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The hair cell, the sensory receptor of the internal ear, releases neurotransmitter at presynaptic active zones studding its basolateral membrane surface. Examination of hair cells by loose-seal patch recording demonstrated that each active zone bears a cluster of voltage-gated Calcium ions (Cav) channels and Ca ions activated potassium ions (KCa) channels. When Cav channels are activated by a hair cell's depolarization in response to acoustical stimulation, the resultant Ca ions influx not only triggers synaptic-vesicle release but also opens KCa channels. The efflux of K ion through these channels causes a rapid repolarization that plays a key role in the electrical oscillation that tunes some hair cells and in fast inhibitory synaptic transmission.

The β subunits of voltage-gated Ca[sup 2+] channels are known to be regulators of the channels' gating properties. Here we report a striking additional function of a β subunit. Screening of chicken cochlear and brain cDNA libraries identified β[sub 4c], a short splice variant of the β[sub 4] subunit. Although β[sub 4c] occurs together with the longer isoforms β[sub 4a] or β[sub 4b] in the brain, eye, heart, and lung, the cochlea expresses exclusively β[sub 4c]. The association of β[sub 4c] with the Ca[sup 2+]-channel α[sub 1] subunit has slight but significant effects on the kinetics of channel activation and inactivation. Yeast two-hybrid and biochemical assays revealed that β[sub 4c] interacts directly with the chromo shadow domain of chromobox protein 2/heterochromatin protein 1γ, (CHCB2/HP1γ), a nuclear protein involved in gene silencing and transcriptional regulation. Coexpression of this protein specifically recruits β[sub 4c] to the nuclei of mammalian cells. Furthermore, β[sub 4c] but not β[sub 4a] dramatically attenuates the genesilencing activity of chromobox protein 2/heterochromatin protein 1γ. The β[sub 4c] subunit is therefore a multifunctional protein that not only constitutes a portion of the Ca[sup 2+] channel but also regulates gene transcription. [ABSTRACT FROM AUTHOR]

The venom peptide maurocalcin (MCa) is atypical among toxins because of its ability to rapidly translocate into cells and potently activate the intracellular calcium channel type 1 ryanodine receptor (RyR1). Therefore, MCa is potentially subjected to posttranslational modifications within recipient cells. Here, we report that MCa Thr(26) belongs to a consensus PKA phosphorylation site and can be phosphorylated by PKA both in vitro and after cell penetration in cellulo. Unexpectedly, phosphorylation converts MCa from positive to negative RyR1 allosteric modulator. Thr(26) phosphorylation leads to charge neutralization of Arg(24), a residue crucial for MCa agonist activity. The functional effect of Thr(26) phosphorylation is partially mimicked by aspartyl mutation. This represents the first case, to our knowledge, of both ex situ posttranslational modification and pharmacological reprogramming of a small natural cystine-rich peptide by target cells. So far, phosphorylated MCa is the first specific negative allosteric modulator of RyR1, to our knowledge, and represents a lead compound for further development of phosphatase-resistant analogs.

The tandem of pore domain in a weak inwardly rectifying K(+) channel (Twik)-related acid-arachidonic activated K(+) channel (TRAAK) and Twik-related K(+) channels (TREK) 1 and TREK2 are active as homodimers gated by stretch, fatty acids, pH, and G protein-coupled receptors. These two-pore domain potassium (K2P) channels are broadly expressed in the nervous system where they control excitability. TREK/TRAAK KO mice display altered phenotypes related to nociception, neuroprotection afforded by polyunsaturated fatty acids, learning and memory, mood control, and sensitivity to general anesthetics. These channels have emerged as promising targets for the development of new classes of anesthetics, analgesics, antidepressants, neuroprotective agents, and drugs against addiction. Here, we show that the TREK1, TREK2, and TRAAK subunits assemble and form active heterodimeric channels with electrophysiological, regulatory, and pharmacological properties different from those of homodimeric channels. Heteromerization occurs between all TREK variants produced by alternative splicing and alternative translation initiation. These results unveil a previously unexpected diversity of K2P channels that will be challenging to analyze in vivo, but which opens new perspectives for the development of clinically relevant drugs.

