Protein Kinase C Isoform Antagonism Controls BNaC2 (ASIC1) Function

Abstract

We explored the involvement of protein kinase C (PKC) and its isoforms in the regulation of BNaC2. Reverse transcriptase PCR evaluation of PKC isoform expression at the level of mRNA revealed the presence of ␣ and ⑀/⑀ in all glioma cell lines analyzed; most, but not all cell lines expressed ␦ and . No messages were found for the ␤I and ␤II isotypes of PKC in the tumor cells. Normal astrocytes expressed ␤ but not ␥. The essential features of these results were confirmed at the protein level by Western analysis. This disproportionate pattern of PKC isoform expression in glioma cell lines was further echoed in the functional effects of these PKC isoforms on BNaC2 activity in bilayers. PKC holoenzyme or the combination of PKC␤I and PKC␤II isoforms inhibited BNaC2. Neither PKC⑀ nor PKC or their combination had any effect on BNaC2 activity in bilayers. The inhibitory effect of the PKC␤I and PKC␤II mixture on BNaC2 activity was abolished by a 5-fold excess of a PKC⑀ and PKC combination. PKC holoenzymes, PKC␤I, PKC␤II, PKC␦, PKC⑀, and PKC phosphorylated BNaC2 in vitro. In patch clamp experiments, the combination of PKC␤I and PKC␤II inhibited the basally activated inward Na ؉ conductance. The variable expression of the PKC isotypes and their functional antagonism in regulating BNaC2 activity support the idea that the participation of multiple PKC isotypes contributes to the overall activity of BNaC2.The recent molecular identification of a class of proton-sensitive ion channels (ASIC; acid-sensitive ion channel (1, 2); also called BNC and BNaC; brain Na ϩ channel (3, 4)) belonging to the degenerin (DEG)/ENaC superfamily of ion channels (5) added a new molecular entity to the already complicated field of nociception. Even though their participation in nociception is controversial, ASICs might underlie some properties of native proton-induced currents (6 -8) and could contribute to the function of nociceptive transduction with many other key constituents including nociceptor-specific voltage-gated Na ϩ channels, ATP-gated channels, and capsaicin receptors (9 -13).The distinguishing feature of this family of ion channels, namely, proton-induced conductance, is exhibited by ASIC1a (14), BNaC2 (4), ASIC1b (15), ASIC␤ (16), ASIC2 (or BNC1) (3), BNaC1 (4), MDEG (17), and ASIC3 (DRASIC; 18), hASIC3 (19,20), and hTNaC1 (21). However, ASIC4 is functionally inactive (22, 23) and may require an association with an accessory protein(s) and/or other subunit(s) of the family, like the splice variant form of ASIC2 (ASIC2b, also called MDEG2) (24) or ASIC␤2, a splice variant of ASIC␤ (25). The multiplicity of current responses to extracellular acid loads in different neurons is consistent with the existence of functionally distinct ASICs in these cells.The tissue distribution of the ASIC members is not limited to the nervous system but also includes many other tissues such as the lung, testis, and intestine (20,21,26,27). Sensory neuron-specific expression of DRASIC has been reported (18), but Chen et al. (16) have found low level trans...

Inhibition of Neuronal Degenerin/Epithelial Na+ Channels by the Multiple Sclerosis Drug 4-Aminopyridine

“…The expression vector encoding YFP-tagged rASIC1b was a gift from Drs. D. J. Benos and C. Fuller (University of Alabama-Birmingham, AL) and has been described previously (34,35). The expression vector encoding constitutively active rat BLINaC harboring the A443T mutation was a gift from Dr. E. Lingueglia (Institut de Pharmacologie Moleculaire et Cellulaire, Valbonne, France) and has been described previously (24).…”

Two PKC consensus sites on human acid-sensing ion channel 1b differentially regulate its function

“…Furthermore, PKA phosphorylates hASIC1b at S479, and this phosphorylation interferes with PICK1 binding (30). Berdiev et al (6) showed that PKC holoenzyme phosphorylates hASIC1b in vitro, and the addition of PKC to the intracellular side of hASIC1b in lipid bilayers decreases its open probability. Generally, the direction of the effect of PKC on ion channels is cell type specific and channel subtype specific, with PKC activation resulting in either an increase or a decrease of the current (11).…”

Two conventional protein kinase C isoforms, α and βI, are involved in the ATP‐induced activation of volume‐regulated anion channel and glutamate release in cultured astrocytes

“…A cooperative role of PKCs and in LDL-induced Akt phosphorylation has recently been found in the THP-1 monocyte cell line (Preiss et al 2007). In glioma cell lines, PKC I and II act cooperatively to inhibit the activity of the proton-sensitive Na + channel ANaC2 (ASIC1) (Berdiev et al 2002). Two interpretations of our data may be considered: (i) PKC and PKC I act as functional homologues, replacing each other after downregulation of their counterpart; (ii) these PKCs work in sequence, and activation of one isoform precedes activation of the second isoform.…”

“…Cultured astrocytes express numerous PKC isoforms from all three PKC subfamilies (Slepko et al 1999;Berdiev et al 2002). Among conventional isoforms, expression of , I, II, but not has been detected (Slepko et al 1999).…”