Properties Of Calcium Channels Research Articles

Alteration in calcium channel properties is responsible for the neurotoxic action of a familial frontotemporal dementia tau mutation.

Tau, a microtubule binding protein, is not only a major component of neurofibrillary tangles in Alzheimer's disease, but also a causative gene for hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We show here that an FTDP-17 tau mutation (V337M) in SH-SY5Y cells reduces microtubule polymerization, increases voltage-dependent calcium current (ICa) density, and decreases ICa rundown. The reduced rundown of ICa by V337M was significantly inhibited by nifedipine (L-type Ca channel blocker), whereas omega-conotoxin GVIA (N-type Ca channel blocker) showed smaller effects, indicating that tau mutations affect L-type calcium channel activity. The depolarization-induced increase in intracellular calcium was also significantly augmented by the V337M tau mutation. Treatment with a microtubule polymerizing agent (taxol), an adenylyl cyclase inhibitor, or a protein kinase A (PKA) inhibitor, counteracted the effects of mutant tau on ICa. Taxol also attenuated the Ca2+ response to depolarization in cells expressing mutant tau. Apoptosis in SH-SY5Y cells induced by serum deprivation was exacerbated by the V337M mutation, and nifedipine, taxol, and a PKA inhibitor significantly protected cells against apoptosis. Our results indicate that a tau mutation which decreases its microtubule-binding ability augments calcium influx by depolymerizing microtubules and activating adenylyl cyclase and PKA.

Ciliary function of polycystins: a new model for cystogenesis.

Autosomal dominant polycystic kidney disease results from loss-of-function mutations in either polycystin-1 (Pc-1) or polycystin-2 (Pc-2). These transmembrane proteins directly interact through cytosolic domains of their C-termini. Pc-1 has been implicated in cell– extracellular matrix (ECM) interactions at focal adhesion contacts, but also in cell–cell interactions at tight junctions, adherens junctions and desmosomes [1]. Pc-2, located to the plasma membrane and/or to the endoplasmic reticulum [2,3], was shown to increase the intracytosolic calcium from both ER stocks and extracellular milieu [4]. The variability of polycystin localization within the cell may be explained by differences in antibody characteristics, cell type, degree of confluence, developmental stage, but also raises the hypothesis of multiple biological functions [1]. The large extracellular domain of Pc-1 is thought to transduce signals from the environment into the cells, through Pc-1-dependent signalling pathways and through activation of PC-2 calcium channel properties. However, the search for polycystin ligands and functions at sites of cell–cell or cell–ECM interactions has remained elusive to date. In fact, the first recently reported biological function of polycystins depends on an additional and unexpected polycystin subcellular localization—the renal primary cilia—and is triggered by an unexpected ligand, the urinary flow.

Mechanisms of Lateral Inhibition in the Olfactory Bulb: Efficiency and Modulation of Spike-Evoked Calcium Influx into Granule Cells

Granule cells are axonless local interneurons that mediate lateral inhibitory interactions between the principal neurons of the olfactory bulb via dendrodendritic reciprocal synapses. This unusual arrangement may give rise to functional properties different from conventional lateral inhibition. Although granule cells spike, little is known about the role of the action potential with respect to their synaptic output. To investigate the signals that underlie dendritic release in these cells, two-photon microscopy in rat brain slices was used to image calcium transients in granule cell dendrites and spines. Action potentials evoked calcium transients throughout the dendrites, with amplitudes increasing with distance from soma and attaining a plateau level within the external plexiform layer, the zone of granule cell synaptic output. Transient amplitudes were, on average, equal in size in spines and adjacent dendrites. Surprisingly, both spine and dendritic amplitudes were strongly dependent on membrane potential, decreasing with depolarization and increasing with hyperpolarization from rest. Both the current-voltage relationship and the time course of inactivation were consistent with the known properties of T-type calcium channels, and the voltage dependence was blocked by application of the T-type calcium channel antagonists Ni2+ and mibefradil. In addition, mibefradil reduced action potential-mediated synaptic transmission from granule to mitral cells. The implication of a transiently inactivating calcium channel in synaptic release from granule cells suggests novel mechanisms for the regulation of lateral inhibition in the olfactory bulb.

