Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-10-26
2002-03-19
Mertz, Prema (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023100, C536S023500, C530S350000, C435S071100, C435S071200, C435S471000, C435S325000, C435S252300, C435S254110, C435S320100
Reexamination Certificate
active
06358706
ABSTRACT:
BACKGROUND OF THE INVENTION
Voltage activated calcium channels play important roles including neuroexcitation, neurotransmitter and hormone secretion, and regulation of gene transcription through Ca-dependent transcription factors. Their functions depend in part on their cellular localization and their gating properties (characteristics of their opening, inactivation, deactivation, and recovery from inactivation). Five general classes of voltage activated calcium channels have been observed in various neuronal and non-neuronal tissues. The complement of channel subunits and the subcellular localization of the expressed voltage activated calcium channels determine the functional cellular properties.
Diversity of Voltage-gated Ca Channels Fall into Two Major Categories: Low Voltage Activated (LVA) and High Voltage Activated (HVA)
A conserved general structure for all cloned voltage-gated calcium channel alpha subunits (the pore-forming subunit) has been identified. It consists of 4 domains with homology to the domains present in voltage-gated K and Na channels. Each domain contains 6 membrane spanning regions (S1-S6) and a pore region (P) located between S5 and S6. The extracellular loops are generally very short; intracellular loops contain sites that are modulated by phosphorylation and can interact with other effectors. However, there are notable differences in the lengths of the S5-S6 loop of domain I and the intracellular loop between domains I and II among alpha subunits.
Different calcium channels are best distinguished by their pharmacological profiles since their electrophysiological properties differ depending on the cell type or tissue in which they are expressed, presumably because of modulation by cellular proteins, for instance kinases, and also auxiliary calcium channel subunits.
The HVA channel classes are thought to be composed of at least 3 or 4 different subunits: &agr;1 (which contains the pore), beta (&bgr;) and &agr;2&dgr;. In skeletal muscle a &ggr; subunit also co-precipitates with the skeletal channel complex. Recently two gamma-like subunits have been cloned from brain—one of which is the gene mutated in the stargazer mutant mouse (Black et al., 1999; Letts et al., 1998). The subunit composition has been proved for only the skeletal L-type (&agr;1 &agr;2&dgr; &bgr; &ggr;) and brain N-type (&agr;1 &agr;2&dgr; &bgr;) channels (Perez-Reyes and Schneider, 1995). These channels generally require large membrane depolarizations for activation (~30 mV from the resting potential (RP)). Four classes of HVA calcium channels have been identified on the basis of electrophysiological, pharmacological and molecular data. These classes include L-type (encoded by at least 4 genes (including a &agr;1 subunits &agr;1S (skeletal muscle), &agr;1C, &agr;1D (neuroendocrine), and &agr;1F (retinal)), N-type (&agr;1B; (Williams et al., 1992)), P/Q-type (&agr;1A) and R-type (encoded by at least the &agr;1E gene).
HVA &agr;1 families are strongly affected by co-expression of the cytoplasmically localized &bgr; subunit, particularly the expression levels of functional cell surface channels and the electrophysiological response of the channel (ie., kinetics). &bgr; subunits interact with a specific sequence in the I-II intracellular loop to increase the number of functional channels and alter the activation and inactivation properties of the channel complex (Furukawa et al., 1998). There are at least 4&bgr; genes that are alternatively spliced (&bgr;1a-c; &bgr;2a-c; &bgr;3; &bgr;4;(Perez-Reyes and Schneider, 1995)); the effect of each of these &bgr;s on &agr;1 function appears to depend on the &agr;1 class. Interestingly, mutants in &bgr; (Cch&bgr;4) produce ataxia and seizures in the lethargic (lh) mouse (Burgess et al., 1997). &agr;2&dgr; subunits also modulate &agr;1 function and the known gene co-segregates with malignant hyperthermia phenotype in certain families (Iles et al., 1994).
