Ciria C. Hernandez

Regulation of neural KCNQ channels: signalling pathways, structural motifs and functional implications

Neural M‐type (KCNQ/Kv7) K+ channels control somatic excitability, bursting and neurotransmitter release throughout the nervous system. Their activity is regulated by multiple signalling pathways. In superior cervical ganglion sympathetic neurons, muscarinic M1, angiotensin II AT1, bradykinin B2 and purinergic P2Y agonists suppress M current (IM). Probes of PLC activity show agonists of all four receptors to induce robust PIP2 hydrolysis. We have grouped these receptors into two related modes of action. One mode involves depletion of phosphatidylinositol 4,5‐bisphosphate (PIP2) in the membrane, whose interaction with the channels is thought necessary for their function. The other involves IP3‐mediated intracellular Ca2+ signals that stimulate PIP2 synthesis, preventing its depletion, and suppress IM via calmodulin. Carbon‐fibre amperometry can evaluate the effect of M channel activity on release of neurotransmitter. Consistent with the dominant role of M current in control of neuronal discharge, M channel openers, or blockers, reduced or augmented the evoked release of noradrenaline neurotransmitter from superior cervical ganglion (SCG) neurons, respectively. We seek to localize the subdomains on the channels critical to their regulation by PIP2. Based on single‐channel recordings from chimeras between high‐PIP2 affinity KCNQ3 and low‐PIP2 affinity KCNQ4 channels, we focus on a 57‐residue domain within the carboxy‐terminus that is a possible PIP2 binding site. Homology modelling of this domain using the published structure of IRK1 channels as a template predicts a structure very similar to an analogous region in IRK1 channels, and shows a cluster of basic residues in the KCNQ2 domain to correspond to those implicated in PIP2 regulation of Kir channels. We discuss some important issues dealing with these topics.

Oxidative modification of M‐type K+ channels as a mechanism of cytoprotective neuronal silencing

Voltage‐gated K+ channels of the Kv7 family underlie the neuronal M current that regulates action potential firing. Suppression of M current increases excitability and its enhancement can silence neurons. We here show that three of five Kv7 channels undergo strong enhancement of their activity by oxidative modification induced by physiological concentrations of hydrogen peroxide. A triple cysteine pocket in the channel S2–S3 linker is critical for this effect. Oxidation‐induced enhancement of M current produced a hyperpolarization and a dramatic reduction of action potential firing frequency in rat sympathetic neurons. As hydrogen peroxide is robustly produced during hypoxia‐induced oxidative stress, we used an oxygen/glucose deprivation neurodegeneration model that showed neuronal death to be severely accelerated by M current blockade. Such blockade had no effect on survival of normoxic neurons. This work describes a novel pathway of M‐channel regulation and suggests a role for M channels in protective neuronal silencing during oxidative stress.

A carboxy-terminal inter-helix linker as the site of phosphatidylinositol 4,5-bisphosphate action on Kv7 (M-type) K+ channels

The regulation of M-type (KCNQ [Kv7]) K+ channels by phosphatidylinositol 4,5-bisphosphate (PIP2) has perhaps the best correspondence to physiological signaling, but the site of action and structural motif of PIP2 on these channels have not been established. Using single-channel recordings of chimeras of Kv7.3 and 7.4 channels with highly differential PIP2 sensitivities, we localized a carboxy-terminal inter-helix linker as the primary site of PIP2 action. Point mutants within this linker in Kv7.2 and Kv7.3 identified a conserved cluster of basic residues that interact with the lipid using electrostatic and hydrogen bonds. Homology modeling of this putative PIP2-binding linker in Kv7.2 and Kv7.3 using the solved structure of Kir2.1 and Kir3.1 channels as templates predicts a structure of Kv7.2 and 7.3 very similar to the Kir channels, and to the seven-β-sheet barrel motif common to other PIP2-binding domains. Phosphoinositide-docking simulations predict affinities and interaction energies in accord with the experimental data, and furthermore indicate that the precise identity of residues in the interacting pocket alter channel–PIP2 interactions not only by altering electrostatic energies, but also by allosterically shifting the structure of the lipid-binding surface. The results are likely to shed light on the general structural mechanisms of phosphoinositide regulation of ion channels.

