Patrick Delmas

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.

Signaling Microdomains Define the Specificity of Receptor-Mediated InsP3 Pathways in Neurons

M(1) muscarinic (M(1)AChRs) and B(2) bradykinin (B(2)Rs) receptors are two PLCbeta-coupled receptors that mobilize Ca(2+) in nonexcitable cells. In many neurons, however, B(2)Rs but not M(1)AChRs mobilize intracellular Ca(2+). We have studied the membrane organization and dynamics underlying this coupling specificity by using Trp channels as biosensors for real-time detection of PLCbeta products. We found that, in sympathetic neurons, although both receptors rapidly produced DAG and InsP(3) as messengers, only InsP(3) formed by B(2)Rs has the ability to activate IP(3)Rs. This exclusive coupling results from spatially restricted complexes linking B(2)Rs to IP(3)Rs, a missing partnership for M(1)AChRs. These complexes allow fast and localized rises of InsP(3), necessary to activate the low-affinity neuronal IP(3)R. Thus, these signaling microdomains are of critical importance for the induction of selective responses, discriminating proinflammatory information associated with B(2)Rs from cholinergic neurotransmission.

Molecular correlates of the M‐current in cultured rat hippocampal neurons

M‐type K+ currents (IK(M)) play a key role in regulating neuronal excitability. In sympathetic neurons, M‐channels are thought to be composed of a heteromeric assembly of KCNQ2 and KCNQ3 K+ channel subunits. Here, we have tried to identify the KCNQ subunits that are involved in the generation of IK(M) in hippocampal pyramidal neurons cultured from 5‐ to 7‐day‐old rats. RT‐PCR of either CA1 or CA3 regions revealed the presence of KCNQ2, KCNQ3, KCNQ4 and KCNQ5 subunits. Single‐cell PCR of dissociated hippocampal pyramidal neurons gave detectable signals for only KCNQ2, KCNQ3 and KCNQ5; where tested, most also expressed mRNA for the vesicular glutamate transporter VGLUT1. Staining for KCNQ2 and KCNQ5 protein showed punctate fluorescence on both the somata and dendrites of hippocampal neurons. Staining for KCNQ3 was diffusely distributed whereas KCNQ4 was undetectable. In perforated patch recordings, linopirdine, a specific M‐channel blocker, fully inhibited IK(M) with an IC50 of 3.6 ± 1.5 μM. In 70 % of these cells, TEA fully suppressed IK(M) with an IC50 of 0.7 ± 0.1 mm. In the remaining cells, TEA maximally reduced IK(M) by only 59.7 ± 5.2 % with an IC50 of 1.4 ± 0.3 mm; residual IK(M) was abolished by linopirdine. Our data suggest that KCNQ2, KCNQ3 and KCNQ5 subunits contribute to IK(M) in these neurons and that the variations in TEA sensitivity may reflect differential expression of KCNQ2, KCNQ3 and KCNQ5 subunits.

Constitutive Activation of G-proteins by Polycystin-1 Is Antagonized by Polycystin-2

Polycystin-1 (PC1), a 4,303-amino acid integral membrane protein of unknown function, interacts with polycystin-2 (PC2), a 968-amino acid α-type channel subunit. Mutations in their respective genes cause autosomal dominant polycystic kidney disease. Using a novel heterologous expression system and Ca2+ and K+ channels as functional biosensors, we found that full-length PC1 functioned as a constitutive activator of Gi/o-type but not Gq-type G-proteins and modulated the activity of Ca2+ and K+ channels via the release of Gβγ subunits. PC1 lacking the N-terminal 1811 residues replicated the effects of full-length PC1. These effects were independent of regulators of G-protein signaling proteins and were lost in PC1 mutants lacking a putative G-protein binding site. Co-expression with full-length PC2, but not a C-terminal truncation mutant, abrogated the effects of PC1. Our data provide the first experimental evidence that full-length PC1 acts as an untraditional G-protein-coupled receptor, activity of which is physically regulated by PC2. Thus, our study strongly suggests that mutations in PC1 or PC2 that distort the polycystin complex would initiate abnormal G-protein signaling in autosomal dominant polycystic kidney disease.

Polycystins: From Mechanosensation to Gene Regulation

Polycystin proteins have been suggested to form mechanosensory transduction complexes involved in a variety of biological functions including sperm fertilization, mating behavior, and asymmetric gene expression in different species. Furthermore, their dysfunction is the cause of cyst formation in human kidney disease. This review focuses on the pros and cons of their candidacy as mechanically gated channels and on recent findings that have significantly advanced our physiological insight.

