Pathophysiology of homocysteine: insights into ion channel dysfunction

Nikola Chmúrčiaková; Robin N Stringer; Leoš Cmarko; Alzbeta Filipova; Lubica Lacinova; Norbert Weiss

doi:10.1007/s00424-026-03165-0

. 2026 Mar 31;478(4):35. doi: 10.1007/s00424-026-03165-0

Pathophysiology of homocysteine: insights into ion channel dysfunction

Nikola Chmúrčiaková ¹, Robin N Stringer ², Leoš Cmarko ^1,², Alzbeta Filipova ³, Lubica Lacinova ³, Norbert Weiss ^2,^3,^✉

PMCID: PMC13038813 PMID: 41915221

Abstract

Homocysteine is a non-proteinogenic amino acid formed during the metabolism of methionine to cysteine and plays a critical role in maintaining cellular homeostasis. Although multiple enzymatic pathways tightly regulate homocysteine levels, their dysfunction can lead to elevated circulating homocysteine, which is recognized as a risk factor for various cardiovascular and neurological disorders. While most evidence linking homocysteine to specific pathologies comes from observational studies, emerging data suggest that dysregulation of ion channels may be an important underlying mechanism. In this review, we summarize the effects of homocysteine on the expression and function of key ion channel families including calcium, sodium, and potassium channels, and discuss their potential pathophysiological implications.

Keywords: Homocysteine, Ion channels, Pathophysiology, Cardiovascular disease, Neurological disease

Introduction

Homocysteine (Hcy) is a non-proteinogenic, sulphur-containing amino acid that serves as an essential intermediate in the metabolism of methionine to cysteine. In plasma, Hcy predominantly exists in oxidized forms, either bound in disulfides (70–80%) or as a mixed disulfide with other thiols (20–30%), whereas less than 1% is present as free thiol [57]. Although methionine is the sole dietary precursor of Hcy, its concentration is tightly regulated by a series of enzymatic reactions that require cofactors from the vitamin B complex, particularly vitamins B6 and B12, as well as folic acid. Accordingly, Hcy can be recycled back to methionine via the remethylation pathway or irreversibly metabolized to cysteine through the transsulfuration pathway [45] (Fig. 1).

Fig. 1 — Overview of homocysteine metabolism. Homocysteine occupies a central position in the methionine cycle and is regulated by remethylation and transsulfuration pathways. Folate-dependent remethylation involves the conversion of homocysteine to methionine via methionine synthase (MS) using 5-methyl-THF generated by MTHFR, with vitamin B12 as a cofactor. Methionine is converted to S-adenosylmethionine (SAM), the universal methyl donor, and subsequently to S-adenosylhomocysteine (SAH), which is hydrolyzed back to homocysteine. Homocysteine can alternatively be remethylated via a folate-independent, betaine-dependent pathway or irreversibly metabolized through the transsulfuration pathway to cysteine and glutathione via cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE)

These pathways maintain physiological plasma Hcy concentrations within a reference range of 5–13 µmol/L [108]. Notably, Hcy levels increase with age and are higher in men and postmenopausal women [33, 143]. Disruption of methionine metabolism, resulting from mutations in genes encoding enzymes involved in Hcy metabolism [121] or from deficiencies in essential cofactors such as vitamin B12 [27, 59], leads to elevated plasma Hcy levels, a condition termed hyperhomocysteinemia (HHcy). Depending on severity, HHcy is classified as mild to moderate (15–30 µmol/L) or severe (> 100 µmol/L) [89], and is recognized as an independent risk factor for a range of cardiovascular and neurological disorders [7, 65, 73, 90, 116, 138].

