC). The concentration of total GnRH was 8.8 ng/g of frozen brain tissue or 21.1 ng per brain. The amino acid sequence of each form of GnRH was determined using automated Edman degradation. The presence of the N-terminal pGlu residue was established by digestion studies with bovine pyroglutamyl aminopeptidase and coelution with synthetic forms of the native peptide. The primary structure of alligator GnRH I is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2 and alligator GnRH II is pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2.