Pathways modulating neural KCNQ/M (Kv7) potassium channels

K(+) channels play a crucial role in regulating the excitability of neurons. Many K(+) channels are, in turn, regulated by neurotransmitters. One of the first neurotransmitter-regulated channels to be identified, some 25 years ago, was the M channel. This was categorized as such because its activity was inhibited through stimulation of muscarinic acetylcholine receptors. M channels are now known to be composed of subunits of the Kv7 (KCNQ) K(+) channel family. However, until recently, the link between the receptors and the channels has remained elusive. Here, we summarize recent developments that have begun to clarify this link and discuss their implications for physiology and medicine.     

Role of calmodulin in neuronal Kv7/KCNQ potassium channels and epilepsy

Neuronal Kv7/KCNQ channels are critical regulators of neuronal excitability since they potently suppress repetitive firing of action potentials. These voltage-dependent potassium channels are composed mostly of Kv7.2 / KCNQ2 and KvT.3 / KCNQ3 subunits that show overlapping distribution throughout the brain and in the peripheral nervous system. They are also called ＇M-channels＇ since their inhibition by muscarinic agonists leads to a profound increase in action potential firing. Consistent with their ability to suppress seizures and attenuate chronic inflammatory and neuropathic pain, mutations in the KCNQ2 and KCNQ3 genes are associated with benign familial neonatal convulsions, a dominantly-inherited epilepsy in infancy. Recently, de novo mutations in the KCNQ2 gene have been linked to early onset epileptic encephalopathy. Notably, some of these mutations are clustered in a region of the intracellular cytoplasmic tail of Kv7.2 that interacts with a ubiquitous calcium sensor, calmodulin. In this review, we highlight the recent advances in understanding the role of calmodulin in modulating physiological function of neuronal Kv7 channels including their biophysical properties, assembly, and trafficking. We also summarize recent studies that have investigated functional impact of epilepsy-associated mutations localized to the calmodulin binding domains of Kv7.2.     

Skeletal muscle Kv7 (KCNQ) channels in myoblast differentiation and proliferation

Voltage-dependent K⁺ channels (Kv) are involved in myocyte proliferation and differentiation by triggering changes in membrane potential and regulating cell volume. Since Kv7 channels may participate in these events, the purpose of this study was to investigate whether skeletal muscle Kv7.1 and Kv7.5 were involved during proliferation and myogenesis. Here we report that, while myotube formation did not regulate Kv7 channels, Kv7.5 was up-regulated during cell cycle progression. Although, Kv7.1 mRNA also increased during the G₁-phase, pharmacological evidence mainly involves Kv7.5 in myoblast growth. Our results indicate that the cell cycle-dependent expression of Kv7.5 is involved in skeletal muscle cell proliferation.     

Expression mechanisms of long-term potentiation in the hippocampus

We have taken a number of different experimental approaches to address whether long-term potentiation (LTP) in hippocampal CA1 pyramidal cells is due primarily to presynaptic or postsynaptic modifications. Examination of miniature EPSCs or EPSCs evoked using minimal stimulation indicate that quantal size increasing during LTP. The conversion of silent to functional synapses may contribute to the LTP-induced changes in mEPSC frequency and failure rate that previously have been attributed to an increase in the probability if transmitter release.     

Presynaptic change associated with long-term potentiation in hippocampus

Long-term potentiation of the hippocampal response to repeated stimulation of rat entorhinal cortex occured concomitantly with a decrease in the excitability of presynaptic terminals. It is, therefore, possible that the long-term potentiation is caused, at least partly, by an enhancement of presynaptic efficacy.     

Made for "anchorin": Kv7.2/7.3 (KCNQ2/KCNQ3) channels and the modulation of neuronal excitability in vertebrate axons

Kv7.2 and Kv7.3 (encoded by KCNQ2 and KCNQ3) are homologous subunits forming a widely expressed neuronal voltage-gated K(+) (Kv) channel. Hypomorphic mutations in either KCNQ2 or KCNQ3 cause a highly penetrant, though transient, human phenotype-epilepsy during the first months of life. Some KCNQ2 mutations also cause involuntary muscle rippling, or myokymia, which is indicative of motoneuron axon hyperexcitability. Kv7.2 and Kv7.3 are concentrated at axonal initial segments (AISs), and at nodes of Ranvier in the central and peripheral nervous system. Kv7.2 and Kv7.3 share a novel ～80 residue C-terminal domain bearing an "anchor" motif, which interacts with ankyrin-G and is required for channel AIS (and likely, nodal) localization. This domain includes the sequence IAEGES/TDTD, which is analogous (not homologous) to the ankyrin-G interaction motif of voltage-gated Na(+) (Na(V)) channels. The KCNQ subfamily is evolutionarily ancient, with two genes (KCNQ1 and KCNQ5) persisting as orthologues in extant bilaterian animals from worm to man. However, KCNQ2 and KCNQ3 arose much more recently, in the interval between the divergence of extant jawless and jawed vertebrates. This is precisely the interval during which myelin and saltatory conduction evolved. The natural selection for KCNQ2 and KCNQ3 appears to hinge on these subunits' unique ability to be coordinately localized with Na(V) channels by ankyrin-G, and the resulting enhancement in the reliability of neuronal excitability.     