Since the first reports of elevated homocysteine levels in humans in 1962 [51, 126], HHcy has attracted sustained interest across multiple scientific disciplines. However, relatively few large-scale epidemiological studies have been conducted, making accurate estimates of its current global prevalence challenging. In 1999, one of the first large population-based studies reported that approximately 5–7% of the general population exhibited mild HHcy [57]. In contrast, more recent studies in the Chinese population suggest substantially higher prevalence rates, ranging from 37.2% [147] to 50.8% [103, 144]. Prevalence rates in other populations vary considerably and are influenced by multiple factors, including advancing age, increased body mass index, smoking status [144], genetic enzyme dysfunction, impaired availability of metabolic cofactors, excessive methionine intake, comorbid diseases, and the use of certain medications [76]. This apparent rise in HHcy prevalence underscores the need for in-depth investigation of the molecular mechanisms underlying its pathophysiological effects, particularly given the growing number of associations reported between HHcy and human disease. HHcy has been linked to a wide range of disorders affecting both the cardiovascular system, such as congestive heart failure and atherosclerosis, and the nervous system, including Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, and epilepsy [62] (Fig. 2).

Fig. 2 — Systemic effects of hyperhomocysteinemia on the cardiovascular and nervous systems. Schematic overview illustrating the major organ systems affected by elevated homocysteine levels. The left side (red) highlights the cardiovascular system, including the heart, large and small blood vessels, and the vascular endothelium, emphasizing homocysteine-associated alterations such as endothelial dysfunction, vascular remodeling, and thrombogenic changes. The right side (blue) depicts the nervous system, including the brain, peripheral nerves, and neuromuscular structures, illustrating the involvement of homocysteine in neurotoxicity, altered neuronal excitability, and peripheral neuropathies. Insets represent selected cellular and tissue-level targets within each system, underscoring the multisystemic impact of hyperhomocysteinemia

Elevated Hcy levels have also been associated with osteoporosis, chronic renal failure, hypothyroidism, insulin-resistant diabetes, polycystic ovarian syndrome, and gastrointestinal disorders [3]. These associations raise an important question: what are the molecular mechanisms through which HHcy contributes to the development of such diverse pathologies? From an etiological perspective, dysregulation of ion-permeable channels, including calcium, sodium, and potassium channels, represents a plausible mechanistic link. These channels play essential roles in maintaining cardiovascular and nervous system homeostasis and are frequently targeted by pharmacological therapies [19, 87]. Moreover, these ion channels are key regulators of cellular electrical excitability, and their dysfunction correlates with many neurological and cardiovascular manifestations associated with HHcy. For instance, neurological symptoms include seizures, particularly in inherited forms of HHcy due to cystathionine β-synthase deficiency [10], as well as anxiety [37], depression [11], psychosis, cognitive impairment, and peripheral neuropathy [86]. In parallel, altered excitability of cardiomyocytes contributes to HHcy-associated arrhythmias and dysregulation of vascular tone [92].

In this review, we focus on three major ion channel families: calcium, sodium, and potassium channels. For each family, we first provide a general overview of the channels, then describe how HHcy affects their function across endothelial, cardiac, and neuronal cells, drawing on evidence from both in vivo animal models and in vitro cellular studies.

Animal models of hyperhomocysteinemia

Animal models are essential tools for investigating the molecular mechanisms underlying human diseases associated with HHcy. Numerous approaches have been developed to induce HHcy in animals, each with specific advantages and limitations in recapitulating impaired homocysteine metabolism observed in humans [32] (Fig. 3).

Fig. 3 — Determinants and experimental models of hyperhomocysteinemia. Schematic representation of the major factors contributing to hyperhomocysteinemia (HHcy) in humans (right, green) and the principal strategies used to induce HHcy in animal models (left, orange). In humans, elevated homocysteine levels arise from a combination of dietary factors (e.g. B-vitamin deficiency, increased methionine intake, limited choline), lifestyle influences (smoking, obesity, alcohol consumption), physiological variables (age, sex), pathological conditions (such as chronic renal failure), genetic mutations affecting one-carbon metabolism (e.g. *MTHFR*, *CBS*), and pharmacological treatments (e.g. levodopa, methotrexate). In experimental animals, HHcy is commonly induced through dietary manipulation, parenteral administration of methionine or homocysteine, maternal HHcy, or drug treatment. The figure highlights the conceptual parallels between clinical and experimental determinants of HHcy

The most common method involves dietary manipulation of one-carbon (C1) metabolism, typically through diets deficient in B vitamins, especially B6, B12, and folic acid, or by L-methionine overconsumption [12, 25, 64]. In some studies, homocysteine itself or methyl group acceptors that interfere with C1 metabolism are administered directly to animals to elevate systemic Hcy levels [101]. Dietary approaches provide continuous exposure, mimicking chronic HHcy conditions.

