The research, led by Ziruo Sun, Jinliang Wang, Zhigao Bu and colleagues at the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, began with a systematic search for the host machinery that rabies virus depends on to invade cells. In a previous genome-wide screen, the team had used 64,755 small interfering RNAs targeting 21,585 human genes in HEK293 cells to identify host factors required for rabies virus replication. Among the hits were sixteen genes related to potassium channels, and the strongest effect came from silencing KCNMA1, the gene that encodes the pore-forming subunit of the large conductance calcium- and voltage-activated potassium channel known as KCa1.1, a seven-transmembrane protein sitting in the plasma membrane.

To confirm that this channel genuinely matters for infection, the researchers knocked down KCa1.1 expression in both mouse neuroblastoma N2a cells and human HEK293 cells using specific siRNAs. Quantitative PCR verified that the channel’s mRNA was substantially reduced, and when the silenced cells were infected with a recombinant rabies virus strain expressing enhanced green fluorescent protein, the amount of infectious virus released into the culture supernatant dropped significantly at both 24 and 48 hours after infection. The team then turned to paxilline, a selective chemical inhibitor of KCa1.1 that binds the inner pore of the channel and blocks potassium conductance. In N2a, HEK293, and human SK-N-SH neuroblastoma cells, paxilline had no effect on cell viability but suppressed rabies virus replication in a clear dose-dependent manner, and time-course experiments confirmed the inhibition persisted across the tested period.

Because KCa1.1 is expressed primarily at the plasma membrane, the investigators hypothesized that it might act during viral entry rather than during later stages of the viral life cycle. Entry involves two separable steps: attachment of virus particles to the cell surface and their subsequent internalization. By incubating cells with virus at 4 degrees Celsius, a temperature that permits binding but prevents internalization, and then quantifying attached viral RNA, the team showed that neither KCa1.1 silencing nor paxilline treatment changed how much virus bound to cells. When the temperature was shifted to 37 degrees to allow entry, however, and surface-bound virus was stripped away with trypsin before analysis, the amount of viral RNA inside treated or silenced cells fell dramatically. A complementary microscopy assay, which labeled viral glycoprotein under conditions that distinguish particles remaining on the surface from those that had entered, showed that paxilline-treated cells accumulated more virus on their surface while their total particle count stayed the same. Together, these experiments demonstrated that activated KCa1.1 is specifically required for the internalization step of rabies virus entry.

A co-immunoprecipitation assay revealed that KCa1.1 does not physically interact with the rabies virus glycoprotein, the viral surface protein that drives attachment and clathrin-mediated endocytosis. That result pointed to an intermediary. Prior work had shown that KCa1.1 assembles into macromolecular complexes with voltage-gated calcium channels, including Cav1.2, and that calcium influx through these channels can activate KCa1.1. Notably, Cav1.2 was the only calcium channel that emerged from the team’s earlier genome-wide screen. Co-immunoprecipitation confirmed a specific interaction between Cav1.2 and KCa1.1, and silencing Cav1.2 in N2a and HEK293 cells significantly reduced rabies virus titers, establishing that the L-type calcium channel is also required for productive infection.

The mechanistic picture became sharper when the researchers examined the role of the viral glycoprotein itself. Co-immunoprecipitation showed that Cav1.2 specifically interacts with the rabies virus glycoprotein, and a pull-down assay using purified virus particles demonstrated that Cav1.2 captures rabies virions in a dose-dependent manner. Binding assays revealed that Cav1.2 silencing or blockade did not reduce how much virus attached to cells, but internalization assays showed far less viral RNA inside treated cells. In other words, the glycoprotein engages Cav1.2, and this engagement is required to activate the channel and trigger the internalization machinery, even though the virus still docks normally on the cell surface when the channel is disabled.