AII Amacrine Cells Express L-Type Calcium Channels at Their Output Synapses

AII amacrine cells play a critical role in the high-fidelity signal transmission pathways involved with nighttime vision. The temporal properties of the light responses strongly depend on the transfer function at different synaptic stages and consequently on presynaptic calcium influx. AII light responses are complex waveforms generated by graded input, they comprise Na+-based spikes as well as a sustained component, and they are transferred to graded cone bipolar cells. It is, therefore, of interest to determine the properties of AII voltage-dependent calcium channels (VDCCs) to establish whether these cells express N-type and/or P/Q-type VDCCs, characteristic of spiking neurons, or whether they are more like graded neurons, which mostly use L-type VDCCs. We combined electrophysiological, molecular biological, and imaging techniques to characterize calcium currents and their sites of origin in mouse AII amacrine cells. Calcium currents activated at potentials more positive than -60 mV (maximally between -50 and -20 mV) and inactivated slowly. These currents were blocked by dihydropyridine (DHP) antagonists and were enhanced by the DHP agonist BayK 8644. Single-cell RT-PCR analysis of mRNA encoding for different calcium channel alpha subunits in AIIs revealed a consistent expression of the alpha1-D subunit. Calcium imaging of AII cells showed that the greatest change in intracellular calcium occurred in the lobular appendages, with minor changes being observed in the arboreal dendrites. Depolarization-induced calcium rises were also modulated by DHPs, suggesting that a particular kind of L-type VDCC, mainly localized to the lobular appendages, enables these spiking-capable neurons to release neurotransmitter in a sustained manner onto OFF-cone bipolar cells.

Influence of the Anx7 (+/-) knockout mutation and fasting stress on the genomics of the mouse adrenal gland.

The Anx7 gene codes for a Ca(2+)/GTPase with calcium channel and membrane fusion properties that has been proposed to regulate exocytotic secretion in chromaffin and other cell types. We have previously reported that the homozygous Anx7 (+/-) knockout mouse has an embryonically lethal phenotype. However, the viable heterozygous Anx7 (+/-) mouse displays a complex phenotype that includes adrenal gland hypertrophy, chromaffin cell hyperplasia, and defective IP(3) receptor (IP(3)R) expression. To search for a molecular basis for this phenotype, we have used cDNA microarray technology and have challenged control and mutant mice with fed or fasting conditions. We report that in the absence of the Anx7/IP(3)R signaling system, the cells in the adrenal gland are unable to discriminate between the fed and fasted states, in vivo. In control chromaffin cells, fasting is accompanied by an increased expression of structural genes for chromaffin cell contents, including chromogranin A and B, and DbetaH. There are also genes whose expression is specifically reduced. However, the Anx7 (+/-) mutation results in sustained expression of these nutritionally sensitive genes. We hypothesize that the calcium signaling defect due to the missing IP(3)R may be responsible for the global effects of the mutation on nutritionally sensitive genes. We further hypothesize that the tonically elevated expression of chromogranin A, a reportedly master control "switch" for dense core granule formation, may contribute to the process driving glandular hypertrophy and chromaffin cell hyperplasia in the Anx7 (+/-) mutant mouse.

The α 1A subunits of rat brain calcium channels are developmentally regulated by alternative RNA splicing

Calcium influx through voltage-gated calcium channels governs important aspects of CNS development. Multiple alternative splicings of the pore-forming α 1 subunits have been evidenced in adult brain but little information about their expression during ontogenesis is presently available. The aim of this study was to focus on the expression of three rat voltage-gated calcium channel α 1A splice variants (α 1A-a, α 1A-b and α 1A-EFe) during brain ontogenesis in vivo. Using a reverse transcription-polymerase chain reaction strategy, we found that the three isoforms have different timings of development throughout the brain: α 1A-b is expressed from embryonic to the adult stage, α 1A-EFe is restricted to the embryonic period whereas α 1A-a is expressed only postnatally. In situ hybridization indicated that α 1A-a and α 1A-b isoforms develop with different regional and cellular patterns. In hippocampus and cerebellum, α 1A-b represented the predominant isoform at all developmental stages. Taken together, these data reveal that alternative RNA splicing may modulate the α 1A calcium channel properties during development.

