The subfamily-specific assembly of Eag and Erg K+ channels is determined by both the amino and the carboxyl recognition domains.

A functional voltage-gated K(+) (Kv) channel comprises four pore-forming α-subunits, and only members of the same Kv channel subfamily may co-assemble to form heterotetramers. The ether-à-go-go family of Kv channels (KCNH) encompasses three distinct subfamilies: Eag (Kv10), Erg (Kv11), and Elk (Kv12). Members of different ether-à-go-go subfamilies, such as Eag and Erg, fail to form heterotetramers. Although a short stretch of amino acid sequences in the distal C-terminal section has been implicated in subfamily-specific subunit assembly, it remains unclear whether this region serves as the sole and/or principal subfamily recognition domain for Eag and Erg. Here we aim to ascertain the structural basis underlying the subfamily specificity of ether-à-go-go channels by generating various chimeric constructs between rat Eag1 and human Erg subunits. Biochemical and electrophysiological characterizations of the subunit interaction properties of a series of different chimeric and truncation constructs over the C terminus suggested that the putative C-terminal recognition domain is dispensable for subfamily-specific assembly. Further chimeric analyses over the N terminus revealed that the N-terminal region may also harbor a subfamily recognition domain. Importantly, exchanging either the N-terminal or the C-terminal domain alone led to a virtual loss of the intersubfamily assembly boundary. By contrast, simultaneously swapping both recognition domains resulted in a reversal of subfamily specificity. Our observations are consistent with the notion that both the N-terminal and the C-terminal recognition domains are required to sustain the subfamily-specific assembly of rat Eag1 and human Erg.

Morphological and physiological evidence of a synaptic connection between the lateral parabrachial nucleus and neurons in the A7 catecholamine cell group in rats.

BACKGROUND: The descending noradrenergic (NAergic) system is one of the important endogenous analgesia systems. It has been suggested that noxious stimuli could activate descending NAergic system; nevertheless, the underlying neuronal circuit remains unclear. As NAergic neurons in the A7 catecholamine cell group (A7) are a part of the descending NAergic system and the lateral parabrachial nucleus (LPB) is an important brainstem structure that relays ascending nociceptive signal, we aimed to test whether LPB neurons have direct synaptic contact with NAergic A7 neurons. RESULTS: Stereotaxic injections of an anterograde tracer, biotinylated dextran-amine (BDA), were administered to LPB in rats. The BDA-labeled axonal terminals that have physical contacts with tyrosine hydroxylase-positive (presumed noadrenergic) neurons were identified in A7. Consistent with these morphological observations, the excitatory synaptic currents (EPSCs) were readily evoked in NAergic A7 neurons by extracellular stimulation of LPB. The EPSCs evoked by LPB stimulation were blocked by CNQX, a non-NMDA receptor blocker, and AP5, a selective NMDA receptor blocker, showing that LPB-A7 synaptic transmission is glutamatergic. Moreover, the amplitude of LPB-A7 EPSCs was significantly attenuated by DAMGO, a selective µ-opioid receptor agonist, which was associated with an increase in paired-pulse ratio. CONCLUSIONS: Taken together, the above results showed direct synaptic connections between LPB and A7 catecholamine cell group, the function of which is subject to presynaptic modulation by µ-opioid receptors.

Sex difference in the associations among hyperuricemia with self-reported peptic ulcer disease in a large Taiwanese population study.

Background: Hyperuricemia may play a role in various systemic diseases. However, few studies have investigated the relationship between hyperuricemia and the risk of peptic ulcer disease (PUD). Therefore, in this population-based study, we enrolled over 120,000 participants from the Taiwan Biobank (TWB) and examined the risk factors for self-reported PUD. In addition, we investigated sex differences in the association between hyperuricemia and self-reported PUD. Methods: Data of 121,583 participants were obtained from the TWB. Male participants with a serum uric acid level >7 mg/dl and female participants with a serum uric acid level >6 mg/dl were classified as having hyperuricemia. Details of self-reported PUD were obtained by questionnaire. The association between hyperuricemia and self-reported PUD in the male and female participants was examined using multivariable logistic regression analysis. Results: The overall prevalence of self-reported PUD was 14.6%, with a higher incidence in males (16.5%) compared to females (13.5%). After multivariable adjustment, male sex [vs. female sex; odds ratio (OR) = 1.139; 95% confidence interval (CI) = 1.084-1.198; p < 0.001], and hyperuricemia (OR = 0.919; 95% CI = 0.879-0.961; p < 0.001) were significantly associated with self-reported PUD. Further, a significant interaction was found between sex and hyperuricemia on self-reported PUD (p = 0.004). Hyperuricemia was associated with a low risk of self-reported PUD in males (OR = 0.890; 95% CI = 0.837-0.947; p < 0.001) but not in females (p = 0.139). Conclusion: The prevalence of self-reported PUD was higher in the male participants than in the female participants. Hyperuricemia was associated with low prevalence of self-reported PUD in males, but not in females. Further studies are needed to clarify the mechanisms behind these observations and verify the potential protective role of hyperuricemia on the development of self-reported PUD.

