Neuronal hyperexcitability is a feature of epilepsy and both inflammatory and neuropathic pain. M currents [IK(M)] play a key role in regulating neuronal excitability, and mutations in neuronal KCNQ2/3 subunits, the molecular correlates of IK(M), have previously been linked to benign familial neonatal epilepsy. Here, we demonstrate that KCNQ/M channels are also present in nociceptive sensory systems. IK(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 microm) and XE991 (IC50, 0.26 microm) and enhanced by retigabine (10 microm). 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 IK(M) plays a key role in controlling the excitability of nociceptors and may represent a novel analgesic target.
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.
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.
Voltage-gated Na(+) currents play critical roles in shaping electrogenesis in neurons. Here, we have identified a TTX-resistant Na(+) current (TTX-R I(Na)) in duodenum myenteric neurons of guinea pig and rat and have sought evidence regarding the molecular identity of the channel producing this current from the expression of Na(+) channel alpha subunits and the biophysical and pharmacological properties of TTX-R I(Na). Whole-cell patch-clamp recording from in situ neurons revealed the presence of a voltage-gated Na(+) current that was highly resistant to TTX (IC(50), approximately 200 microm) and selectively distributed in myenteric sensory neurons but not in interneurons and motor neurons. TTX-R I(Na) activated slowly in response to depolarization and exhibited a threshold for activation at -50 mV. V(1/2) values of activation and steady-state inactivation were -32 and -31 mV in the absence of fluoride, respectively, which, as predicted from the window current, generated persistent currents. TTX-R I(Na) also had prominent ultraslow inactivation, which turns off 50% of the conductance at rest (-60 mV). Substituting CsF for CsCl in the intracellular solution shifted the voltage-dependent parameters of TTX-R I(Na) leftward by approximately 20 mV. Under these conditions, TTX-R I(Na) had voltage-dependent properties similar to those reported previously for NaN/Na(V)1.9 in dorsal root ganglion neurons. Consistent with this, reverse transcription-PCR, single-cell profiling, and immunostaining experiments indicated that Na(V)1.9 transcripts and subunits, but not Na(V)1.8, were expressed in the enteric nervous system and restricted to myenteric sensory neurons. TTX-R I(Na) may play an important role in regulating subthreshold electrogenesis and boosting synaptic stimuli, thereby conferring distinct integrative properties to myenteric sensory neurons.
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