An alternative strategy employs parenteral administration of homocysteine, usually via subcutaneous or intraperitoneal injection [78]. Unlike dietary methods, the chronicity and intensity of HHcy depend on injection frequency and dosage, offering a more controlled temporal elevation of Hcy.

Genetic approaches represent another major category of animal model. Mutations are introduced into genes encoding key enzymes in C1 metabolism, most notably cystathionine beta-synthase (CBS), which converts homocysteine to cystathionine in a vitamin B6-dependent manner [2, 131]. Disruption of CBS function leads to Hcy accumulation, recapitulating HHcy observed in humans with CBS deficiency [40]. Similarly, methylenetetrahydrofolate reductase (MTHFR), required for vitamin B12-dependent remethylation of homocysteine to methionine, has been targeted to generate genetic models of HHcy [2, 26].

Some studies focus on the maternal HHcy effect, where elevated Hcy levels in pregnant females result in increased exposure in their offspring, either through the placenta or via lactation. By increasing maternal homocysteine before, during, or after pregnancy, Hcy crosses the placenta or is transferred via breast milk, affecting developing embryos and neonates without directly manipulating the pups [141]. This approach allows investigation of developmental and early-life effects of HHcy.

A minority of studies combine multiple methods to induce HHcy, such as pairing dietary manipulation with genetic modifications, or employ alternative strategies designed to simulate human lifestyle influences. In addition, primary cultures of animal cells treated with Hcy have been widely used to study cellular and molecular effects in a controlled in vitro environment [44, 107].

Collectively, these animal and cellular models provide a robust framework for exploring the mechanistic consequences of HHcy, particularly its modulation of calcium, sodium, and potassium channels in endothelial, cardiac, and neuronal cells, which are discussed in the following sections.

Calcium channels

General overview

Calcium channels constitute a major superfamily of proteins that allow the influx of calcium ions from both intracellular stores and the extracellular milieu [8, 28]. Calcium influx plays a dual role: it contributes to cellular excitability by enhancing positive charge inside the cell, and it acts as a second messenger regulating numerous signaling cascades [23, 146]. Through these mechanisms, calcium channels maintain intracellular calcium homeostasis and tightly control several cellular processes, including heart and muscle contractions, neurotransmission, learning and memory, embryonic formation and development, cell proliferation and apoptosis, cell division and differentiation, energy metabolism, protein phosphorylation and dephosphorylation, and gene expression and regulation [112, 132, 145].

Two families of calcium-permeable channels play a role in HHcy: voltage-gated calcium channels (VGCCs, Ca_v) [21, 24, 42, 146] and ligand-gated calcium channels (LGCCs). Voltage-gated calcium channels comprise several subtypes that are commonly classified according to their activation properties into high-voltage-activated (HVA) channels, which require strong membrane depolarization, and low-voltage-activated (LVA) channels, which open at comparatively more negative membrane potentials. Among VGCCs, high-voltage-activated L- and P/Q-types, and low-voltage-activated T-type channels are particularly relevant to HHcy, whereas other VGCCs have not been documented. HVA L-type VGCCs are characterized by slow activation and inactivation kinetics and play pivotal roles in several organ systems, including the smooth muscle of the intestine and vasculature, the heart, and the central nervous system (CNS). In neurons, L-type channels contribute to excitability, neurotransmitter release, learning, and memory. Dysregulation of these channels has been linked to various cardiac and neuronal disorders, including Timothy syndrome, hypokalemic periodic paralysis, and bipolar affective disorder [20]. L-type channels carry calcium influx predominantly during the plateau phase of the action potential (AP), a contribution that is particularly pronounced in cardiomyocytes and also affects the frequency of AP firing in neuronal cells [9].

