Colocalization and coassembly of two human brain M-type potassium channel subunits that are mutated in epilepsy

Abstract

Acetylcholine excites many central and autonomic neurons through inhibition of M-channels, slowly activating, noninactivating voltage-gated potassium channels. We here provide information regarding the in vivo distribution and biochemical characteristics of human brain KCNQ2 and KCNQ3, two channel subunits that form M-channels when expressed in vitro, and, when mutated, cause the dominantly inherited epileptic syndrome, benign neonatal familial convulsions. KCNQ2 and KCNQ3 proteins are colocalized in a somatodendritic pattern on pyramidal and polymorphic neurons in the human cortex and hippocampus. Immunoreactivity for KCNQ2, but not KCNQ3, is also prominent in some terminal fields, suggesting a presynaptic role for a distinct subgroup of M-channels in the regulation of action potential propagation and neurotransmitter release. KCNQ2 and KCNQ3 can be coimmunoprecipitated from brain lysates. Further, KCNQ2 and KCNQ3 are coassociated with tubulin and protein kinase A within a Triton X-100-insoluble protein complex. This complex is not associated with low-density membrane rafts or with N-methyl-Daspartate receptors, PSD-95 scaffolding proteins, or other potassium channels tested. Our studies thus provide a view of a signaling complex that may be important for cognitive function as well as epilepsy. Analysis of this complex may shed light on the unknown transduction pathway linking muscarinic acetylcholine receptor activation to M-channel inhibition. M utations in ion channel genes have been shown to cause a large number of forms of epilepsy and other episodic disorders in humans and inbred mice (1-3). Independent efforts led by Leppert (4-6) and Steinlein (7,8) to identify genes responsible for benign neonatal familial convulsions (BNFC), a dominantly inherited epilepsy associated with frequent seizures in the first weeks of life and a 10-16% risk of seizure recurrence after infancy, resulted in the cloning of two potassium channel subunits, KCNQ2 and KCNQ3. KCNQ2 and KCNQ3 have been characterized electrophysiologically with heterologous expression systems (8-12). Coexpression of KCNQ2 and KCNQ3 in Xenopus oocytes resulted in currents that were much larger than those observed when either subunit was expressed alone, leading to the suggestion that heteromultimeric KCNQ2͞KCNQ3 channels were expressed in vivo (9, 10). In oocytes or HEK cells, currents mediated by these heterologously expressed KCNQ2͞ KCNQ3 channels were enhanced by activation of the cAMPdependent protein kinase (PKA); this effect was abolished by site-directed mutations that eliminated a single PKA consensus site near the amino terminus of the KCNQ2 polypeptide (10). Mutations causing BNFC were found to cause only slight reductions in current compared with wild-type controls, suggesting that small differences in the activity of these KCNQ channels in vivo might be sufficient to cause epilepsy (10). When KCNQ2 and KCNQ3 cDNAs were coexpressed in oocytes, currents were observed that exhibited kinetic and voltagedependent properties similar to M-currents...

Distinct enzyme combinations in AKAP signalling complexes permit functional diversity

“…For example, we show that downregulation of AMPA currents requires anchored PP2B, but we are unable to demonstrate a role for the same enzyme in the repression of muscarine-dependent M-current inhibition. This observation is supported by a detailed biochemical analysis showing that PP2B is not present in KCNQ2-AKAP79 complexes that are isolated from mammalian neurons 28 . It is, therefore, reasonable to propose that parallel AKAP signalling complexes may recruit different combinations of signalling enzymes.…”

Identification by mass spectrometry and functional characterization of two phosphorylation sites of KCNQ2/KCNQ3 channels

“…Guinea pig anti-KCNQ2 antibodies (17) were crosslinked to Protein A beads (Dynal) using dimethyl pimelimidate (Sigma). Human embryonic kidney 293 (HEK) cells, maintained in DMEM with 10% FBS at 37°C in 5% CO 2 , were transfected with KCNQ2 and KCNQ3 cDNAs in pcDNA3 in a 1:1 ratio by using FuGENE-6 (Roche), harvested 2 days later in lysis buffer [150 mM NaCl͞50 mM Tris⅐HCl͞1% Triton X-100͞50 mM NaF͞10 mM sodium pyrophosphate͞1ϫ Complete protease inhibitor mixture (Roche)͞2 mM sodium orthovanadate͞100 nM calyculin A͞1 mM DTT], kept on ice for 30 min, and centrifuged at 4°C at 20,800 ϫ g for 30 min.…”

“…Beads were washed four times with lysis buffer, resuspended in SDS sample buffer (Pierce), and heated for 2 min to 85-90°C. After electrophoresis on 7% SDS polyacrylamide gels, proteins were either stained with SYPRO Ruby protein gel stain (Molecular Probes), or subjected to electrotransfer and Western blotting (3,17,24). Bands at Ϸ89 (KCNQ2) and Ϸ97 kDa (KCNQ3) were excised, minced, washed, dried on a centrifugal concentrator, rehydrated on ice for 10 min in 10 l of 5 ng͞ l sequencing grade trypsin (Promega) in 25 mM NH 4 HCO 3 , covered with an additional 25 l NH 4 HCO 3 solution and incubated overnight at 37°C.…”

“…PIP 2 depletion is the likely mechanism mediating the classic muscarinic inhibition of M-current (14-16), but other mechanisms, including phosphorylation, may contribute to channel modulation by other neurotransmitters (16). Indeed, KCNQ1 and KCNQ2 subunits associate with a variety of kinases and phosphatases via A-kinase anchor proteins (17)(18)(19)(20), and mutational studies implicate KCNQ2͞KCNQ3 phosphorylation sites for channel regulation by protein kinase A (PKA), protein kinase C (PKC), and src tyrosine kinase (10, 19, 21, 22). KCNQ2 and KCNQ3 subunits possess large intracellular domains containing many additional potential phosphorylation sites (Fig.…”

Down-regulation of BK channel expression in the pilocarpine model of temporal lobe epilepsy

“…1a,*) and in the lateral portion of the stratum lacunosum/moleculare near the CA2-CA3 area (Fig. 1a,2a) indicating that BK channels may be localized on perforant path axon terminals ( Fig.1, 2b) Because the granule cell dendrites extend radially through both inner and outer molecular layers, while the mossy cell axons and terminals are restricted to the middle layer, the BK staining in the inner molecular layer most likely represents presynaptic localization (Cooper et al, 2000). Less intense and widespread staining was detected in the stratum radiatum and stratum oriens in CA2-CA3 (Fig.1 2a).…”