Kv7 and Kv11 channels in myometrial regulation.

Ion channels play a key role in defining myometrial contractility. Modulation of ion channel populations is proposed to underpin gestational changes in uterine contractility associated with the transition from uterine quiescence to active labour. Of the myriad ion channels present in the uterus, this article will focus upon potassium channels encoded by the KCNQ genes and ether-à-go-go-related (ERG) genes. Voltage-gated potassium channels encoded by KCNQ and ERG (termed Kv7 and Kv11, respectively) are accepted as major determinants of neuronal excitability and the duration of the cardiac action potential. However, there is now growing appreciation that these ion channels have a major functional impact in vascular and non-vascular smooth muscle. Moreover, Kv7 channels may be potential therapeutic targets for the treatment of preterm labour

Two Types of K⁺ Channel Subunit, Erg1 and KCNQ2/3, Contribute to the M-Like Current in a Mammalian Neuronal Cell

The potassium M current was originally identified in sympathetic ganglion cells, and analogous currents have been reported in some central neurons and also in some neural cell lines. It has recently been suggested that the M channel in sympathetic neurons comprises a heteromultimer of KCNQ2 and KCNQ3 (Wang et al., 1998) but it is unclear whether all other M-like currents are generated by these channels. Here we report that the M-like current previously described in NG108–15 mouse neuroblastoma x rat glioma cells has two components, “fast” and “slow”, that may be differentiated kinetically and pharmacologically. We provide evidence from PCR analysis and expression studies to indicate that these two components are mediated by two distinct molecular species of K+ channel: the fast component resembles that in sympathetic ganglia and is probably carried byKCNQ2/3 channels, whereas the slow component appears to be carried by merg1a channels. Thus, the channels generating M-like currents in different cells may be heterogeneous in molecular composition

KCNQ/M currents in sensory neurons: Significance for pain therapy

Neuronal hyperexcitability is a feature of epilepsy and both inflammatory and neuropathic pain. M currents [I-K(M)] play a key role in regulating neuronal excitability, and mutations in neuronal KCNQ2/3 subunits, the molecular correlates of I-K(M), have previously been linked to benign familial neonatal epilepsy. Here, we demonstrate that KCNQ/M channels are also present in nociceptive sensory systems. I-K(M) was identified, on the basis of biophysical and pharmacological properties, in cultured neurons isolated from dorsal root ganglia (DRGs) from 17-d-old rats. Currents were inhibited by the M-channel blockers linopirdine (IC50, 2.1 muM) and XE991 (IC50, 0.26 muM) and enhanced by retigabine (10 muM). The expression of neuronal KCNQ subunits in DRG neurons was confirmed using reverse transcription-PCR and single-cell PCR analysis and by immunofluorescence. Retigabine, applied to the dorsal spinal cord, inhibited C and Adelta fiber-mediated responses of dorsal horn neurons evoked by natural or electrical afferent stimulation and the progressive "windup" discharge with repetitive stimulation in normal rats and in rats subjected to spinal nerve ligation. Retigabine also inhibited responses to intrapaw application of carrageenan in a rat model of chronic pain; this was reversed by XE991. It is suggested that I-K(M) plays a key role in controlling the excitability of nociceptors and may represent a novel analgesic target

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

Coexpression and activation of TRPV1 suppress the activity of the KCNQ2/3 channel

Transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated nonselective cation channel expressed predominantly in peripheral nociceptors. By detecting and integrating diverse noxious thermal and chemical stimuli, and as a result of its sensitization by inflammatory mediators, the TRPV1 receptor plays a key role in inflammation-induced pain. Activation of TRPV1 leads to a cascade of pro-nociceptive mechanisms, many of which still remain to be identified. Here, we report a novel effect of TRPV1 on the activity of the potassium channel KCNQ2/3, a negative regulator of neuronal excitability. Using ion influx assays, we revealed that TRPV1 activation can abolish KCNQ2/3 activity, but not vice versa, in human embryonic kidney (HEK)293 cells. Electrophysiological studies showed that coexpression of TRPV1 caused a 7.5-mV depolarizing shift in the voltage dependence of KCNQ2/3 activation compared with control expressing KCNQ2/3 alone. Furthermore, activation of TRPV1 by capsaicin led to a 54% reduction of KCNQ2/3-mediated current amplitude and attenuation of KCNQ2/3 activation. The inhibitory effect of TRPV1 appears to depend on Ca2+ influx through the activated channel followed by Ca2+-sensitive depletion of phosphatidylinositol 4,5-bisphosphate and activation of protein phosphatase calcineurin. We also identified physical interactions between TRPV1 and KCNQ2/3 coexpressed in HEK293 cells and in rat dorsal root ganglia neurons. Mutation studies established that this interaction is mediated predominantly by the membrane-spanning regions of the respective proteins and correlates with the shift of KCNQ2/3 activation. Collectively, these data reveal that TRPV1 activation may deprive neurons from inhibitory control mediated by KCNQ2/3. Such neurons may thus have a lower threshold for activation, which may indirectly facilitate TRPV1 in integrating multiple noxious signals and/or in the establishment or maintenance of chronic pain