The team also identified the downstream cellular process that the channel axis manipulates. Rabies virus enters cells through clathrin-mediated endocytosis with the help of actin, and previous work had shown that KCa1.1 promotes influenza virus entry by regulating F-actin polymerization. Using phalloidin staining to visualize filamentous actin, the researchers found that rabies virus entry induces F-actin polymerization in HEK293 cells, and that this polymerization was markedly reduced when Cav1.2 or KCa1.1 were knocked down or when cells were treated with diltiazem or paxilline. The authors therefore propose a sequential model: rabies virus glycoprotein binds Cav1.2 and triggers its activation; activated Cav1.2 then interacts with and activates KCa1.1; and activated KCa1.1 drives F-actin polymerization that powers the internalization of the virus particle.

The therapeutic implications rest on diltiazem, a benzothiazepine-class L-type calcium channel blocker approved in the United States in 1982 and long prescribed for hypertension, angina, and atrial arrhythmias. In cell culture, diltiazem blocked rabies virus infection in a dose-dependent manner without affecting viability, and four other FDA-approved Cav1.2 antagonists, the phenylalkylamine verapamil and the dihydropyridines nifedipine, nicardipine, and nimodipine, also inhibited the virus in vitro. The decisive test came in mice. Six-week-old C57BL/6J mice were inoculated intramuscularly with ten 50 percent mouse lethal doses of the rabies street virus GX/09, and six hours later they began receiving daily intramuscular injections of diltiazem at 25 or 50 milligrams per kilogram, or a phosphate buffer control. All 19 control mice died within 9 to 16 days with typical rabies signs such as body tremors and hind limb paralysis. At the lower dose, 5 of 19 mice survived, roughly 26 percent, while at 50 milligrams per kilogram, 13 of 19 mice survived, approximately 70 percent, and the survivors remained completely asymptomatic throughout a 28-day observation period.

Measurements of viral burden supported the idea that diltiazem works by blocking entry before the virus reaches the brain. No viral RNA was detectable in the brains of treated or control mice at day 4, but by day 6, viral RNA was present in all six control animals examined and in only two of six diltiazem-treated animals, and immunohistochemistry at day 8 detected rabies antigen in all control brains but only one of three treated brains. When the researchers delayed diltiazem administration until days 6 and 7, after viral replication was already underway in the brain, they observed no significant reduction in brain viral RNA, indicating that the drug’s protection is unlikely to stem from direct suppression of established central nervous system infection. Notably, the 50 milligram per kilogram mouse dose corresponds, after body surface area normalization under FDA guidelines, to a human equivalent dose of roughly 4 milligrams per kilogram, or about 240 milligrams per day for a 60-kilogram adult, which falls squarely within the range already used clinically for cardiovascular indications.

The authors argue that diltiazem could serve as an affordable, mass-produced complement to existing post-exposure prophylaxis, offering a stopgap where rabies immunoglobulin is unavailable and a potential enhancement where it is not, since it is estimated that only 1 to 10 percent of category III exposure patients currently receive the recommended treatment. The findings also extend a growing body of evidence that ion channels are conserved entry factors for diverse viruses: Cav1.2 has been implicated in SARS-CoV-2 and influenza virus entry, KCa1.1 in influenza virus endocytosis, and diltiazem has shown activity against rotavirus, porcine reproductive and respiratory syndrome virus, and porcine deltacoronavirus. With no approved drugs currently targeting KCa1.1, the potassium channel remains a target for future drug development, while Cav1.2, already served by three structural classes of clinically validated antagonists, stands out as a promising foundation for broad-spectrum antiviral strategies. Further studies will be needed to evaluate the in vivo efficacy of the other calcium channel blockers and to determine whether diltiazem concentrations in the central nervous system reach antiviral levels, but the prospect of repurposing a decades-old cardiovascular pill against one of humanity’s most lethal pathogens gives the global effort to eliminate rabies deaths a genuinely new weapon to test.