Formation of a new receptor-operated channel by heteromeric assembly of TRPP2 and TRPC1 subunits

Although several protein–protein interactions have been reported between transient receptor potential (TRP) channels, they are all known to occur exclusively between members of the same group. The only intergroup interaction described so far is that of TRPP2 and TRPC1; however, the significance of this interaction is unknown. Here, we show that TRPP2 and TRPC1 assemble to form a channel with a unique constellation of new and TRPP2/TRPC1‐specific properties. TRPP2/TRPC1 is activated in response to G‐protein‐coupled receptor activation and shows a pattern of single‐channel conductance, amiloride sensitivity and ion permeability distinct from that of TRPP2 or TRPC1 alone. Native TRPP2/TRPC1 activity is shown in kidney cells by complementary gain‐of‐function and loss‐of‐function experiments, and its existence under physiological conditions is supported by colocalization at the primary cilium and by co‐immunoprecipitation from kidney membranes. Identification of the heteromultimeric TRPP2/TRPC1 channel has implications in mechanosensation and cilium‐based Ca2+ signalling.

The versatile nature of the calcium‐permeable cation channel TRPP2

TRPP2 is a member of the transient receptor potential (TRP) superfamily of cation channels, which is mutated in autosomal dominant polycystic kidney disease (ADPKD). TRPP2 is thought to function with polycystin 1—a large integral protein—as part of a multiprotein complex involved in transducing Ca2+‐dependent information. TRPP2 has been implicated in various biological functions including cell proliferation, sperm fertilization, mating behaviour, mechanosensation and asymmetric gene expression. Although its function as a Ca2+‐permeable cation channel is well established, its precise role in the plasma membrane, the endoplasmic reticulum and the cilium is controversial. Recent studies suggest that TRPP2 function is highly dependent on the subcellular compartment of expression, and is regulated by many interactions with adaptor proteins. This review summarizes the most pertinent evidence about the properties of TRPP2 channels, focusing on the compartment‐specific functions of mammalian TRPP2.

Gating and modulation of presumptive NaV1.9 channels in enteric and spinal sensory neurons.

The NaV1.9 subunit is expressed in nociceptive dorsal root ganglion (DRG) neurons and sensory myenteric neurons in which it generates persistent tetrodotoxin-resistant (TTX-R) Na+ currents of yet unknown physiological functions. Here, we have analyzed these currents in details by combining single-channel and whole-cell recordings from cultured rat DRG and myenteric neurons. Comparison of single-channel with whole-cell data indicates that recording using internal CsCl best reflects the basic electrical features of NaV1.9 currents. Inclusion of fluoride in the pipette solution caused a negative shift in the activation and inactivation gates of NaV1.9 but not NaV1.8. Fluoride acts by promoting entry of NaV1.9 channels into a preopen closed state, which causes a strong bias towards opening and enhances the ability of sensory neurons to sustain spiking. Thus, the modulation of the resting-closed states of NaV1.9 channels strongly influences nociceptor excitability and may provide a mechanism by which inflammatory mediators alter pain threshold.

The alpha subunit of Gq contributes to muscarinic inhibition of the M-type potassium current in sympathetic neurons.

Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (IK(M)), which can be inhibited by activation of M1 muscarinic receptors. This inhibition occurs via pertussis toxin-insensitive G-proteins belonging to the Gαq family (Caulfield et al., 1994). We have used DNA plasmids encoding antisense sequences against the 3′ untranslated regions of Gα subunits (antisense plasmids) to investigate the specific G-protein subunits involved in muscarinic inhibition of IK(M). These antisense plasmids specifically reduced levels of the target G-protein 48 hr after intranuclear injection. In cells depleted of Gαq, muscarinic inhibition of IK(M) was attenuated compared both with uninjected neurons and with neurons injected with an inappropriate GαoAantisense plasmid. In contrast, depletion of Gα11 protein did not alter IK(M) inhibition. To determine whether the α or βγ subunits of the G-protein mediated this inhibition, we have overexpressed the C terminus of β adrenergic receptor kinase 1 (βARK1), which binds free βγ subunits. βARK1 did not reduce muscarinic inhibition of IK(M) at a concentration of plasmid that can reduce βγ-mediated inhibition of calcium current (Delmas et al., 1998a). Also, expression of β1γ2 dimers did not alter the IK(M) density in SCG neurons. In contrast, IK(M) was virtually abolished in cells expressing GTPase-deficient, constitutively active forms of Gαq and Gα11. These data suggest that Gαq is the principal mediator of muscarinic IK(M) inhibition in rat SCG neurons and that this more likely results from an effect of the α subunit than the βγ subunits of the Gqheterotrimer.

Muscarinic mechanisms in nerve cells

The receptor subtype and transduction mechanisms involved in the regulation of various neuronal ionic currents are reviewed, with some recent observations on sympathetic neurons, hippocampal cell membranes and basal forebrain cells.