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
2000-03-03
2003-04-15
Romeo, David S. (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...
C435S325000, C435S320100, C536S023500, C530S350000
Reexamination Certificate
active
06548270
ABSTRACT:
BACKGROUND OF THE INVENTION
Tissue acidosis (increased concentrations of extracellular protons or decreased pH) is associated with a number of painful physiological (e.g., cramps) and pathological (e.g., intermittent claudication, inflammation, ischemia, myocardial infarction). The extracellular pH may decrease by more than 2 log units during tissue acidosis (Reeh and Steen, 1996). The chemoreception of acid (protons) plays a critical role in the detection of nociceptive pH imbalances that occur in a number of conditions including camps, trauma, inflammation and hypoxia (Lindahl, 1974). Local tissue acidosis (increases in extracellular H
+
concentration) arises from changes in the extracellular space during inflammatory or ischaemic conditions (Wall and Melzack, 1994). External acidification is a major factor in pain associated with inflammation, hematomas, cardiac or muscle ischemia, or cancer. Noxious chemical stimuli excite peripheral nerve endings of small diameter sensory neurons (nociceptors) in sensory ganglia (eg., dorsal root, nodose and trigeminal ganglia) and initiate signals that are perceived as pain. For instance, there is evidence that the sensation of pain parallels pH decreases (Steen et al., 1995). Prolonged intradermal infusion of low pH solutions can cause sensations that are similar to that felt during hyperalgesia, or chronic pain. Acid evoked currents in cardiac sensory neurons may mediate the sensation of myocardial ischemia (Benson et al., 1999). While decreasing the pH may cause a myriad of effects through a variety of mechanisms, the existence of ion channels that are directly gated (activated) by protons provides a means to pharmacologically manipulate specific pathways. A number of conductances in sensory and central neurons have been shown to be gated by low pH (Bevan and Yeats, 1991; Varming, 1999). Proton-gated cation channels with different pH sensitivities and kinetics were reported in sensory (trigeminal, cardiac and DRG) neurons (Benson et al., 1999; Bevan and Yeats, 1991; Kovalchuk et al., 1990; Krishtal and Pidoplichko, 1981), in neurons of the central nervous system (Grantyn and Lux, 1988; Ueno et al., 1992) and in oligodendrocytes (Sontheimer et al., 1989). The native proton-gated currents in DRG appear to vary among cells and include rapidly inactivating, non-inactivating and biphasic (both rapidly inactivating and subsequent non-inactivating) currents. A number of proton-gated channels have been cloned since 1996 and include the VR1 capsaicin-activated receptor (Tominaga et al., 1999) and a family of receptors that have homology to the nematode degenerin/mammalian amiloride-sensitive sodium channels (epithelial or brain Na channels; ENaC or BNaC). At least 4 of the latter have been shown to be expressed in sensory ganglia (see TABLE), suggesting a molecular correlate to the diversity of observed currents.
The cloned acid-sensing ion channels (ASICs) are structurally related to the Caenorhabditis elegans degenerins and mammalian epithelial sodium channels and are composed of 2 putative transmembrane domains and a large extracellular domain. The first member of this superfamily was the
C. elegans
deg-1 gene. A gain of function mutation of this gene (ie., the gene product was more active than wildtype) induced neuronal swellinng and degeneration (Chalfie and Wolinsky, 1990). This gene as well as a gene encoding a related protein MEC-4 (Driscoll and Chalfie, 1991) were called “degenerins” since they could mutate to toxic forms. There is a functional diversity of channels in this superfamily from H+ activated (ASICs), constitutively active (ENaC, Epithelial Na channel), peptide-gated (FMRF-receptor) in the snail to possibly stretch activation (degenerins of
C. elegans
). However, all of these family members appear to be sensitive to amiloride, are permeable to Na+, and are voltage insensitive (Waldmann and Lazdunski, 1998). With the exception of ASIC2b, all ASIC subunits form functional homomers when expressed in heterologous expression systems.