Subject of Research: Repurposing the calcium channel blocker diltiazem to block rabies virus entry via the Cav1.2-KCa1.1 ion channel axis

Article Title: Targeting the Cav1.2-KCa1.1 axis prevents rabies virus internalization and protects against lethal infection

Article References: Sun, Z., Wang, J., Wen, Z., Shuai, L., Sun, W., Yang, M., Wang, J., Chen, J., Mi, T., Zhou, X., Zhou, P., Lv, N., Zhao, Z., Ge, J., Chen, W., Wang, X., Wang, C., Bu, Z., & Wang, J. (2026). Targeting the Cav1.2-KCa1.1 axis prevents rabies virus internalization and protects against lethal infection. PLOS Pathogens, 22(10), e1014675. https://doi.org/10.1371/journal.ppat.1014675

Image Credits: AI Generated

DOI: 10.1371/journal.ppat.1014675

Keywords: rabies virus, diltiazem, Cav1.2, KCa1.1, ion channels, viral entry, endocytosis, drug repurposing, F-actin polymerization, post-exposure prophylaxis, PLOS Pathogens, antiviral therapy

Juvenile neuronal ceroid lipofuscinosis, also known as juvenile Batten disease, is caused by mutations in the CLN3 gene and represents a major form of juvenile dementia. Children with the condition typically develop vision loss in early childhood, followed by seizures, motor decline, and progressive cognitive deterioration, usually dying in their late twenties or thirties. The disease belongs to a family of lysosomal storage disorders in which cellular waste-processing machinery fails, leading to the accumulation of fatty waste materials called ceroid lipofuscin inside cells. Despite decades of research into the underlying genetics, effective therapies that slow the relentless neurodegeneration remain painfully out of reach.

The Korean team, led by Hyungkuen Kim, Eunmi Hwang, and Sung-Jo Kim, focused their investigation on parthanatos, a distinctive form of programmed cell death that has been extensively studied in Alzheimer’s disease but never before systematically characterized in JNCL. Parthanatos takes its name from poly(ADP-ribose), or PAR, a polymer produced by the enzyme PARP1. When cells experience excessive DNA damage, PARP1 becomes hyperactivated and floods the nucleus with PAR chains. These polymers eventually trigger the release of apoptosis-inducing factor from mitochondria, which migrates to the nucleus and orchestrates large-scale DNA fragmentation, killing the cell in a way that is distinct from classical apoptosis.

Using patient-derived lymphoblasts, immortalized white blood cells generated from JNCL patients, the researchers employed quantitative immunofluorescence microscopy to compare the disease cells with healthy controls. The results were striking. JNCL cells accumulated significantly higher levels of 8-oxoguanine, a pre-mutagenic DNA lesion produced when reactive oxygen species attack guanine bases, as well as elevated levels of PAR, the molecular signature of parthanatos. The accumulation of 8-oxoguanine is particularly insidious because it impairs DNA repair pathways, creating a vicious cycle in which oxidative damage begets repair failure, which begets further PARP1 overactivation and ultimately cell death.

The mechanistic logic connecting these observations to calcium is central to the study. JNCL is known to exhibit elevated intracellular calcium levels, a disturbance that has been documented in previous work on CLN3-deficient cells. Calcium overload is a well-established activator of PARP1, and studies in cardiomyocytes and other cell types have shown that calcium influx can directly stimulate PAR polymer production. In JNCL, this calcium-PARP1 axis appeared to be running unchecked, pushing vulnerable cells toward the parthanatos threshold. The researchers reasoned that if excess calcium was fueling the PARP1 fire, then dampening calcium entry might extinguish it.

Enter diltiazem. This L-type calcium channel blocker, which has been on pharmacy shelves since the 1970s, works by blocking voltage-gated calcium channels in heart and vascular smooth muscle. When the team treated JNCL patient-derived lymphoblasts with diltiazem, PAR accumulation dropped markedly. Critically, the protective effect persisted even when the cells were simultaneously exposed to a calcium ionophore, a chemical that forcibly floods cells with calcium. This suggested that diltiazem was not merely plugging calcium channels at the membrane but was intervening in the downstream signaling cascade that translates calcium excess into PARP1 activation, suppressing the expression of PARP1 itself and thereby limiting the raw material for parthanatos.

