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The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the kidney participate in reabsorbing potassium (K+) and ammonium (NH4+) in the nephron, contributing to the acid–base balance. Acidosis is a metabolic condition of renal tubular acidosis and chronic kidney disease. Acidosis stimulates the production of mitochondrial reactive oxygen species (mROS), activating protective mechanisms dependent on mitochondrial membrane potential (Δψm) such as autophagy. The HCN3 channel is expressed in the plasma membrane, mitochondria (mitoHCN3), and lysosomes (lysoHCN3) of the rat proximal tubule. In this work we aimed to investigate the role of HCN3 in autophagy, mROS production, and Δψm in cultured rat proximal tubule cells (NRK-52E) exposed to ammonium chloride (NH4Cl). NH4Cl arrested autophagic flux and produced extracellular acidosis and, under this condition, mitoHCN3 and lysoHCN3 were up-regulated. NH4Cl or/and ZD7288, a specific blocker of HCN channels, enhanced mROS. ZD7288 in NH4Cl conditions at 24 h stimulated autophagy by reducing Beclin1, LC3BII, p62, and Parkin in an mROS- or Δψm independent pathway. Therefore, ZD7288 reverted NH4Cl inhibited autophagy through lysoHCN3 inhibition. Oxidative stress induced by H2O2 up-regulated mitoHCN3 expression, while Tiron had the opposite effect. In conclusion, inhibition of mito- and lysoHCN3 channels by ZD7288 can protect against mitochondrial oxidative stress and stimulate the lysosome–autophagy pathway in response to NH4Cl treatment.

Objective This case study investigates the role of hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) ion channels, which are integral membrane proteins crucial for regulating neuronal excitability. HCN channels are composed of four subunits (HCN1‐4), with HCN1, HCN2, and HCN4 previously linked to epilepsy. However, the role of the HCN3 in epileptogenesis remains underexplored. Methods We recruited a cohort of 298 epilepsy patients to screen for genetic variants in the HCN3 (NM_020897.3) using Sanger sequencing. We identified rare variants and conducted functional assays to evaluate their pathogenicity. Results We identified three rare heterozygous variants in HCN3: c.1370G > A (R457H), c.1982G > A (R661Q), and c.1982G > A(P630L). In vitro functional analyses demonstrated that these variants affected the expression level of HCN3 protein without altering its membrane localization. Whole‐cell voltage‐clamp experiments showed that two variants (R457H and R661Q) significantly reduced current density in cells, while P630L has no effect on ion channel current. Significance Our findings suggest that the identified HCN3 genetic variants disrupt HCN ion channel function, highlighting HCN3 as a novel candidate gene involved in epileptic disorders. This expands the genetic landscape of epilepsy and provides new insights into its molecular underpinnings. Plain Language Summary Epilepsy is a brain disease that can be caused by mutations in specific genes. We found three rare variants in HCN3 gene in 298 patients with epilepsy, and two of the three mutations could be pathogenic and cause epilepsy and another one is single‐nucleotide polymorphism, which could have no effect and no contribution to the development of epilepsy.

The kidney controls body fluids, electrolyte and acid–base balance. Previously, we demonstrated that hyperpolarization-activated and cyclic nucleotide-gated (HCN) cation channels participate in ammonium excretion in the rat kidney. Since acid–base balance is closely linked to potassium metabolism, in the present work we aim to determine the effect of chronic metabolic acidosis (CMA) and hyperkalemia (HK) on protein abundance and localization of HCN3 in the rat kidney. CMA increased HCN3 protein level only in the outer medulla (2.74 ± 0.31) according to immunoblot analysis. However, immunofluorescence assays showed that HCN3 augmented in cortical proximal tubules (1.45 ± 0.11) and medullary thick ascending limb of Henle’s loop (4.48 ± 0.45) from the inner stripe of outer medulla. HCN3 was detected in brush border membranes (BBM) and mitochondria of the proximal tubule by immunogold electron and confocal microscopy in control conditions. Acidosis did not alter HCN3 levels in BBM and mitochondria but augmented them in lysosomes. HCN3 was also immuno-detected in mitoautophagosomes. In the distal nephron, HCN3 was expressed in principal and intercalated cells from cortical to medullary collecting ducts. CMA did not change HCN3 abundance in these nephron segments. In contrast, HK doubled HCN3 level in cortical collecting ducts and favored its basolateral localization in principal cells from the inner medullary collecting ducts. These findings further support HCN channels contribution to renal acid–base and potassium balance.