A model of the mechanisms of long-term potentiation in the hippocampus

Long-Term Potentiation (LTP) in the hippocampus has been considered to be a phenomenon closely related to learning and memory in the brain. In this paper, an integrated model of LTP is constructed based on hypotheses about both the mechanism of LTP induction and that of LTP maintenance, that is, the NMDA-receptor channel, protein phosphorylation and protein turnover. The validity of the model is discussed based on the results of computer simulations.     

Two mechanisms of K(+)-dependent potentiation in Kv2.1 potassium channels

Elevation of external [K(+)] potentiates outward K(+) current through several voltage-gated K(+) channels. This increase in current magnitude is paradoxical in that it occurs despite a significant decrease in driving force. We have investigated the mechanisms involved in K(+)-dependent current potentiation in the Kv2.1 K(+) channel. With holding potentials of -120 to -150 mV, which completely removed channels from the voltage-sensitive inactivated state, elevation of external [K(+)] up to 10 mM produced a concentration-dependent increase in outward current magnitude. In the absence of inactivation, currents were maximally potentiated by 38%. At more positive holding potentials, which produced steady-state inactivation, K(+)-dependent potentiation was enhanced. The additional K(+)-dependent potentiation (above 38%) at more positive holding potentials was precisely equal to a K(+)-dependent reduction in steady-state inactivation. Mutation of two lysine residues in the outer vestibule of Kv2.1 (K356 and K382), to smaller, uncharged residues (glycine and valine, respectively), completely abolished K(+)-dependent potentiation that was not associated with inactivation. These mutations did not influence steady-state inactivation or the K(+)-dependent potentiation due to reduction in steady-state inactivation. These results demonstrate that K(+)-dependent potentiation can be completely accounted for by two independent mechanisms: one that involved the outer vestibule lysines and one that involved K(+)-dependent removal of channels from the inactivated state. Previous studies demonstrated that the outer vestibule of Kv2.1 can be in at least two conformations, depending on the occupancy of the selectivity filter by K(+) (Immke, D., M. Wood, L. Kiss, and S. J. Korn. 1999. J. Gen. Physiol. 113:819-836; Immke, D., and S. J. Korn. 2000. J. Gen. Physiol. 115:509-518). This change in conformation was functionally defined by a change in TEA sensitivity. Similar to the K(+)-dependent change in TEA sensitivity, the lysine-dependent potentiation depended primarily (>90%) on Lys-356 and was enhanced by lowering initial K(+) occupancy of the pore. Furthermore, the K(+)-dependent changes in current magnitude and TEA sensitivity were highly correlated. These results suggest that the previously described K(+)-dependent change in outer vestibule conformation underlies the lysine-sensitive, K(+)-dependent potentiation mechanism.     

Imaging spatio-temporal patterns of long-term potentiation in mouse hippocampus

Spatio-temporal patterns of neuronal activity before and after the induction of long-term potentiation in mouse hippocampal slices were studied using a real-time high-resolution optical recording system. After staining the slices with voltage-sensitive dye, transmitted light images and extracellular field potentials were recorded in response to stimuli applied to CA1 stratum radiatum. Optical and electrical signals in response to single test pulses were enhanced for at least 30 minutes after brief high-frequency stimulation at the same site. In two-pathway experiments, potentiation was restricted to the tetanized pathway. The optical signals demonstrated that both the amplitude and area of the synaptic response were increased, in patterns not predictable from the initial, pretetanus, pattern of activation. Optical signals will be useful for investigating spatio-temporal patterns of synaptic enhancement underlying information storage in the brain.     