TABLE 1
Alternate
Genbank
Name
name
pH 0.5
Vrev (mV)
(species)
References
ASIC1
BNaC2
6.2
+25 (Ena = 73)
U78180, U78181
(Chen et al., 1998;
(2 splice
(Waldmann
(Chen et al.,
(human);
Garcia Anoveros et al.,
variants,
et al., 1997)
1998)
U94403 (rat)
1997; Waldmann et
A and B)
5.9 (Chen et
al., 1997)
al., 1998)
(Chen et al., 1998)
ASIC2a
BNC1;
4.05
Highly
U50352 (human);
(Price et al., 1996;
MDEG;
selective for
U53211
Waldmann et al.,
MDEG
Na+
(rat)
1996; Welsh and
(Pna:PK:Pca =
Price, 1999; Welsh
20:10:1)
and Price,)
ASIC2b*
MDEG2
na
Y14635
(Lingueglia et al.,
(rat); Y14634
1997)
(mouse)
hASIC3
++
3.66 (fast);
+33; +48
AF095897,
(Babinski et al., 1999;
3.82
AF057711,
de Weille et al., 1998;
(sustained)
AB010575
Ishibashi and Marumo,
[Seguela];
(human)
1998; Seguela and
6.2; 4.3
Babinski, 1999)
(DeWeille et
al., 1998)
ASIC3
BNaC3;
6.5 (fast); 3.5
+32; +32
AF013598
(Waldmann et al.,
DRASIC
(sustained)
(rat)
1997)
pH 0.5 is the pH at which the induced current is half maximally activated.
*Splice variant of ASIC2 in rat; does not appear to form functional homomeric channels. There is no evidence for the existence of this splice variant in humans (BLAST search of EST databases).
++
This may not be the ortholog of the rat ASIC3 since it is only 83% identical compared to >97% identity between rat and human ASIC1 and rat and human ASIC2.
The kinetic properties of the homomeric channels vary. ASIC1 activates and desensitizes in the continued presence of acid and thus induces only a transient response (Waldmann et al., 1997). ASIC2 activates and inactivates more slowly than ASIC1 (Bassilana et al., 1997). DRASIC produces a biphasic response to extracellular acidification (Babinski et al., 1999; Waldmann et al., 1997). The sensitivity to acid depends on the subunit (see TABLE 1) and the pH producing the half-maximal current varies from about 6.5 to near 3.
Members of the degenerin superfamily form heteromers. This has been clearly shown with the ENaC family (Canessa, 1996; Fyfe and Canessa, 1998). ASIC subunits also appear to form heteromers. ASIC subunits may co-localize. At a tissue level, ASIC1A, ASIC2a and b, and ASIC3 mRNA are expressed the brain and ASIC1A and B, ASIC2b and ASIC3 mRNA are expressed in DRG (TABLE 2). For instance, ASIC2 and ASIC1 are co-expressed in almost all regions of mouse and human brain (Garcia Anoveros et al., 1997). Immunocytochemistry using polyclonal antisera against rat ASIC1 reveals ASIC1 protein in superficial dorsal horn, DRG and spinal trigeminal nucleus and peripheral nerve fibers (Olson et al., 1998). Functional co-expression of multiple ASIC subunits reveal channels with properties that differ from either homomeric channel, particularly in terms of the pH0.5, relative ion permabilities, and kinetics (Bassilana et al., 1997). ASIC2b is capable of modifying the kinetic and permeability properties of ASIC2a and DRASIC (Lingueglia et al., 1997). Furthermore, co-expression of ASIC family members as well as hASIC3 and the structurally similar P
2
X
2
(Seguela and Babinski, 1999) produce receptors with properties that differ from either homomer. Messenger RNA encoding the present invention is localized to regions that express other ASIC subunits and suggest that hBNaC4 likely forms heteromers as well.
Most members of the amiloride sensitive Na channel/degenerin family appear to be highly selective for Na over K (Bassilana et al., 1997; Chen et al., 1998; Waldmann et al., 1997; Waldmann et al., 1997). The sustained component of DRASIC shifts from being largely mediated by Na to being less so in the presence of MDEG2 (Lingueglia et al., 1997). Chimers of MDEG1 and MDEG2 revealed nine amino acids in the putative N terminal cytoplasmic region adjacent to the first transmembrane region (TM1) are critically important determinants of the ion selectivity of these channels. Ile19, Phe-20 and Thr-25 appear to be particularly important since mutations in these residues discriminated poorly
Dubin Adrienne Elizabeth
Erlander Mark G.
Huvar Rene
Pyati Jayashree
Murphy Joseph F.
Ortho-McNeil Pharmaceutical , Inc.
Romeo David S.
Wallen III John W.
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