Membrane lipids serve as second messengers and docking sites for proteins and play central roles in cell signaling. A major question about lipid signaling is whether diffusible lipids can selectively target specific proteins. One family of lipid-regulated membrane proteins is the TWIK-related K channel (TREK) subfamily of K2P channels: TREK1, TREK2, and TWIK-related arachdonic acid stimulated K(+) channel (TRAAK). We investigated the regulation of TREK channels by phosphatidic acid (PA), which is generated by phospholipase D (PLD) via hydrolysis of phosphatidylcholine. Even though all three of the channels are sensitive to PA, we found that only TREK1 and TREK2 are potentiated by PLD2 and that none of these channels is modulated by PLD1, indicating surprising selectivity. We found that PLD2, but not PLD1, directly binds to the C terminus of TREK1 and TREK2, but not to TRAAK. The results have led to a model for selective lipid regulation by localization of phospholipid enzymes to specific effector proteins. Finally, we show that regulation of TREK channels by PLD2 occurs natively in hippocampal neurons.

Maintaining wakefulness is associated with a progressive increase in the need for sleep. This phenomenon has been linked to changes in synaptic function. The synaptic adhesion molecule Neuroligin-1 (NLG1) controls the activity and synaptic localization of N-methyl-d-aspartate receptors, which activity is impaired by prolonged wakefulness. We here highlight that this pathway may underlie both the adverse effects of sleep loss on cognition and the subsequent changes in cortical synchrony. We found that the expression of specific Nlg1 transcript variants is changed by sleep deprivation in three mouse strains. These observations were associated with strain-specific changes in synaptic NLG1 protein content. Importantly, we showed that Nlg1 knockout mice are not able to sustain wakefulness and spend more time in nonrapid eye movement sleep than wild-type mice. These changes occurred with modifications in waking quality as exemplified by low theta/alpha activity during wakefulness and poor preference for social novelty, as well as altered delta synchrony during sleep. Finally, we identified a transcriptional pathway that could underlie the sleep/wake-dependent changes in Nlg1 expression and that involves clock transcription factors. We thus suggest that NLG1 is an element that contributes to the coupling of neuronal activity to sleep/wake regulation.

TWIK1 belongs to the family of background K(+) channels with two pore domains. In native and transfected cells, TWIK1 is detected mainly in recycling endosomes. In principal cells in the kidney, TWIK1 gene inactivation leads to the loss of a nonselective cationic conductance, an unexpected effect that was attributed to adaptive regulation of other channels. Here, we show that TWIK1 ion selectivity is modulated by extracellular pH. Although TWIK1 is K(+) selective at neutral pH, it becomes permeable to Na(+) at the acidic pH found in endosomes. Selectivity recovery is slow after restoration of a neutral pH. Such hysteresis makes plausible a role of TWIK1 as a background channel in which selectivity and resulting inhibitory or excitatory influences on cell excitability rely on its recycling rate between internal acidic stores and the plasma membrane. TWIK1(-/-) pancreatic β cells are more polarized than control cells, confirming a depolarizing role of TWIK1 in kidney and pancreatic cells.

Task2 K(+) channel expression in the central nervous system is surprisingly restricted to a few brainstem nuclei, including the retrotrapezoid (RTN) region. All Task2-positive RTN neurons were lost in mice bearing a Phox2b mutation that causes the human congenital central hypoventilation syndrome. In plethysmography, Task2(-/-) mice showed disturbed chemosensory function with hypersensitivity to low CO(2) concentrations, leading to hyperventilation. Task2 probably is needed to stabilize the membrane potential of chemoreceptive cells. In addition, Task2(-/-) mice lost the long-term hypoxia-induced respiratory decrease whereas the acute carotid-body-mediated increase was maintained. The lack of anoxia-induced respiratory depression in the isolated brainstem-spinal cord preparation suggested a central origin of the phenotype. Task2 activation by reactive oxygen species generated during hypoxia could silence RTN neurons, thus contributing to respiratory depression. These data identify Task2 as a determinant of central O(2) chemoreception and demonstrate that this phenomenon is due to the activity of a small number of neurons located at the ventral medullary surface.