Changes in cardiac myocyte morphology alter the properties of voltage-gated ion channels.

The goal of this study was to determine if the properties of the transient outward potassium (I(to)), TTX-resistant sodium (I(Na)) and L-type calcium (I(Ca)) currents are altered during changes in cardiac cell shape. Ventricular myocytes were isolated from 3- to 4-day-old neonatal rats and cultured on either non-aligned or aligned collagen thin gels. In contrast to the flat, stellar-shaped myocytes obtained when the cells are plated on non-aligned collagen gels, myocytes plated on aligned gels display an elongated, rod-like shape. Ion channel expression was measured using the whole-cell arrangement of the patch clamp technique and Western blot analysis. Peak values for I(to), I(Na) and I(Ca) were 9+/-1, 71+/-13 and 7+/-1 pA/pF, respectively, in the flat cells, and increased to 21+/-2, 190+/-26 and 13+/-1 pA/pF, respectively, in the aligned cells. Application of forskolin (2 microM) and 3-isobutyl-1-methylxanthine (100 microM) resulted in a 101+/-18% increase in I(Ca) in the flat cells, but increased the current by only 43+/-9% in the aligned cells. Internal dialysis of the myocytes with cAMP strongly increased the peak I(Ca) in the flat cells, but caused no significant change in the aligned cells. While both basal and forskolin-stimulated levels of cAMP were the same in the two cell morphologies, the expression of the calcium channel alpha(1C) subunit was increased in the aligned cells. The expression and regulatory properties of voltage-gated calcium channels are modified during changes in neonatal rat myocyte shape.

α1H mRNA in single skeletal muscle fibres accounts for T‐type calcium current transient expression during fetal development in mice

Calcium channels are essential for excitation-contraction coupling and muscle development. At the end of fetal life, two types of Ca(2+) currents can be recorded in muscle cells. Whereas L-type Ca(2+) channels have been extensively studied, T-type channels have been poorly characterized in skeletal muscle. We describe here the functional and molecular properties of T-type calcium channels in developing mouse skeletal muscle. The T-type current density increased transiently during prenatal myogenesis with a maximum at embryonic day E16 followed by a drastic decrease until birth. This current showed similar electrophysiological and pharmacological properties at all examined stages. It displayed a wide window current centred at about -35 and -55 mV in 10 and 2 mM external Ca(2+), respectively. Activation and inactivation kinetics were fast (3 and 16 ms, respectively). The current was inhibited by nickel and amiloride with an IC(50) of 5.4 and 156 microM, respectively, values similar to those described for cloned T-type alpha(1H) channels. Whole muscle tissue RT-PCR analysis revealed mRNAs corresponding to alpha(1H) and alpha(1G) subunits in the fetus but not in the adult. However, single-fibre RT-PCR demonstrated that only alpha(1H) mRNA was present in prenatal fibres, suggesting that the alpha(1G) transcript present in muscle tissue must be expressed by non-skeletal muscle cells. Altogether, these results demonstrate that the alpha(1H) subunit generates functional T-type calcium channels in developing skeletal muscle fibres and suggest that these channels are involved in the early stages of muscle differentiation.

Decrease in calbindin content significantly alters LTP but not NMDA receptor and calcium channel properties