P/Q-type calcium channels are another class of HVA channels, highly expressed in the CNS, predominantly in cerebellar Purkinje and granule cells. They are primarily localized in presynaptic terminals, where they mediate fast neurotransmission through vesicular exocytosis driven by calcium influx [38], but they also influence postsynaptic calcium signaling in Purkinje cells [139]. The CACNA1A gene encoding P/Q channels undergoes extensive alternative splicing, producing a wide array of P/Q channels with distinct electrophysiological properties [13]. Dysregulation of P/Q channels has been associated with disorders such as episodic ataxia type 2 (EA2), spinocerebellar ataxia type 6 (SCA6), and familial hemiplegic migraine-1 (FHM1) [70, 96, 136, 137].

T-type channels, classified as LVA channels, exhibit fast activation and inactivation kinetics and a low activation threshold near the resting membrane potential of many neurons [100, 133, 135]. These channels are widely distributed throughout the body, including the brain, heart, muscles, endocrine cells, and bones. They play crucial roles in regulating membrane oscillations and pacemaking [67], initiating low-threshold calcium spikes and AP bursts in neuronal cells, and mediating hormone secretion. Dysregulation of T-type channels has been implicated in several pathophysiological conditions, including pain, atrial fibrillation, epilepsy, hypertension, cancer, and congestive heart failure, although most of these disorders involve multiple contributing factors beyond T-type channel activity [68, 134].

In addition to VGCCs, a diverse group of ligand-gated and receptor operated channels can also mediate calcium entry. These include ionotropic glutamate receptors such as NMDA receptors [39], as well as other calcium-permeable channels including acid-sensing ion channels (ASICs) [104] and several members of the transient receptor potential (TRP) channel family [148], which contribute to neuronal excitability and sensory signaling in multiple physiological and pathological contexts, such as in the pain pathways [14]. Ligand-gated calcium channels, notably NMDA receptors of the glutamate receptor family, mediate excitatory neurotransmission in the CNS by allowing the influx of sodium and calcium ions, triggering depolarization that activates voltage-gated sodium channels and AP firing. NMDA receptors are extensively expressed throughout brain development and are central to normal brain function, including neuronal development and synaptic plasticity [98]. Consequently, alterations in NMDA receptor activity have been associated with neurological and psychiatric disorders such as Alzheimer’s disease, ischemic stroke, epilepsy, schizophrenia, and mood disorders, many of which overlap with conditions linked to HHcy [30].

Effects of HHcy on calcium channels

Endothelial cells

HHcy profoundly affects calcium homeostasis in endothelial cells and vascular smooth muscle cells (VSMC) (Fig. 4; Table 1).

Table 1.

Effects of homocysteine on ion channels across different cell types

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Summary of reported effects of homocysteine on calcium (blue), sodium (beige), and potassium (green) channels in epithelial, neuronal, and cardiac cell types

In VSMCs from healthy rats, Hcy induces a transient rise of intracellular calcium primarily through a release of intracellular calcium stores, such as the endoplasmic reticulum (ER). The suggested mechanism involves the activation of the phosphoinositide signaling pathway by Hcy followed by IP3 (inositol 1,4,5-trisphosphate)-mediated calcium release [93]. Similar effects have been observed in human umbilical VSMCs, where, in contrast, Hcy enhances extracellular calcium influx through voltage-gated calcium channels, contributing to vasospasm and the suppression of endothelial nitric oxide (NO) synthesis [95]. This increase in cytosolic calcium enhances cellular sensitivity to angiotensin II, which triggers intracellular signaling cascades producing reactive oxygen species (ROS). ROS, in combination with Hcy, further suppress NO production [124], a known vasodilator and anti-thrombogenic factor in vascular endothelial cells [72, 102]. These mechanisms play a major role in promoting hypercoagulation and atherosclerosis, with studies demonstrating a positive correlation between plasma Hcy levels and the progression of atherosclerotic lesions [115]. In human endothelial progenitor cells, elevated Hcy induces apoptosis and ER stress-mediated caspase-3 activation, accompanied by calcium release from intracellular stores [83]. Together, these effects explain, at least in part, the contribution of HHcy to vascular dysfunction and suggest potential impacts on neurological disorders involving the vascular system, such as ischemic stroke, headaches, and vascular dementia.