A persistent postsynaptic modification mediates long-term potentiation in the hippocampus

Long-term potentiation (LTP) is a long-lasting enhancement of synaptic transmission that can be induced by brief repetitive stimulation of excitatory pathways in the hippocampus. One of the most controversial points is whether the process underlying the enhanced synaptic transmission occurs pre- or postsynaptically. To examine this question, we have taken advantage of the novel physiological properties of excitatory synaptic transmission in the CA1 region of the hippocampus. Synaptically released glutamate activates both NMDA and non-NMDA receptors on pyramidal cells, resulting in an excitatory postsynaptic potential (EPSP) with two distinct components. A selective increase in the non-NMDA component of the EPSP was observed with LTP. This result suggests that the enhancement of synaptic transmission during LTP is caused by an increased sensitivity of the postsynaptic neuron to synaptically released glutamate.     

Mechanisms underlying induction and maintenance of long-term potentiation in the hippocampus

Long-term potentiation (LTP) in the hippocampus is accompanied by a number of changes on both sides of the synapse. It is now generally considered that the trigger for initiating LTP is the entry of calcium into the postsynaptic area through the NMDA-associated channel while the mechanism(s) underlying the maintenance of LTP are less well understood and probably involve contributions from both sides of the synapse.     

Effects of KCNQ2 Gene Truncation on M-Type Kv7 Potassium Currents

The KCNQ2 gene product, Kv7.2, is a subunit of the M-channel, a low-threshold voltage-gated K⁺ channel that regulates mammalian and human neuronal excitability. Spontaneous mutations one of the KCNQ2 genes cause disorders of neural excitability such as Benign Familial Neonatal Seizures. However there appear to be no reports in which both human KCNQ2 genes are mutated. We therefore asked what happens to M-channel function when both KCNQ2 genes are disrupted. We addressed this using sympathetic neurons isolated from mice in which the KCNQ2 gene was truncated at a position corresponding to the second transmembrane domain of the Kv7.2 protein. Since homozygote KCNQ2−/− mice die postnatally, experiments were largely restricted to neurons from late embryos. Quantitative PCR revealed an absence of KCNQ2 mRNA in ganglia from KCNQ2−/− embryos but 100–120% increase of KCNQ3 and KCNQ5 mRNAs; KCNQ2+/− ganglia showed ∼30% less KCNQ2 mRNA than wild-type (+/+) ganglia but 40–50% more KCNQ3 and KCNQ5 mRNA. Neurons from KCNQ2−/− embryos showed a complete absence of M-current, even after applying the Kv7 channel enhancer, retigabine. Neurons from heterozygote KCNQ2+/− embryos had ∼60% reduced M-current. In contrast, M-currents in neurons from adult KCNQ2+/− mice were no smaller than those in neurons from wild-type mice. Measurements of tetraethylammonium block did not indicate an increased expression of Kv7.5-containing subunits, implying a compensatory increase in Kv7.2 expression from the remaining KCNQ2 gene. We conclude that mouse embryonic M-channels have an absolute requirement for Kv7.2 subunits for functionality, that the reduced M-channel activity in heterozygote KCNQ2+/− mouse embryos results primarily from a gene-dosage effect, and that there is a compensatory increase in Kv7.2 expression in adult mice.     

Posttetanic potentiation and presynaptically induced long-term potentiation at the mossy fiber synapse in rat hippocampus

A form of long-term potentiation (LTP) is induced at the mossy fiber (MF) synapse in the hippocampus by highfrequency presynaptic stimulation (HFS). It is generally accepted that induction of this form of LTP (MF LTP) does not depend on postsynaptic Ca²⁺ current gated by N-methyl-D -aspartate receptors, but it has remained controversial whether induction depends on postsynaptic depolarization and voltage-gated entry of Ca²⁺. There are also contradictory data on the time course of both LTP and post-tetanic potentiation (PTP), a shorter duration form of potentiation observed at MF synapses immediately following HFS. It has been proposed that some of these differences in results may have arisen because of difficulties in isolating monosynaptic responses to MF input. In the present study, whole cell recording was used to observe excitatory postsynaptic currents (EPSCs) elicited in CA3 pyramidal cells by input from MFs. Postsynaptic cells were dialyzed with 1,2-bis(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and F^? to inhibit postsynaptic mechanisms that required Ca²⁺, cells were under voltage clamp during HFS, and conditions were selected to minimize the likelihood of polysynaptic contamination. Under these conditions, HFS nevertheless induced robust LTP (mean magnitude, 62%). The possibility that EPSCs were contaminated by polysynaptic components was investigated by exposing the slices to a suppressing medium (one that partially blocked neurotransmission). EPSC waveforms did not change shape during suppression, indicating that contamination was absent. The LTP observed always was accompanied by prominent PTP that lasted through the first 5 to 15 min following HFS (mean decay time constant, 3.2 min). Induction of this LTP was not cooperative; there was no relationship between the size of responses and the magnitude of the LTP induced. LTP magnitude also was unrelated to the extent to which postsynaptic cells depolarized during HFS. These results show that high rates of presynaptic MF activity elicit robust LTP whether or not there is accompanying postsynaptic depolarization or increase in the concentration of postsynaptic Ca²⁺. High-frequency MF activity also results in a PTP that is unusually large and long. © 1995 John Wiley & Sons, Inc.     