The contribution of the cytosolic calcium binding protein calbindin D 28K (CaBP) to the synaptic plasticity was investigated in hippocampal CA1 area of wild-type and antisense transgenic CaBP-deficient mice. We showed that long-term potentiation (LTP) induced by tetanic stimulation in CaBP-deficient mice was impaired. The fundamental biophysical properties of NMDA receptors and their number were not modified in CaBP-deficient mice. We also demonstrated that the physiological properties of calcium channels were identical between genotypes. An insufficient Ca 2+ entry through NMDA receptors or calcium channels, or a decrease in NMDA receptor density are unlikely to explain this impairment of LTP. Interestingly, we showed that the loss of LTP was not prevented by glycine but was restored in the presence of a low concentration of the NMDA receptor antagonist D-APV (5 μM) and of the calcium chelator BAPTA-AM (5 μM). Moreover, we observed a loss of LTP in the wild-type mice when the postsynaptic tetanic-induced [Ca 2+] i rise is excessively increased. Conversely, a weaker tetanus stimulation allowed LTP induction and maintenance in CaBP-deficient mice. These results suggest that a higher cytosol [Ca 2+] i, due to the decrease of CaBP expression may impair LTP induction and maintenance mechanisms without affecting the mechanisms of calcium entry. Thus, CaBP plays a critical role in long term synaptic plasticity by limiting the elevation of calcium rise in the cytosol to some appropriate spatio-temporal pattern.

Biophysical and Pharmacological Properties of Voltage-gated Calcium Channels in Osteoblastic MC3T3-E1 Cells.

The electrophysiological and pharmacological properties of the voltage-gated Ca2+ channels in MC3T3-E1 cells were analyzed using the perforated whole-cell patch-clamp technique. When the voltage was depolarized by step pulses from a holding potential of -104mV, the cells displayed transient inward currents (-4.25±0.25pA/pF, n=16) in 10mM Ba2+ solution. The activation threshold for the inward Ba2+current was about -60mV and the peak existed between -40 and -20mV. The steady state activation and inactivation properties of the inward Ba2+ current generated a window current in the range of -70 to -40 mV. Gd2+ (0.1mM) inhibited the inward Ba2+ currents by about 60%. Ni2+ (0.1mM, a blocker for T-type and R-type Ca2+ channels at this concentration), nifedipine (5μM, L-type Ca2+ channel blocker), ω-conotoxin GVIA (3μM, N-type Ca2+ channel blocker) and ω-agatoxin TK (200nM, a P/Q-type Ca2+ channel blocker) did not inhibit the currents. Bay K 8644 (0.5μM, a dihydropyridine agonist for L-type Ca2+ channel) also did not affect the Ba2+ currents. The results suggest that Ca2+ channels with novel properties are expressed in MC3T3-E1 cells.

Increased calcium influx is responsible for the sustained mechanical tone in colon from dystrophic (mdx) mice.

Proximal colon from dystrophic mice develops spontaneous tone increment, but the mechanisms involved in its development have not been investigated. This study examined whether alterations in the properties of cell membrane calcium channels and/or sarcoplasmic reticular (SR) Ca2+-adenosine triphosphatase (ATPase) contribute to tone development. Effects of calcium-free solution, nifedipine, pinaverium (calcium channel blockers), and cyclopiazonic acid (CPA; SR Ca2+-ATPase inhibitor) on the contractile activity of colon from mdx and control mice were determined. Calcium-free solution abolished spontaneous contractions in both preparations, but decreased the tone only in mdx mice. Nifedipine or pinaverium abolished phasic contractions, acting with different sensitivities on the 2 preparations. They also decreased the tone in colons of mdx mice, and Ca2+-free solution did not cause any further loss of tone. CPA, after an early contractile effect, abolished spontaneous contractions in control animals. It did not suppress the contractile activity in mdx mice. CPA inhibited the repletion of intracellular calcium stores in both tissues to the same degree. Increased Ca2+ influx through L-type voltage-dependent Ca2+ channels seems to be responsible for the sustained mechanical tone of proximal colon from mdx mice. The mechanisms for sequestering calcium appear to be unaltered.