The physiological roles of HVA channels depend on subcellular location of the channel and tissue type. Subcellular location varies among tissues but have been shown to be important in neurotransmitter and hormone release, action potential duration, excitation-contraction coupling in muscle cells, and gene expression (Miller, 1987).
There are at least three genes in the T-type family of LVA calcium channels (&agr;1G, &agr;1H, and &agr;1I) (Perez-Reyes, 1998). Their structure differs from that of the HVA channels in a number of important ways. The I-II intracellular linker is much longer (~400 amino acids) than that of the known HVA channels. The Domain I S5-P extracellular linker is longer than that of the HVA channels and may be a good target for drug interactions with this channel. &bgr; does not appear to be associated with &agr;1 in this class and they lack the canonical sequence that is known to be crucial for beta subunit binding (Lambert et al., 1997; Leuranguer et al., 1998). Anti-sense experiments directed against all known beta's show a decrease in the expression of HVA calcium channels but not LVA calcium channels in nodose ganglion neurons (Lambert et al., 1997).
Other proteins or cellular environments may be required for robust T-channel expression since &agr;1G expressed in oocytes or HEK293 cells produces dramatically different current magnitudes in these two cell types (Perez-Reyes, 1998).
T-type calcium currents have been observed in vivo in many cell types in the peripheral and central nervous systems including thalamus, inferior olive, cerebellar Purkinje cells, lateral habenular cells, dorsal horn neurons, sensory neurons (DRG, nodose), cholinergic forebrain neurons, hippocampal intemeurons, CA1, CA3 dentate gyrus pyramidal cells, basal forebrain neurons, amygdaloid neurons (Talley et al., 1999). T-type channels are prominent in the soma and dendrites of neurons that reveal robust Ca-dependent burst firing behaviors such as the thalamic relay neurons and cerebellar Purkinje cells (Huguenard, 1996).
Physiological Roles and Therapeutic Areas
T-type calcium channels are involved in the generation of low threshold spikes to produce burst firing (Huguenard, 1996). These channels differ from HVA channels in that they have some probability of opening at the resting membrane potential. Because their steady state inactivation curve is shifted toward negative voltages compared to HVA channels (ie., half the channels are not inactivated and are able to be opened by a depolarizing voltage step at voltages more negative than the resting membrane potential (RP)), there is a window current near the RP (ie., a portion of the T-channels are open at RP). Low threshold spikes and rebound burst firing is prominent in neurons from inferior olive, thalamus, hippocampus and neocortex (Huguenard, 1996).
T-type channels promote oscillatory behavior which has important consequences for epilepsy. The ability of a cell to fire low threshold spikes is critical in the genesis of oscillatory behavior and increased burst firing (groups of action potentials separated by about 50-100 ms). T-type calcium channels are thought to play a significant role in absence epilepsy, a type of generalized non-convulsive seizure. The evidence that voltage-gated calcium currents contribute to the epileptogenic discharge, including seizure maintenance and propagation includes 1) a specific enhancement of T-type currents in the reticular thalamic (nRT) neurons which are hypothesized to be involved in the genesis of epileptic seizures in a rat genetic model (GAERS) for absence epilepsy (Tsakiridou et al., 1995); 2) antiepileptics against absence petit mal epilepsy (ethosuximide and dimethadione) have been shown at physiologically relevant doses to partially depress T-type currents in thalamic (ventrobasal complex) neurons (Coulter et al., 1989; Kostyuk et al., 1992); and 3) T-type calcium channels underlie the intrinsic bursting properties of particular neurons that are hypothesized to be involved in epilepsy (nRT, thalamic relay and hippocampal pyramidal cells) (Huguenard, 1996). The rat &agr;1G is highly expressed in thala
Dubin Adrienne E.
Erlander Mark G.
Galindo Jose E.
Pyati Jayashree
Zhu Jessica Y.
Mertz Prema
Ortho-McNeil Pharmaceutical , Inc.
Wallan, III John W.
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