Ca2+/Calmodulin Disrupts AKAP79/150 Interactions with KCNQ (M-Type) K+ Channels

M-type channels are localized to neuronal, cardiovascular, and epithelial tissues, where they play critical roles in control of excitability and K+ transport, and are regulated by numerous receptors via Gq/11-mediated signals. One pathway shown for KCNQ2 and muscarinic receptors uses PKC, recruited to the channels by A-kinase anchoring protein (AKAP)79/150. As M-type channels can be variously composed of KCNQ1-5 subunits, and M current is known to be regulated by Ca2+/calmodulin (CaM) and PIP2, we probed the generality of AKAP79/150 actions among KCNQ1-5 channels, and the influence of Ca2+/CaM and PIP2 on AKAP79/150 actions. We first examined which KCNQ subunits are targeted by AKAP79 in Chinese hamster ovary (CHO) cells heterologously expressing KCNQ1-5 subunits and AKAP79, using fluorescence resonance energy transfer (FRET) under total internal reflection fluorescence (TIRF) microscopy, and patch-clamp analysis. Donor-dequenching FRET between CFP-tagged KCNQ1-5 and YFP-tagged AKAP79 revealed association of KCNQ2-5, but not KCNQ1, with AKAP79. In parallel with these results, CHO cells stably expressing M1 receptors studied under perforated patch-clamp showed cotransfection of AKAP79 to “sensitize” KCNQ2/3 heteromers and KCNQ2-5, but not KCNQ1, homomers to muscarinic inhibition, manifested by shifts in the dose–response relations to lower concentrations. The effect on KCNQ4 was abolished by the T553A mutation of the putative PKC phosphorylation site. We then probed the role of CaM and PIP2 in these AKAP79 actions. TIRF/FRET experiments revealed cotransfection of wild-type, but not dominant-negative (DN), CaM that cannot bind Ca2+, to disrupt the interaction of YFP-tagged AKAP791-153 with CFP-tagged KCNQ2-5. Tonic depletion of PIP2 by cotransfection of a PIP2 phosphatase had no effect, and sudden depletion of PIP2 did not delocalize GFP-tagged AKAP79 from the membrane. Finally, patch-clamp experiments showed cotransfection of wild-type, but not DN, CaM to prevent the AKAP79-mediated sensitization of KCNQ2/3 heteromers to muscarinic inhibition. Thus, AKAP79 acts on KCNQ2-5, but not KCNQ1-containing channels, with effects disrupted by calcified CaM, but not by PIP2 depletion.

Homomeric and heteromeric assembly of KCNQ (Kv7) K+ channels assayed by total internal reflection fluorescence/fluorescence resonance energy transfer and patch clamp analysis.

M-type K+ channels, consisting of KCNQ1–5 (Kv7.1–7.5) subunits, form a variety of homomeric and heteromeric channels. Whereas all the subunits can assemble into homomeric channels, the ability of the subunits to assemble into heteromultimers is highly variable. KCNQ3 is widely thought to co-assemble with several other KCNQ subtypes, whereas KCNQ1 and KCNQ2 do not. However, the existence of other subunit assemblies is not well studied. To systematically explore the heteromeric assembly of KCNQ channels in individual living cells, we performed fluorescence resonance energy transfer (FRET) between cyan fluorescent protein- and yellow fluorescent protein-tagged KCNQ subunits expressed in Chinese hamster ovary cells under total internal reflection fluorescence microscopy in which excitation light only penetrates several hundred nanometers into the cell, thus isolating membrane events. We found significant FRET between homomeric subunits as expected from their functional expression in heterologous expression systems. Also as expected from previous work, robust FRET was observed between KCNQ2 and KCNQ3. KCNQ3 and KCNQ4 also showed substantial FRET as did KCNQ4 and KCNQ5. To determine functional assembly of KCNQ4/KCNQ5 heteromers, we performed two types of experiments. In the first, we constructed a mutant tetraethylammonium ion-sensitive KCNQ4 subunit and tested its assembly with KCNQ5 by patch clamp analysis of the tetraethylammonium ion sensitivity of the resulting current; however, those data were not conclusive. In the second, we co-expressed a KCNQ4 (G285S) pore mutant with KCNQ5 and found the former to act as a dominant negative, suggesting co-assembly of the two types of subunits. These data confirm that among the allowed assembly conformations are KCNQ3/4 and KCNQ4/5 heteromers.

Affinity for phosphatidylinositol 4,5-bisphosphate determines muscarinic agonist sensitivity of Kv7 K+ channels

