Functional characterization of ether-à-go-go-related gene potassium channels in midbrain dopamine neurons - implications for a role in depolarization block.

Bursting activity by midbrain dopamine neurons reflects the complex interplay between their intrinsic pacemaker activity and synaptic inputs. Although the precise mechanism responsible for the generation and modulation of bursting in vivo has yet to be established, several ion channels have been implicated in the process. Previous studies with nonselective blockers suggested that ether-à-go-go-related gene (ERG) K(+) channels are functionally significant. Here, electrophysiology with selective chemical and peptide ERG channel blockers (E-4031 and rBeKm-1) and computational methods were used to define the contribution made by ERG channels to the firing properties of midbrain dopamine neurons in vivo and in vitro. Selective ERG channel blockade increased the frequency of spontaneous activity as well as the response to depolarizing current pulses without altering spike frequency adaptation. ERG channel block also accelerated entry into depolarization inactivation during bursts elicited by virtual NMDA receptors generated with the dynamic clamp, and significantly prolonged the duration of the sustained depolarization inactivation that followed pharmacologically evoked bursts. In vivo, somatic ERG blockade was associated with an increase in bursting activity attributed to a reduction in doublet firing. Taken together, these results show that dopamine neuron ERG K(+) channels play a prominent role in limiting excitability and in minimizing depolarization inactivation. As the therapeutic actions of antipsychotic drugs are associated with depolarization inactivation of dopamine neurons and blockade of cardiac ERG channels is a prominent side effect of these drugs, ERG channels in the central nervous system may represent a novel target for antipsychotic drug development.

Tmem16A encodes the Ca2+-activated Cl- channel in mouse submandibular salivary gland acinar cells.

Activation of an apical Ca(2+)-dependent Cl(-) channel (CaCC) is the rate-limiting step for fluid secretion in many exocrine tissues. Here, we compared the properties of native CaCC in mouse submandibular salivary gland acinar cells to the Ca(2+)-gated Cl(-) currents generated by Tmem16A and Best2, members from two distinct families of Ca(2+)-activated Cl(-) channels found in salivary glands. Heterologous expression of Tmem16A and Best2 transcripts in HEK293 cells produced Ca(2+)-activated Cl(-) currents with time and voltage dependence and inhibitor sensitivity that resembled the Ca(2+)-activated Cl(-) current found in native salivary acinar cells. Best2(-/-) and Tmem16A(-/-) mice were used to further characterize the role of these channels in the exocrine salivary gland. The amplitude and the biophysical footprint of the Ca(2+)-activated Cl(-) current in submandibular gland acinar cells from Best2-deficient mice were the same as in wild type cells. Consistent with this observation, the fluid secretion rate in Best2 null mice was comparable with that in wild type mice. In contrast, submandibular gland acinar cells from Tmem16A(-/-) mice lacked a Ca(2+)-activated Cl(-) current and a Ca(2+)-mobilizing agonist failed to stimulate Cl(-) efflux, requirements for fluid secretion. Furthermore, saliva secretion was abolished by the CaCC inhibitor niflumic acid in wild type and Best2(-/-) mice. Our results demonstrate that both Tmem16A and Best2 generate Ca(2+)-activated Cl(-) current in vitro with similar properties to those expressed in native cells, yet only Tmem16A appears to be a critical component of the acinar Ca(2+)-activated Cl(-) channel complex that is essential for saliva production by the submandibular gland.

Cav1.3 channel voltage dependence, not Ca2+ selectivity, drives pacemaker activity and amplifies bursts in nigral dopamine neurons.

Ca(v)1.3 (alpha 1D) L-type Ca(2+) channels have been implicated in substantia nigra (SN) dopamine (DA) neuron pacemaking and vulnerability to Parkinson's disease. These effects may arise from the depolarizing current and cytoplasmic Ca(2+) elevation produced by Ca(v)1.3 channels at subthreshold membrane potentials. However, the assumption that the Ca(2+) selectivity of Ca(v)1.3 channels is essential has not been tested. In this study the properties of SN DA neuron L-type Ca(2+) channels responsible for driving pacemaker activity in juvenile rat brain slices were probed by replacing native channels blocked with the dihydropyridine nimodipine with virtual channels generated by dynamic clamp. Surprisingly, virtual L-type channels that mimic native and recombinant Ca(v)1.3 channels supported pacemaker activity even though dynamic clamp currents are not carried by Ca(2+). This effect is specific because pacemaker activity could not be restored by tonic current injection, virtual nonselective leak channels or virtual NMDA receptors, which share with L-type channels a negative slope conductance region in their current-voltage (I-V) curve. Altering virtual channels showed that the production of pacemaker activity depended on the characteristic voltage dependence of DA neuron L-type channels, while activation kinetics and reversal potential were not critical parameters. Virtual L-type channels also supported slow oscillatory potentials and enhanced firing rate during evoked bursts. Thus, Ca(v)1.3 channel voltage dependence, rather than Ca(2+) selectivity, drives pacemaker activity and amplifies bursts in SN DA neurons.