Histidine Phosphorylation of Annexin I in Airway Epithelia

Although [Cl(-)](i) regulates many cellular functions including cell secretion, the mechanisms governing these actions are not known. We have previously shown that the apical membrane of airway epithelium contains a 37-kDa phosphoprotein (p37) whose phosphorylation is regulated by chloride concentration. Using metal affinity (chelating Fe(3+)-Sepharose) and anion exchange (POROS HQ 20) chromatography, we have purified p37 from ovine tracheal epithelia to electrophoretic homogeneity. Sequence analysis and immunoprecipitation using monoclonal and specific polyclonal antibodies identified p37 as annexin I, a member of a family of Ca(2+)-dependent phospholipid-binding proteins. Phosphate on [(32)P]annexin I, phosphorylated using both [gamma-(32)P]ATP and [gamma-(32)P]GTP, was labile under acidic but not alkaline conditions. Phosphoamino acid analysis showed the presence of phosphohistidine. The site of phosphorylation was localized to a carboxyl-terminal fragment of annexin I. Our data suggest that cAMP and AMP (but not cGMP) may regulate annexin I histidine phosphorylation. We propose a role for annexin I in an intracellular signaling system involving histidine phosphorylation.

Specific Properties of T-type Calcium Channels Generated by the Human α1I Subunit

We have cloned and expressed a human alpha(1I) subunit that encodes a subtype of T-type calcium channels. The predicted protein is 95% homologous to its rat counterpart but has a distinct COOH-terminal region. Its mRNA is detected almost exclusively in the human brain, as well as in adrenal and thyroid glands. Calcium currents generated by the functional expression of human alpha(1I) and alpha(1G) subunits in HEK-293 cells were compared. The alpha(1I) current activated and inactivated approximately 10 mV more positively. Activation and inactivation kinetics were up to six times slower, while deactivation kinetics was faster and showed little voltage dependence. A slower recovery from inactivation, a lower sensitivity to Ni(2+) ions (IC(50) approximately 180 micrometer), and a larger channel conductance (approximately 11 picosiemens) were the other discriminative features of the alpha(1I) current. These data demonstrate that the alpha(1I) subunit encodes T-type Ca(2+) channels functionally distinct from those generated by the human alpha(1G) or alpha(1H) subunits and point out that human and rat alpha(1I) subunits have species-specific properties not only in their primary sequence, but also in their expression profile and electrophysiological behavior.

Developmental regulation of the L-type calcium channel alpha1C subunit expression in heart.

We used Northern analyses, RNase protection assays and immunoblot analyses to examine the relationship among developmental age of the heart, abundance of mRNA and L-type calcium channel alpha1C subunit protein, and to establish the size of the native protein in heart. Northern analysis, RNase protection assays, and immunoblots were used to study RNA and protein from rat heart of various ages. In fetal and adult ventricles there was a predominant 8.3-kb transcript for the alpha1C subunit with no change in transcript size during development. RNase protection assays demonstrated a 2-fold increase in abundance of the DHP receptor message during postnatal development. Immunoblots identified a 240 kD protein, corresponding to the predicted molecular mass of the full length alpha1C subunit. No change in size of protein for the alpha1C subunit was observed at any developmental stage and there was no evidence for a truncated isoform. There was an approximate 2-fold increase in alpha1C subunit protein in ventricular homogenates during postnatal development. Thus, in the developing rat heart, alterations in calcium channel properties during development appear to result neither from alternative splicing that produces a smaller transcript for the alpha1C subunit nor from expression of a truncated protein, but at least in part from transcriptionally-regulated expression of the 240 kDa polypepde.

The human polycystic kidney disease 2-like (PKDL) gene: exon/intron structure and evidence for a novel splicing mechanism.

Polycystin-L is a member of the expanding family of polycystins. Mutations in polycystin-1 or -2 cause human autosomal dominant polycystic kidney disease (ADPKD). The mouse ortholog of PKDL, Pkdl, is deleted in a mouse line with renal and retinal defects. We recently have shown that polycystin-L has calcium channel properties. In the current study, we determined the exon/intron organization of the PKDL gene and its alternative splicing. We show that PKDL has 16 exons. All splice acceptor/donor sites for these exons conform to the GT-AG rule. The positions of introns and the sizes of exons in the PKDL gene are very similar to those of PKD2, except for the last two 3' end exons. RT-PCR demonstrates the existence of at least three polycystin-L splice variants: PKDL(Delta5), PKDL(Delta456), and PKDL(Delta15) that are expressed in a tissue-specific manner. In addition, we have localized polymorphic marker D10S603 to intron 4 and exon 5 of PKDL. Elucidation of the gene structure, exact location, and alternative splicing patterns of PKDL will facilitate its evaluation as a candidate gene in cystic or other genetic disorders.

