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

Abstract

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 M(1), angiotensin II AT(1), bradykinin B(2) and purinergic P2Y agonists suppress M current (I(M)). Probes of PLC activity show agonists of all four receptors to induce robust PIP(2) hydrolysis. We have grouped these receptors into two related modes of action. One mode involves depletion of phosphatidylinositol 4,5-bisphosphate (PIP(2)) in the membrane, whose interaction with the channels is thought necessary for their function. The other involves IP(3)-mediated intracellular Ca(2+) signals that stimulate PIP(2) synthesis, preventing its depletion, and suppress I(M) 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 PIP(2). Based on single-channel recordings from chimeras between high-PIP(2) affinity KCNQ3 and low-PIP(2) affinity KCNQ4 channels, we focus on a 57-residue domain within the carboxy-terminus that is a possible PIP(2) 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 PIP(2) regulation of Kir channels. We discuss some important issues dealing with these topics.

Figure 1. The PLCδ-PH probe reports PIP₂ hydrolysis upon stimulation of multiple G_q/11-coupled receptors

A, confocal images from a SCG neuron sequentially stimulated by UTP, bradykinin (BK), and oxotremorine (Oxo-M). In the unstimulated cell (control), the probe was concentrated mainly in the membrane, bound to PIP₂. Upon stimulation by agonist, activation of PLC hydrolyses PIP₂ and the probe translocates to the cytoplasm, bound to IP₃. B, summarized data of the average fluorescence intensity, F, in a cytoplasmic region, normalized to the average intensity over 30 s before agonist application, F_o, quantified as the percentage increase in F (ΔF/F_o) induced by the four agonists. C, a schematic depiction of movement of the PLCδ-PH probe before (left panel) and after (right panel) activation of PLC via stimulation G_q/11-coupled receptors. D, an example of M current regulation under whole-cell clamp before (a, c) and after (b, d) application of either bradykinin or Oxo-M, and after complete blockade after application of linopirdine (e). GPCR, G protein coupled receptor; PLC, phospholipase C; DAG, diacylglycerol; BK or brady, bradykinin; Oxo-M or oxo, oxotremorine-M; LP, linopirdine. (G. P. Tolstykh, O. Zaika and M.S. Shapiro, unpublished observations.)

Figure 2. Modes of M channel regulation in sympathetic neurons

Shown are mechanisms of inhibition of M channels used by two types of G_q/11-coupled receptors in the SCG. Both types activate PLCβ, which in turn hydrolyses PIP₂ to IP₃ and diacylglycerol. The first is used by M₁ muscarinic acetylcholine and AT₁ angiotensin II receptors (left). These agonists are ineffective in producing cytoplasmic Ca²⁺ signals, probably because the receptors are too far away from ER IP₃ receptors, and the IP₃ produced dissipates away (thick red arrow). Thus, much PIP₂ is consumed, PIP₂ unbinds from M channels down the [PIP₂] gradient, and M channels are suppressed. The second is used by bradykinin B₂ and purinergic P2Y₆ receptors (right). Due to the spatial colocalization of these receptors to ER IP₃ receptors, cytoplasmic Ca²⁺ signals are elicited. The released Ca²⁺ binds to neuronal Ca²⁺ sensor-1 (NCS-1) and to calmodulin (CaM). NCS-1 promotes PIP₂ synthesis via acceleration of PI4-kinase, providing PIP₂ to the membrane (purple arrows), and stabilizing PIP₂ levels in the face of PLC activity. CaM binds to carboxy-terminal domains of the channel and likely acts by reducing its affinity for PIP₂, which then unbinds from the channel since tonic [PIP₂] is now insufficient to maintain association with the channel, and M current is suppressed. PLC, phospholipase C; PIP₂, phosphatidylinositol 4,5-bisphosphate; CaM, calmodulin; PI4-kinase, phosphoinositide 4-kinase; NCS-1, neuronal calcium sensor-1 protein; IP₃R, inositol trisphosphate receptor; ER, endoplasmic reticulum.

Figure 3. M channels affect the release of NA from cultured sympathetic neurons detected by carbon-fibre amperometry

A, schematic illustration of experimental conditions. A carbon-fibre electrode (CFE) is placed proximal to a cluster of SCG neurons, and the cells excited by 18 1 ms shocks from an extracellular field electrode (e-stim). The applied shocks induce many action potentials, causing the release of NA, both from the soma, and from varicosities in the neurites that are symbolized as small spots. A multibarrelled solution exchange system for perfusion of experimental solutions completes the set-up. In control conditions (B and C), the train of shocks elicited a total amperometric current that is proportional to the concentration of NA. The effects on NA release of retigabine (B) and XE991 (C) are shown. Consistent with the effects of these compounds on somatic excitability and action potential firing, retigabine caused a strong decrease in released NA, and XE991 caused an increase in released NA, suggesting that the effects of altering M channel activity on neuronal excitability may be paralleled by their effects on neurotransmitter release at nerve terminals. D, bars show the summarized data from these experiments. In the presence of retigabine, the train of shocks evoked a total charge of 68 ± 22% (P < 0.05) of control, and after XE991, the train of shocks evoked a total charge of 148 ± 19% (P < 0.05) of control. The effects of both drugs were fully reversible (O. Zaika and M. S. Shapiro, unpublished observations.)

Figure 4. Homology modelling of a putative PIP₂-binding domain within the C terminus of KCNQ2 shows structural similarity to a similar domain in IRK1

Shown are the results of homology modelling using the program SWISS-MODEL. A, superimposed are the structure of residues 186–245 of IRK1 (Kir2.1) and the predicted structure of residues 428–484 of KCNQ2 using the IRK1 structure as a template. B, shown individually are the homology model of the linker domain of KCNQ2 (blue) and the homologous region of IRK1 (red), with elements of secondary structure indicated. Shown are clusters of basic residues (stick model) located in a putative PIP₂ binding site on IRK1 (Logothetis et al. 2007a), as well as a cluster of basic residues located in the C-terminal domain of KCNQ currently under investigation as critical for PIP₂ regulation. (C. C. Hernandez and M. S. Shapiro, unpublished.)