Activity-dependent vesicular monoamine transporter-mediated depletion of the nucleus supports somatic release by serotonin neurons.

Packaging by the vesicular monoamine transporter (VMAT) is essential for mood-controlling serotonin transmission but has not been assayed during activity. Here, two-photon imaging of the fluorescent serotonin analog 5,7-dihydroxytryptamine and three-photon imaging of endogenous serotonin were used to study vesicular packaging as it supports release from the soma of serotonin neurons. Glutamate receptor activation in dorsal raphe brain slice evoked somatic release that was mediated solely by vesicle exocytosis. This release was accompanied by VMAT-mediated serotonin depletion from the nucleus, a large compartment free of monoaminergic degradation pathways that has not been implicated in neurotransmission previously. Finally, while some monoamine packaged at rest was held in reserve, monoamine packaged during stimulation was released completely. Hence, somatic vesicles loaded by VMAT during activity rapidly undergo exocytosis. In the absence of active zones and with limited neurotransmitter reuptake, somatic release by serotonin neurons is supported by recruitment from a large pool of extravesicular serotonin in the nucleus and cytoplasm, and preferential release of the newly packaged transmitter.

Dopamine neuron responses depend exponentially on pacemaker interval.

Midbrain dopamine neuron activity results from the integration of the responses to metabo- and ionotropic receptors with the postsynaptic excitability of these intrinsic pacemakers. Interestingly, intrinsic pacemaker rate varies greatly between individual dopamine neurons and is subject to short- and long-term regulation. Here responses of substantia nigra dopamine neurons to defined dynamic-clamp stimuli were measured to quantify the impact of cell-to-cell variation in intrinsic pacemaker rate. Then this approach was repeated in single dopamine neurons in which pacemaker rate was altered by activation of muscarinic receptors or current injection. These experiments revealed a dramatic exponential dependence on pacemaker interval for the responses to voltage-gated A-type K+ channels, voltage-independent cation channels and ionotropic synapses. Likewise, responses to native metabotropic (GABAb and mGluR1) inhibitory synapses depended steeply on pacemaker interval. These results show that observed variations in dopamine neuron pacemaker rate are functionally significant because they produce a >10-fold difference in responses to diverse stimuli. Both the magnitude and the mathematical form of the relationship between pacemaker interval and responses were not previously anticipated.

Looking chloride channels straight in the eye: bestrophins, lipofuscinosis, and retinal degeneration.

Recent evidence suggests that Cl(-) ion channels are important for retinal integrity. Bestrophin Cl(-) channel mutations in humans are genetically linked to a juvenile form of macular degeneration, and disruption of some ClC Cl(-) channels in mice leads to retinal degeneration. In both cases, accumulation of lipofuscin pigment is a key feature of the cellular degeneration. Because Cl(-) channels regulate the ionic environment inside organelles in the endosomal-lysosomal pathway, retinal degeneration may result from defects in lysosomal trafficking or function.

Calcium-activated chloride channels.

Calcium-activated chloride channels (CaCCs) play important roles in cellular physiology, including epithelial secretion of electrolytes and water, sensory transduction, regulation of neuronal and cardiac excitability, and regulation of vascular tone. This review discusses the physiological roles of these channels, their mechanisms of regulation and activation, and the mechanisms of anion selectivity and conduction. Despite the fact that CaCCs are so broadly expressed in cells and play such important functions, understanding these channels has been limited by the absence of specific blockers and the fact that the molecular identities of CaCCs remains in question. Recent status of the pharmacology and molecular identification of CaCCs is evaluated.

Chloride accumulation in mammalian olfactory sensory neurons.

The generation of an excitatory receptor current in mammalian olfactory sensory neurons (OSNs) involves the sequential activation of two distinct types of ion channels: cAMP-gated Ca(2+)-permeable cation channels and Ca(2+)-gated Cl(-) channels, which conduct a depolarizing Cl(-) efflux. This unusual transduction mechanism requires an outward-directed driving force for Cl(-), established by active accumulation of Cl(-) within the lumen of the sensory cilia. We used two-photon fluorescence lifetime imaging microscopy of the Cl(-)-sensitive dye 6-methoxy-quinolyl acetoethyl ester to measure the intracellular Cl(-) concentration in dendritic knobs of OSNs from mice and rats. We found a uniform intracellular Cl(-) concentration in the range of 40-50 mm, which is indicative of active Cl(-) accumulation. Functional assays and PCR experiments revealed that NKCC1-mediated Cl(-) uptake through the apical membrane counteracts Cl(-) depletion in the sensory cilia, and thus maintains the responsiveness of OSNs to odor stimulation. To permit Cl(-) accumulation, OSNs avoid the "chloride switch": they do not express KCC2, the main Cl(-) extrusion cotransporter operating in neurons of the adult CNS. Cl(-) accumulation provides OSNs with the driving force for the depolarizing Cl(-) current that is the basis of the low-noise receptor current in these neurons.