Mechanosensitive K(+) channels TREK1 and TREK2 form a subclass of two P-domain K(+) channels. They are potently activated by polyunsaturated fatty acids and are involved in neuroprotection, anesthesia, and pain perception. Here, we show that acidification of the extracellular medium strongly inhibits TREK1 with an apparent pK near to 7.4 corresponding to the physiological pH. The all-or-none effect of pH variation is steep and is observed within one pH unit. TREK2 is not inhibited but activated by acidification within the same range of pH, despite its close homology with TREK1. A single conserved residue, H126 in TREK1 and H151 in TREK2, is involved in proton sensing. This histidine is located in the M1P1 extracellular loop preceding the first P domain. The differential effect of acidification, that is, activation for TREK2 and inhibition for TREK1, involves other residues located in the P2M4 loop, linking the second P domain and the fourth membrane-spanning segment. Structural modeling of TREK1 and TREK2 and site-directed mutagenesis strongly suggest that attraction or repulsion between the protonated side chain of histidine and closely located negatively or positively charged residues in P2M4 control outer gating of these channels. The differential sensitivity of TREK1 and TREK2 to external pH variations discriminates between these two K(+) channels that otherwise share the same regulations by physical and chemical stimuli, and by hormones and neurotransmitters.

The acid- and volume-sensitive TASK2 K+ channel is strongly expressed in renal proximal tubules and papillary collecting ducts. This study was aimed at investigating the role of TASK2 in renal bicarbonate reabsorption by using the task2 -/- mouse as a model. After backcross to C57BL6, task2 -/- mice showed an increased perinatal mortality and, in adulthood, a reduced body weight and arterial blood pressure. Patch-clamp experiments on proximal tubular cells indicated that TASK2 was activated during HCO3- transport. In control inulin clearance measurements, task2 -/- mice showed normal NaCl and water excretion. During i.v. NaHCO3 perfusion, however, renal Na+ and water reabsorption capacity was reduced in -/- animals. In conscious task2 -/- mice, blood pH, HCO3- concentration, and systemic base excess were reduced but urinary pH and HCO3- were increased. These data suggest that task2 -/- mice exhibit metabolic acidosis caused by renal loss of HCO3-. Both in vitro and in vivo results demonstrate the specific coupling of TASK2 activity to HCO3- transport through external alkalinization. The consequences of the task2 gene inactivation in mice are reminiscent of the clinical manifestations seen in human proximal renal tubular acidosis syndrome.

The beta subunits of voltage-gated Ca(2+) channels are known to be regulators of the channels' gating properties. Here we report a striking additional function of a beta subunit. Screening of chicken cochlear and brain cDNA libraries identified beta(4c), a short splice variant of the beta(4) subunit. Although beta(4c) occurs together with the longer isoforms beta(4a) or beta(4b) in the brain, eye, heart, and lung, the cochlea expresses exclusively beta(4c). The association of beta(4c) with the Ca(2+)-channel alpha(1) subunit has slight but significant effects on the kinetics of channel activation and inactivation. Yeast two-hybrid and biochemical assays revealed that beta(4c) interacts directly with the chromo shadow domain of chromobox protein 2heterochromatin protein 1gamma (CHCB2HP1gamma), a nuclear protein involved in gene silencing and transcriptional regulation. Coexpression of this protein specifically recruits beta(4c) to the nuclei of mammalian cells. Furthermore, beta(4c) but not beta(4a) dramatically attenuates the gene-silencing activity of chromobox protein 2heterochromatin protein 1gamma. The beta(4c) subunit is therefore a multifunctional protein that not only constitutes a portion of the Ca(2+) channel but also regulates gene transcription.