Cardiac cells

In cardiac tissues, HHcy has been shown to alter calcium dynamics in atrial myocytes (Fig. 5; Table 1).

Mice fed a high Hcy diet exhibit increased L-type calcium current, enhanced late sodium current, and elevated intracellular calcium concentrations [56]. This increase may be partly due to Hcy-induced upregulation of the IP3 receptor type 1 (IP3R1). Prolongation of the AP duration has also been observed, which may elevate the risk of atrial fibrillation. While increased calcium influx contributes to this effect, enhanced late sodium current and inhibition of repolarizing transient outward and delayed rectifier potassium currents also play important roles in prolonging the AP [17]. Moreover, Hcy induces calcium influx through T-type channels in platelets from healthy human volunteers, enhancing platelet activation and aggregation, which may contribute to thrombogenic effects seen in HHcy patients [6, 35, 81]. In cardiac neural crest cells isolated from chick embryos, Hcy triggers IP3-mediated release of calcium from the ER, increasing cell attachment while inhibiting migration, resulting in neurocristopathy [60]. Collectively, these calcium-dependent effects in endothelial cells, VSMCs, and cardiac neural crest cells may contribute to the congestive heart failure frequently observed in patients with HHcy, although this is a multifactorial condition and direct causality remains complex to establish. Nevertheless, plasma Hcy levels serve as a useful diagnostic marker for future cardiac complications [29].

Neuronal cells

In neuronal cells, elevated Hcy concentrations affect both the release of calcium from intracellular stores and extracellular influx (Fig. 6 and Table1)

In cultured human neuroblastoma cells, Hcy directly stimulates calcium influx through NMDA receptors, which leads to neurotoxic effects upon exposure to high concentrations of Hcy [62]. Subsequent studies confirm NMDA receptor involvement in Hcy-mediated calcium signaling, using cortical neurons from embryonic mice and rats [63, 75]. Blockade of NMDA channels with MK-801 prevents this calcium influx, demonstrating that Hcy acts as an agonist. Subunit-specific effects have been observed, with GluN1/2A-composed NMDA receptors preferentially activated in cortical neurons and GluN2C/D-composed receptors in cerebellar neurons [110, 111].

In dorsal root ganglion (DRG) neurons from wild-type chicks, Hcy primarily increases calcium influx through L-type VGCCs possibly by affecting channel trafficking, preceding calcium release from internal stores [123]. This calcium overload promotes ROS generation, which further stimulates P/Q-type voltage-dependent channels, amplifying intracellular calcium overload [94]. Such dysregulation induces oxidative stress and apoptosis in neuronal cells, contributing to neurological disorders associated with HHcy, including age-related macular degeneration, Alzheimer’s disease, Parkinson’s disease, autism, schizophrenia, bipolar disorder, vascular dementia, peripheral neuritis, stroke, multiple sclerosis, epilepsy, and headaches [30, 46, 47, 117].

Through a mechanism involving activation of protein kinase C-dependent signaling pathway, Hcy enhances T-type currents in DRG neurons, by promoting the recycling of Ca_v3.2 channels back to the plasma membrane [50]. Consequently, in a rat model of prenatal hyperhomocysteinemia, chronic elevation of homocysteine leads to peripheral neuropathy, consistent with the role of Ca_v3.2 channels in pain transmission. Pharmacological blockade of T-type channels restores normal mechanical sensation, confirming their involvement in Hcy-induced neuropathic phenotypes [50].