High-voltage-activated calcium channel messenger RNA expression in the 140-3 neuroblastoma–glioma cell line

Expression of calcium channel α 1 subunits in oocytes or cell lines has proven to be a powerful method in the analysis of structure–function relations, but these experimental systems are of limited value in the examination of neuron-specific functions such as transmitter release. Cell lines derived from neurons are often capable of these functions, but their intrinsic calcium channel α 1 subunits are complicating factors in experimental design. We have examined the biophysical and molecular properties of calcium channels in a little studied neuroblastoma–glioma hybrid cell line, 140-3, a close relative of the NG108-15 cell line, to test whether this cell line might serve a role as an expression system for neural mechanisms. This cell was selected as it contains an intact transmitter release mechanism yet secretes little in response to depolarization. Patch-clamp recording revealed only a prominent low-threshold, rapidly inactivating calcium current with a single-channel conductance of ∼7 pS that was identified as T type. A search for calcium channel α 1 subunit messenger RNA in the 140-3 cells with three different tests only revealed α 1C, whereas α 1A–α 1C were present in the parent NG108-15 line. We made a particular effort to search for α 1E, since this subunit has been associated with a low-voltage-activated current. Our findings suggest that, since the principal nerve terminal-associated calcium channels (α 1A, α 1B, α 1E) are absent in the 140-3 cell, this cell line may prove a particularly useful model for the analysis of the role of high-voltage-activated calcium channels in complex functions of neuronal cells.

Properties of Q-Type Calcium Channels in Neostriatal and Cortical Neurons are Correlated with β Subunit Expression

In brain neurons, P- and Q-type Ca(2+) channels both appear to include a class A alpha1 subunit. In spite of this similarity, these channels differ pharmacologically and biophysically, particularly in inactivation kinetics. The molecular basis for this difference is unclear. In heterologous systems, alternative splicing and ancillary beta subunits have been shown to alter biophysical properties of channels containing a class A alpha1 subunit. To test the hypothesis that similar mechanisms are at work in native systems, P- and Q-type currents were characterized in acutely isolated rat neostriatal, medium spiny neurons and cortical pyramidal neurons using whole-cell voltage-clamp techniques. Cells were subsequently aspirated and subjected to single-cell RT-PCR (scRT-PCR) analysis of calcium channel alpha(1) and beta (beta(1-4)) subunit expression. In both cortical and neostriatal neurons, P- and Q-type currents were found in cells expressing class A alpha(1) subunit mRNA. Although P-type currents in cortical and neostriatal neurons were similar, Q-type currents differed significantly in inactivation kinetics. Notably, Q-type currents in neostriatal neurons were similar to P-type currents in inactivation rate. The variation in Q-type channel biophysics was correlated with beta subunit expression. Neostriatal neurons expressed significantly higher levels of beta(2a) mRNA and lower levels of beta(1b) mRNA than cortical neurons. These findings are consistent with the association of beta(2a) and beta(1b) subunits with slow and fast inactivation, respectively. Analysis of alpha(1A) splice variants in the linker between domains I and II failed to provide an alternative explanation for the differences in inactivation rates. These findings are consistent with the hypothesis that the biophysical properties of Q-type channels are governed by beta subunit isoforms and are separable from toxin sensitivity.