14-3-3 proteins regulate cullin 7-mediated Eag1 degradation.

BACKGROUND: Mutations in the human gene encoding the neuron-specific Eag1 (KV10.1; KCNH1) potassium channel are linked to congenital neurodevelopmental diseases. Disease-causing mutant Eag1 channels manifest aberrant gating function and defective protein homeostasis. Both the E3 ubiquitin ligase cullin 7 (Cul7) and the small acid protein 14-3-3 serve as binding partners of Eag1. Cul7 mediates proteasomal and lysosomal degradation of Eag1 protein, whereas over-expression of 14-3-3 notably reduces Eag1 channel activity. It remains unclear whether 14-3-3 may also contribute to Eag1 protein homeostasis. RESULTS: In human cell line and native rat neurons, disruptions of endogenous 14-3-3 function with the peptide inhibitor difopein or specific RNA interference up-regulated Eag1 protein level in a transcription-independent manner. Difopein hindered Eag1 protein ubiquitination at the endoplasmic reticulum and the plasma membrane, effectively promoting the stability of both immature and mature Eag1 proteins. Suppression of endogenous 14-3-3 function also reduced excitotoxicity-associated Eag1 degradation in neurons. Difopein diminished Cul7-mediated Eag1 degradation, and Cul7 knock-down abolished the effect of difopein on Eag1. Inhibition of endogenous 14-3-3 function substantially perturbed the interaction of Eag1 with Cul7. Further structural analyses suggested that the intracellular Per-Arnt-Sim (PAS) domain and cyclic nucleotide-binding homology domain (CNBHD) of Eag1 are essential for the regulatory effect of 14-3-3 proteins. Significantly, suppression of endogenous 14-3-3 function reduced Cul7-mediated degradation of disease-associated Eag1 mutant proteins. CONCLUSION: Overall these results highlight a chaperone-like role of endogenous 14-3-3 proteins in regulating Eag1 protein homeostasis, as well as a therapeutic potential of 14-3-3 modulators in correcting defective protein expression of disease-causing Eag1 mutants.

Cholinergic and glutamatergic transmission at synapses between pedunculopotine tegmental nucleus axonal terminals and A7 catecholamine cell group noradrenergic neurons in the rat.

We characterized transmission from the pedunculopotine tegmental nucleus (PPTg), which contains cholinergic and glutamatergic neurons, at synapses with noradrenergic (NAergic) A7 neurons. Injection of an anterograde neuronal tracer, biotinylated-dextran amine, into the PPTg resulted in labeling of axonal terminals making synaptic connection with NAergic A7 neurons. Consistent with this, extracellular stimulation using a train of 10 pulses at 100 Hz evoked both fast and slow excitatory synaptic currents (EPSCs) that were blocked, respectively, by DNQX, a non-N-methyl-d-aspartate receptor blocker, or atropine, a cholinergic muscarinic receptor (mAChR) blocker. Interestingly, many spontaneous-like, but stimulation-dependent, EPSCs, were seen for up to one second after the end of stimulation and were blocked by DNQX and decreased by EGTA-AM, a membrane permeable form of EGTA, showing they are glutamatergic EPSCs causing by asynchronous release of vesicular quanta. Moreover, application of atropine or carbachol, an mAChR agonist, caused, respectively, an increase in the number of asynchronous EPSCs or a decrease in the frequency of miniature EPSCs, showing that mAChRs mediated presynaptic inhibition of glutamatergic transmission of the PPTg onto NAergic A7 neurons. In conclusion, our data show direct synaptic transmission of PPTg afferents onto pontine NAergic neurons that involves cooperation of cholinergic and glutamatergic transmission. This dual-transmitter transmission drives the firing rate of NAergic neurons, which may correlate with axonal and somatic/dendritic release of NA.