Kv7 K+-channel subunits differ in their apparent affinity for PIP2 and are differentially expressed in nerve, muscle, and epithelia in accord with their physiological roles in those tissues. To investigate how PIP2 affinity affects the response to physiological stimuli such as receptor stimulation, we exposed homomeric and heteromeric Kv7.2, 7.3, and 7.4 channels to a range of concentrations of the muscarinic receptor agonist oxotremorine-M (oxo-M) in a heterologous expression system. Activation of M1 receptors by oxo-M leads to PIP2 depletion through Gq and phospholipase C (PLC). Chinese hamster ovary cells were transiently transfected with Kv7 subunits and M1 receptors and studied under perforated-patch voltage clamp. For Kv7.2/7.3 heteromers, the EC50 for current suppression was 0.44 ± 0.08 µM, and the maximal inhibition (Inhibmax) was 74 ± 3% (n = 5–7). When tonic PIP2 abundance was increased by overexpression of PIP 5-kinase, the EC50 was shifted threefold to the right (1.2 ± 0.1 µM), but without a significant change in Inhibmax (73 ± 4%, n = 5). To investigate the muscarinic sensitivity of Kv7.3 homomers, we used the A315T pore mutant (Kv7.3T) that increases whole-cell currents by 30-fold without any change in apparent PIP2 affinity. Kv7.3T currents had a slightly right-shifted EC50 as compared with Kv7.2/7.3 heteromers (1.0 ± 0.8 µM) and a strongly reduced Inhibmax (39 ± 3%). In contrast, the dose–response curve of homomeric Kv7.4 channels was shifted considerably to the left (66 ± 8 nM), and Inhibmax was slightly increased (81 ± 6%, n = 3–4). We then studied several Kv7.2 mutants with altered apparent affinities for PIP2 by coexpressing them with Kv7.3T subunits to boost current amplitudes. For the lower affinity (Kv7.2 (R463Q)/Kv7.3T) or higher affinity (Kv7.2 (R463E)/Kv7.3T) channels, the EC50 and Inhibmax were similar to Kv7.4 or Kv7.3T homomers (0.12 ± 0.08 µM and 79 ± 6% [n = 3–4] and 0.58 ± 0.07 µM and 27 ± 3% [n = 3–4], respectively). The very low-affinity Kv7.2 (R452E, R459E, and R461E) triple mutant was also coexpressed with Kv7.3T. The resulting heteromer displayed a very low EC50 for inhibition (32 ± 8 nM) and a slightly increased Inhibmax (83 ± 3%, n = 3–4). We then constructed a cellular model that incorporates PLC activation by oxo-M, PIP2 hydrolysis, PIP2 binding to Kv7-channel subunits, and K+ current through Kv7 tetramers. We were able to fully reproduce our data and extract a consistent set of PIP2 affinities.

Atypical Gating Of M-Type Potassium Channels Conferred by Mutations in Uncharged Residues in the S4 Region of KCNQ2 Causing Benign Familial Neonatal Convulsions

Heteromeric assembly of KCNQ2 and KCNQ3 subunits underlie the M-current (IKM), a slowly activating and noninactivating neuronal K+ current. Mutations in KCNQ2 and KCNQ3 genes cause benign familial neonatal convulsions (BFNCs), a rare autosomal-dominant epilepsy of the newborn. In the present study, we describe the identification of a novel KCNQ2 heterozygous mutation (c587t) in a BFNC-affected family, leading to an alanine to valine substitution at amino acid position 196 located at the N-terminal end of the voltage-sensing S4 domain. The consequences on KCNQ2 subunit function prompted by the A196V substitution, as well as by the A196V/L197P mutation previously described in another BFNC-affected family, were investigated by macroscopic and single-channel current measurements in CHO cells transiently transfected with wild-type and mutant subunits. When compared with KCNQ2 channels, homomeric KCNQ2 A196V or A196V/L197P channels showed a 20 mV rightward shift in their activation voltage dependence, with no concomitant change in maximal open probability or single-channel conductance. Furthermore, current activation kinetics of KCNQ2 A196V channels displayed an unusual dependence on the conditioning prepulse voltage, being markedly slower when preceded by prepulses to more depolarized potentials. Heteromeric channels formed by KCNQ2 A196V and KCNQ3 subunits displayed gating changes similar to those of KCNQ2 A196V homomeric channels. Collectively, these results reveal a novel role for noncharged residues in the N-terminal end of S4 in controlling gating of IKM and suggest that gating changes caused by mutations at these residues may decrease IKM function, thus causing neuronal hyperexcitability, ultimately leading to neonatal convulsions.

GABRB3 mutation, G32R, associated with childhood absence epilepsy alters α1β3γ2L γ-aminobutyric acid type A (GABAA) receptor expression and channel gating.