Voltage-Activated Calcium Currents in Rat Retinal Ganglion CellsIn Situ: Changes during Prenatal and Postnatal Development

Voltage-activated calcium currents (ICa) are one way by which calcium influx into neurons is mediated. To investigate changes in kinetic properties of ICa during neuronal development and to correlate possible kinetic changes with specific differentiation processes, the ICa of retinal ganglion cells (RGCs) was recorded with the perforated patch-clamp technique in rat retinal slices and in whole mounts at different prenatal and postnatal stages. ICa density increased between embryonic day (E) 20 and the adult stage, paralleled by a shift in activation of the omega-conotoxin GVIA-sensitive ICa toward more negative membrane potentials. Furthermore, developmental alterations were observed in ICa inactivation rate during a 120 msec test pulse and in steady-state inactivation of ICa. The most striking feature in ICa kinetics was a transient slowing of calcium current deactivation, which peaked at postnatal day (P)3-5 and affected all ICa subtypes. Although the shift in activation and the decreased inactivation rate of ICa can be explained by differential regulation of distinct calcium channel subtypes, it is more likely that a more general alteration of the cells' functional state was the underlying factor in alterations in steady-state inactivation and current deactivation of ICa. Alterations in the omega-conotoxin GVIA-sensitive and the toxin-resistant currents temporarily coincide with dendritic differentiation, and it is tempting to speculate about their role in network formation in the inner retina. In contrast, alterations in steady-state inactivation and current deactivation may be involved in the regulation of RGC survival, because they occur during the period of programmed cell death in the ganglion cell layer. In conclusion, distinct time windows of alterations in calcium channel properties were found, and this study has provided a basis for performing functional assays to clarify in detail the developmental process to which these alterations are related.

Protein Fate in Neurodegenerative Proteinopathies: Polyglutamine Diseases Join the (Mis)Fold

Protein misfolding is now recognized to be a central feature of neurodegenerative diseases. Alzheimer disease (AD), Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), prion diseases, and the polyglutamine (polygln) diseases all represent proteinopathies—diseases in which a particular protein or set of proteins misfolds and aggregates. In the past decade, mutations have been identified in an increasing number of genes underlying specific forms of neurodegeneration—for example, genes encoding amyloid precursor protein (APP) and presenilins 1 and 2 in AD, parkin and alpha-synuclein in PD, Cu/Zn superoxide dismutase in ALS, tau in frontotemporal dementia, prion protein in Creutzfeldt-Jacob and Gerstmann-Straussler diseases, and at least eight different proteins in the polygln diseases (Hardy and GwinnHardy 1998; Price et al. 1998). Identification of these mutations was a first step in understanding the disease process; next, it will be necessary to explain how specific changes in disease proteins may alter the neuron’s physiology and to further question how neuronal responses to the mutant protein influence the disease process. This review examines the fate of mutant protein in neurons and the potential role of posttranslational events in pathogenesis, with particular focus on the polygln diseases. Posttranslational events have already been implicated

Gating of single N-type calcium channels recorded from bullfrog sympathetic neurons.

For many neurons, N-type calcium channels provide the primary pathway for calcium influx during an action potential. We investigated the gating properties of single N-type calcium channels using the cell-attached patch technique. With 100 mM Ba2+ in the pipet, mean N-channel open probability (Po, measured over 100 ms) increased with depolarization, but the range at a single voltage was large (e.g., Po at +40 mV ranged from 0.1 to 0.8). The open dwell time histograms were generally well fit by a single exponential with mean open time (tauo) increasing from 0.7 ms at +10 mV to 3.1 ms at +40 mV. Shut time histograms were well fit by two exponentials. The brief shut time component (taush1 = 0.3 ms) did not vary with the test potential, while the longer shut time component (taush2) decreased with voltage from 18.9 ms at +10 mV to 2.3 ms at +40 mV. Although N-channel Po during individual sweeps at +40 mV was often high ( approximately 0.8), mean Po was reduced by null sweeps, low Po gating, inactivation, and slow activation. The variability in mean Po across patches resulted from differences in the frequency these different gating processes were expressed by the channels. Runs analysis showed that null sweeps tended to be clustered in most patches, but that inactivating and slowly activating sweeps were generally distributed randomly. Low Po gating (Po = 0.2, tauo = 1 ms at +40 mV) could be sustained for approximately 1 min in some patches. The clustering of null sweeps and sweeps with low Po gating is consistent with the idea that they result from different modes of N-channel gating. While Po of the main N-channel gating state is high, the net Po is reduced to a maximum value of close to 0.5 by other gating processes.