Oxytocin, a hypothalamic neuropeptide essential for breastfeeding, is mainly produced in oxytocin neurons in the supraoptic nucleus (SON) and paraventricular nucleus. However, mechanisms underlying oxytocin secretion, specifically the involvement of hyperpolarization-activated cyclic nucleotide-gated channel 3 (HCN3) in oxytocin neuronal activity, remain unclear. Using a rat model of intermittent and continuous pup deprivation (PD) at the middle stage of lactation, we analyzed the contribution of HCN3 in oxytocin receptor (OTR)-associated signaling cascade to oxytocin neuronal activity in the SON. PD caused maternal depression, anxiety, milk shortage, involution of the mammary glands, and delays in uterine recovery, particularly in continuous PD. PD increased hypothalamic but not plasma oxytocin levels in enzyme-linked immunosorbent assay. In the SON, PD increased c-Fos expression but reduced expressions of cyclooxygenase-2 and HCN3 in Western blots and/or immunohistochemistry. Moreover, PD significantly increased the molecular association of OTR with HCN3 in coimmunoprecipitation. In brain slices, inhibition of HCN3 activity with DK-AH269 blocked prostaglandin E2-evoked increase in the firing activity and burst discharge in oxytocin neurons in patch-clamp recordings. In addition, oxytocin-evoked increase in the molecular association between OTR and HCN3 in brain slices of the SON was blocked by pretreatment with indomethacin, an inhibitor of cyclooxygenase-2. These results indicate that normal activity of oxytocin neurons is under the regulation of an oxytocin receptor–cyclooxygenase-2–HCN3 pathway and that PD disrupts maternal behavior through increasing intranuclear oxytocin secretion in the SON but likely reducing bolus oxytocin release into the blood through inhibition of HCN3 activity.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) in the nervous system are implicated in a variety of neuronal functions including learning and memory, regulation of vigilance states and pain. Dysfunctions or genetic loss of these channels have been shown to cause human diseases such as epilepsy, depression, schizophrenia, and Parkinson's disease. The physiological functions of HCN1 and HCN2 channels in the nervous system have been analyzed using genetic knockout mouse models. By contrast, there are no such genetic studies for HCN3 channels so far. Here, we use a HCN3-deficient (HCN3 ) mouse line, which has been previously generated in our group to examine the expression and function of this channel in the CNS. Specifically, we investigate the role of HCN3 channels for the regulation of circadian rhythm and for the determination of behavior. Contrary to previous suggestions we find that HCN3 mice show normal visual, photic, and non-photic circadian function. In addition, HCN3 mice are impaired in processing contextual information, which is characterized by attenuated long-term extinction of contextual fear and increased fear to a neutral context upon repeated exposure.

Coordinated ureteric peristalsis propels urine from the kidney to the bladder. Cells in the renal pelvis and ureter spontaneously generate and propagate electrical activity to control this process. Recently, c-kit tyrosine kinase and hyperpolarization-activated cyclic nucleotide-gated channel 3 (HCN3) were identified in the upper urinary tract. Both of these proteins are required for coordinated proximal to distal contractions in the ureter. Alterations in pacemaker cell expression are present in multiple congenital kidney diseases, suggesting a functional contribution by these cells to pathologic states. In contrast to gut and heart pacemaker cells, the developmental biology of ureteric pacemaker cells, including cell lineage and signaling mechanisms, is undefined. Here, we review pacemaker cell identify and function in the urinary pelvis and ureter and the control of pacemaker function by Hedgehog-GLI signaling. Next, we highlight current knowledge of gut and heart pacemaker cells that is likely to provide insight into developmental mechanisms that could control urinary pacemaker cells.