Background: A GABRB3 mutation has been associated with childhood absence epilepsy and β3 subunit hyperglycosylation. Results: The mutation altered subunit expression and reduced GABAA receptor function independent of N-glycosylation. Conclusion: The mutation introduced a charged residue predicted to alter subunit interactions. Significance: The distal N terminus of GABAA receptor subunits may play an unexpected role in receptor assembly and channel gating. A GABAA receptor β3 subunit mutation, G32R, has been associated with childhood absence epilepsy. We evaluated the possibility that this mutation, which is located adjacent to the most N-terminal of three β3 subunit N-glycosylation sites, might reduce GABAergic inhibition by increasing glycosylation of β3 subunits. The mutation had three major effects on GABAA receptors. First, coexpression of β3(G32R) subunits with α1 or α3 and γ2L subunits in HEK293T cells reduced surface expression of γ2L subunits and increased surface expression of β3 subunits, suggesting a partial shift from ternary αβ3γ2L receptors to binary αβ3 and homomeric β3 receptors. Second, β3(G32R) subunits were more likely than β3 subunits to be N-glycosylated at Asn-33, but increases in glycosylation were not responsible for changes in subunit surface expression. Rather, both phenomena could be attributed to the presence of a basic residue at position 32. Finally, α1β3(G32R)γ2L receptors had significantly reduced macroscopic current density. This reduction could not be explained fully by changes in subunit expression levels (because γ2L levels decreased only slightly) or glycosylation (because reduction persisted in the absence of glycosylation at Asn-33). Single channel recording revealed that α1β3(G32R)γ2L receptors had impaired gating with shorter mean open time. Homology modeling indicated that the mutation altered salt bridges at subunit interfaces, including regions important for subunit oligomerization. Our results suggest both a mechanism for mutation-induced hyperexcitability and a novel role for the β3 subunit N-terminal α-helix in receptor assembly and gating.

Human chagasic IgGs bind to cardiac muscarinic receptors and impair L-type Ca2+ currents

OBJECTIVES Antibodies against cardiac G protein-coupled receptors have been reported in sera from chronic chagasic patients (CChP) and other non-parasitic cardiomyopathies, but the effects and underlying mechanism of interaction between these antibodies and heart cells are not fully established. To address this point, binding of antibodies purified from sera of CChP patients and normal blood donors (NBD) to cardiac muscarinic acetylcholine receptors (mAChR) and their effect on L-type Ca(2+) currents were examined. METHODS AND RESULTS Saturation [3H]NMS binding experiments with porcine atrial membranes showed that B(max) in the presence of CChP-immunoglobulin G (IgG) decreased from 280.2+/-16.08 fmol/mg (control) to 91.00+/-5.98 fmol/mg, with no apparent change in K(D), while NBD-IgG did not significantly alter these parameters. At the single channel level, CChP-IgG decreased both the fast and slow mean open times and P(o) (from 0.074+/-0.023 to 0.025+/-0.007) without changes in single channel conductance. I/V plots of isoproterenol-stimulated whole-cell L-type Ca(2+) currents (I(Ca)) from rabbit ventricular cardiomyocytes showed a significant reduction in peak I(Ca) during perfusion with CChP-IgG (at 0 mV: from 10.61+/-2.97 to 8.45+/-2.54 pA/pF). NBD-IgGs had no effect on I(Ca). A CChP-IgG purified against a peptide corresponding to the second extracellular loop of the M(2) receptor also impaired L-type Ca(2+) currents. All effects of CChP-IgG were blocked by atropine. CONCLUSIONS Our results show that antibodies from CChP bind to mAChR in a non-competitive manner and are able to activate the receptor in an agonist-like form resulting in L-type Ca(2+) current inhibition.

Glycosylation of β2 Subunits Regulates GABAA Receptor Biogenesis and Channel Gating

γ-Aminobutyric acid type A (GABAA) receptors are heteropentameric glycoproteins. Based on consensus sequences, the GABAA receptor β2 subunit contains three potential N-linked glycosylation sites, Asn-32, Asn-104, and Asn-173. Homology modeling indicates that Asn-32 and Asn-104 are located before the α1 helix and in loop L3, respectively, near the top of the subunit-subunit interface on the minus side, and that Asn-173 is located in the Cys-loop near the bottom of the subunit N-terminal domain. Using site-directed mutagenesis, we demonstrated that all predicted β2 subunit glycosylation sites were glycosylated in transfected HEK293T cells. Glycosylation of each site, however, produced specific changes in α1β2 receptor surface expression and function. Although glycosylation of Asn-173 in the Cys-loop was important for stability of β2 subunits when expressed alone, results obtained with flow cytometry, brefeldin A treatment, and endo-β-N-acetylglucosaminidase H digestion suggested that glycosylation of Asn-104 was required for efficient α1β2 receptor assembly and/or stability in the endoplasmic reticulum. Patch clamp recording revealed that mutation of each site to prevent glycosylation decreased peak α1β2 receptor current amplitudes and altered the gating properties of α1β2 receptor channels by reducing mean open time due to a reduction in the proportion of long open states. In addition to functional heterogeneity, endo-β-N-acetylglucosaminidase H digestion and glycomic profiling revealed that surface β2 subunit N-glycans at Asn-173 were high mannose forms that were different from those of Asn-32 and N104. Using a homology model of the pentameric extracellular domain of α1β2 channel, we propose mechanisms for regulation of GABAA receptors by glycosylation.