Short- and long-term potentiation in afferent pathways of the neonatal rabbit hippocampus

Vocal potentials were recorded in hippocampal area CA₁ and dentate fascia in unanesthetized rabbits aged from 1 to 50 days during stimulation of Schaffer's collaterals and the perforant path, respectively, with paired (interval 15–100 msec) and repetitive (20–40 Hz for 3–5 sec) electric pulses. Short-term potentiation of focal potentials during paired stimulation and post-tetanic potentiation lasting from a few minutes to 3 h were shown to be reproduced in the hippocampus from the first days after birth, whereas in the dentate fascia, which matures later, reproduction began on the 8th–10th day, when neurons first began to respond to stimulation of the corresponding afferent pathways.     

K(V)7/KCNQ channels are functionally expressed in oligodendrocyte progenitor cells

Background

K_V7/KCNQ channels are widely expressed in neurons and they have multiple important functions, including control of excitability, spike afterpotentials, adaptation, and theta resonance. Mutations in KCNQ genes have been demonstrated to associate with human neurological pathologies. However, little is known about whether K_V7/KCNQ channels are expressed in oligodendrocyte lineage cells (OLCs) and what their functions in OLCs.

Methods and Findings

In this study, we characterized K_V7/KCNQ channels expression in rat primary cultured OLCs by RT-PCR, immunostaining and electrophysiology. KCNQ2-5 mRNAs existed in all three developmental stages of rat primary cultured OLCs. K_V7/KCNQ proteins were also detected in oligodendrocyte progenitor cells (OPCs, early developmental stages of OLCs) of rat primary cultures and cortex slices. Voltage-clamp recording revealed that the I_M antagonist XE991 significantly reduced K_V7/KCNQ channel current (I_K(Q)) in OPCs but not in differentiated oligodendrocytes. In addition, inhibition of K_V7/KCNQ channels promoted OPCs motility in vitro.

Conclusions

These findings showed that K_V7/KCNQ channels were functionally expressed in rat primary cultured OLCs and might play an important role in OPCs functioning in physiological or pathological conditions.     

Suppression of associative long-term potentiation in the hippocampus after tetanization of reinforcing afferents

Field potentials (FP) induced in area C1 by gentle orthodromic stimulation were recorded in murine hippocampal slices and associative long-term potentiation (ALTP) produced by C2 tetanization associated with intensive tetanization of another group of fibers (C1) was investigated. A comparison was made between the effects of additional C1 tetanization produced at 50–300 msec before and after combined tetanization of both afferents. Where these intervals measured 50–200 msec, preliminary tetanization of C1 suppressed ALTP (rise in FP amplitude: 10.4±5.2%) in comparison with the regimen whereby additional C1 tetanization came later (giving a rise of 32.4±5.3%); no significant difference was noted at an interval of 300 msec. The three possible reasons for ALTP suppression are discussed, namely: inactivation of "fast" calcium channels, post-activation hyperpolarization of postsynaptic neurons, and synaptic inhibition. The ALTP suppression mechanism is thought to resemble that underlying the relative inefficacy of "reversible" combinations in the shaping of behavioral conditioned reflexes.Institute for Brain Research, Academy of Medical Sciences of the USSR, Moscow. Institute of Chemical Physics, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 21, No. 5, pp. 636–643, September–October, 1989.     

Kv1.3/Kv1.5 heteromeric channels compromise pharmacological responses in macrophages

Voltage-dependent K(+) (Kv) channels are involved in the immune response. Kv1.3 is highly expressed in activated macrophages and T-effector memory cells of autoimmune disease patients. Macrophages are actively involved in T-cell activation by cytokine production and antigen presentation. However, unlike T-cells, macrophages express Kv1.5, which is resistant to Kv1.3-drugs. We demonstrate that mononuclear phagocytes express different Kv1.3/Kv1.5 ratios, leading to biophysically and pharmacologically distinct channels. Therefore, Kv1.3-based treatments to alter physiological responses, such as proliferation and activation, are impaired by Kv1.5 expression. The presence of Kv1.5 in the macrophagic lineage should be taken into account when designing Kv1.3-based therapies.