Discovery of a Novel Activator of KCNQ1-KCNE1 K+ Channel Complexes

KCNQ1 voltage-gated K+ channels (Kv7.1) associate with the family of five KCNE peptides to form complexes with diverse gating properties and pharmacological sensitivities. The varied gating properties of the different KCNQ1-KCNE complexes enables the same K+ channel to function in both excitable and non excitable tissues. Small molecule activators would be valuable tools for dissecting the gating mechanisms of KCNQ1-KCNE complexes; however, there are very few known activators of KCNQ1 channels and most are ineffective on the physiologically relevant KCNQ1-KCNE complexes. Here we show that a simple boronic acid, phenylboronic acid (PBA), activates KCNQ1/KCNE1 complexes co-expressed in Xenopus oocytes at millimolar concentrations. PBA shifts the voltage sensitivity of KCNQ1 channel complexes to favor the open state at negative potentials. Analysis of different-sized charge carriers revealed that PBA also targets the permeation pathway of KCNQ1 channels. Activation by the boronic acid moiety has some specificity for the Kv7 family members (KCNQ1, KCNQ2/3, and KCNQ4) since PBA does not activate Shaker or hERG channels. Furthermore, the commercial availability of numerous PBA derivatives provides a large class of compounds to investigate the gating mechanisms of KCNQ1-KCNE complexes

Kv7 Channels Can Function without Constitutive Calmodulin Tethering

M-channels are voltage-gated potassium channels composed of Kv7.2-7.5 subunits that serve as important regulators of neuronal excitability. Calmodulin binding is required for Kv7 channel function and mutations in Kv7.2 that disrupt calmodulin binding cause Benign Familial Neonatal Convulsions (BFNC), a dominantly inherited human epilepsy. On the basis that Kv7.2 mutants deficient in calmodulin binding are not functional, calmodulin has been defined as an auxiliary subunit of Kv7 channels. However, we have identified a presumably phosphomimetic mutation S511D that permits calmodulin-independent function. Thus, our data reveal that constitutive tethering of calmodulin is not required for Kv7 channel function

How Noisy Adaptation of Neurons Shapes Interspike Interval Histograms and Correlations

Channel noise is the dominant intrinsic noise source of neurons causing variability in the timing of action potentials and interspike intervals (ISI). Slow adaptation currents are observed in many cells and strongly shape response properties of neurons. These currents are mediated by finite populations of ionic channels and may thus carry a substantial noise component. Here we study the effect of such adaptation noise on the ISI statistics of an integrate-and-fire model neuron by means of analytical techniques and extensive numerical simulations. We contrast this stochastic adaptation with the commonly studied case of a fast fluctuating current noise and a deterministic adaptation current (corresponding to an infinite population of adaptation channels). We derive analytical approximations for the ISI density and ISI serial correlation coefficient for both cases. For fast fluctuations and deterministic adaptation, the ISI density is well approximated by an inverse Gaussian (IG) and the ISI correlations are negative. In marked contrast, for stochastic adaptation, the density is more peaked and has a heavier tail than an IG density and the serial correlations are positive. A numerical study of the mixed case where both fast fluctuations and adaptation channel noise are present reveals a smooth transition between the analytically tractable limiting cases. Our conclusions are furthermore supported by numerical simulations of a biophysically more realistic Hodgkin-Huxley type model. Our results could be used to infer the dominant source of noise in neurons from their ISI statistics

P2Y receptors and pain transmission

It is widely accepted that the most important ATP receptors involved in pain transmission belong to the P2X3 and P2X2/3 subtypes, selectively expressed in small diameter dorsal root ganglion (DRG) neurons. However, several types of the metabotropic ATP (P2Y) receptors have also been found in primary afferent neurons; P2Y1 and P2Y2 receptors are typically expressed in small, nociceptive cells. Here we review the results available on the involvement of P2Y receptors in the modulation of pain transmission