The drug’s benefits did not stop there. Diltiazem also reduced the accumulation of 8-oxoguanine in the JNCL cells, though intriguingly, this effect appeared to be largely independent of its calcium-blocking activity. Subcellular fractionation followed by immunoblotting, a technique that separates cellular compartments and identifies which proteins reside where, revealed that diltiazem enhanced the nuclear recruitment of OGG1, the primary enzyme responsible for excising 8-oxoguanine from DNA, along with its partner proteins. In other words, the drug was not just reducing the damage signal but was actively bolstering the cell’s repair crew, delivering the fix-it enzymes to the nucleus where the oxidative lesions accumulate.

To confirm that OGG1 was genuinely central to the drug’s protective mechanism, the researchers performed knockdown experiments, silencing the OGG1 gene in the treated cells. The results were unambiguous: when OGG1 was removed, diltiazem’s protective effects vanished entirely. This dependency establishes a dual role for the drug, acting simultaneously on calcium signaling to suppress PARP1 expression and on the OGG1-dependent base excision repair pathway to clear oxidative DNA damage. The finding also underscores a broader biological principle that has emerged from studies in Alzheimer’s disease and aging: OGG1-initiated repair is a double-edged sword, essential for genome maintenance but capable of exacerbating parthanatos when repair intermediates overstimulate PARP1.

The therapeutic implications are considerable. Diltiazem is an approved, well-characterized medication with decades of clinical safety data, which means that repurposing it for JNCL could potentially bypass some of the lengthy development timelines that plague novel drug candidates. Previous studies have already hinted at the drug’s relevance to neurodegeneration, with earlier work showing that L-type calcium channel blockers significantly lower intracellular calcium in CLN3-deficient neuroblastoma cells, and animal research demonstrating protective effects of diltiazem in chemically induced dementia models in mice. The new study adds a specific molecular mechanism, parthanatos suppression via PARP1 and OGG1 modulation, to this growing body of evidence.

Significant caveats remain before patients can benefit. The current findings derive from patient-derived lymphoblasts, which are accessible but imperfect proxies for the neurons that actually die in JNCL, and the researchers note that their data identify parthanatos as a novel therapeutic target rather than a validated clinical intervention. Whether diltiazem can cross the blood-brain barrier at sufficient concentrations, whether it will slow neurodegeneration in living organisms, and what dosing regimens would be appropriate for children all require further study. Nevertheless, by pinpointing PAR accumulation as a hallmark of JNCL pathology and demonstrating that an existing cardiovascular drug can interrupt this deadly cascade at two distinct points, the Hoseo University team has given the Batten disease research community something it has long needed: a concrete, pharmacologically tractable target in a disease where hope has been in short supply.

Subject of Research: Parthanatos and DNA repair dysfunction in juvenile neuronal ceroid lipofuscinosis and its modulation by the calcium channel blocker diltiazem

Article Title: The calcium channel blocker diltiazem ameliorates parthanatos in juvenile neuronal ceroid lipofuscinosis

Article References: Kim, H., Hwang, E., & Kim, S.-J. (2026). The calcium channel blocker diltiazem ameliorates parthanatos in juvenile neuronal ceroid lipofuscinosis. Journal of Molecular Medicine, 104(1), Article 118. https://doi.org/10.1007/s00109-026-02726-1

Image Credits: AI Generated

DOI: 10.1007/s00109-026-02726-1

Keywords: juvenile neuronal ceroid lipofuscinosis, Batten disease, CLN3, parthanatos, PARP1, diltiazem, 8-oxoguanine, OGG1, calcium signaling, DNA repair, drug repurposing, neurodegeneration