The regulation of renal function is strongly coupled with cardiac sensory input (Feng et al., 2025; Johns, 2013). Stretch-sensitive receptors located in the atrial walls and great vessels detect changes in cardiac filling and wall tension and transmit this information via afferent neural pathways to central autonomic control centers (van Weperen and Vaseghi, 2023). Through this cardiopulmonary afferent signaling, the heart exerts a profound influence on renal sympathetic nerve activity and thereby modulates sodium and water excretion (Evans and Bie, 2016). In the setting of structural cardiac remodeling—such as in heart failure without overt systolic dysfunction (Hamo et al., 2024)—this reflex arc may become impaired, leading to dysregulated renal sympathetic outflow and inappropriate fluid retention (DiBona et al., 1996).

We recently demonstrated that in heart failure without overt systolic dysfunction, the regulation of sympathetic renal nerve activity via cardiac afferent pathways is impaired (Pickny et al., 2023). This disturbance appears to originate from dysfunctional sensory nerve endings embedded in the altered myocardial tissue (Chapleau et al., 2001; Moore, 2024; Pauziene et al., 2023). Primary afferent neurons located in the nodose ganglion, which relay signals from the heart, displayed increased excitability and elevated firing rates in vitro. However, this enhanced neuronal responsiveness did not restore afferent-mediated reflex function in vivo, suggesting that other mechanisms—beyond simple excitability—are involved in the breakdown of afferent control.

Cardiac afferents are predominantly unmyelinated C-fibers with chemo- and mechanosensitive (MS) properties (Veelken et al., 2003), and their excitability depends on the coordinated activity of ion channels mediating depolarization and repolarization. Although voltage-gated sodium channels such as Nav1.7 and Nav1.8 initiate action potentials (Catterall, 2023; Vasylyev et al., 2024; Ditting et al., 2016; Kwong et al., 2008), they are not the primary focus of the present study. Rather, we concentrated on the repolarization phase, which is shaped largely by potassium conductance and may be influenced by mechanical stimuli. MS potassium channels are strong candidates for dynamically adjusting afferent excitability in response to changes in cardiac wall tension.

Despite MS Piezo1 and Piezo2 channels having emerged as highly prominent targets of research (Lacroix and Wijerathne, 2025; Wang and Xiao, 2018) and having advanced our understanding of cardiovascular mechanotransduction and baroreceptor signaling (Zeng et al., 2018; McGrane et al., 2025), these non-selective cation channels primarily conducting calcium play only an indirect role in neuronal repolarization. In contrast, potassium channels activated by mechanical stress are directly suited to terminate action potentials and reset neuronal excitability.

However, while Piezo channels are an essential element for mechanoelectrical coupling, their role in neuronal repolarization is likely indirect. The role of Ca²⁺-sensitive potassium channels in action potential generation is considered modulatory in terms of neuronal excitability (Orfali and Albanyan, 2023). In contrast, potassium channels activated by mechanical stress are directly suited to terminate action potentials and reset neuronal excitability (Kanda et al., 2023). This distinction underlies the rationale of the present study, which focuses on identifying and characterizing MS potassium channels as potential key modulators of cardiac afferent function.

The present study was, therefore, designed to test whether MS potassium channels are functionally expressed in nodose ganglion neurons with cardiac projections. We used whole-cell patch-clamp recordings from primary cultures of nodose ganglion neurons and applied gadolinium—a known blocker of stretch-activated ion fluxes (Zhang et al., 2018)—to assess the presence and functional contribution of stretch-sensitive potassium conductance in these neurons. We focused specifically on cardiac afferent neurons because of their central role in modulating renal sympathetic nerve activity via cardiopulmonary reflex pathways (Feng et al., 2025; Johns, 2013; van Weperen and Vaseghi, 2023). Hence, a special method was used, as previously described, to identify neurons with cardiac axons in the nodose ganglion (Pickny et al., 2023). Understanding how cardiac sensory neurons process mechanical input is essential for elucidating the mechanisms that couple cardiac filling states to renal sodium and water excretion.

For the experiments, male Sprague–Dawley rats (Ivanovas, Kisslegg, Germany) weighting 250 g–300 g were maintained in cages at 24 ± 2 °C. They were fed a standard rat diet (no. C 1000, Altromin, Lage, FRG) containing 0.2% sodium by weight and were allowed free access to tap water. All procedures performed on the animals were carried out in accordance with the guidelines of the American Physiological Society and in compliance with the NIH Guide for Animal Care and Use in Laboratory Practice. They were approved by the local government agency (Regierung von Unterfranken). We retrogradely labeled cardiac neurons in twelve rats. The preparation failed in 5% of the animals. A total of 753 successful excised inside-out patch recordings out of a total of 820 patches were included in the data analysis.

We hypothesized that neurons from the nodose ganglion with cardiac afferents should contain MS potassium channels that, among other effects, will support proper repolarization and post-hyperpolarization phases during action potential generation.

This paper could eventually demonstrate that in nodose ganglion neurons with cardiac afferents, there must exist at least two types of stretch-activated MS ion channels with predominant potassium conductance besides other proposed putative mechanotransducing mechanisms (Zeng et al., 2018). One exhibited a slope conductance of 116 pS and was blocked by 100 μM Gd³⁺, a known inhibitor of MS currents (Zhang et al., 2018). It has likely been described using cell-attached measurements previously (Hamill and Martinac, 2001). The author showed that in a co-culture with aortic endothelial cells, the number of patches on neurons containing these MS channels increased This points to a diffusible factor from endothelial cells, which might regulate the expression of these channels. The author was able to block MS activity when 20 µM of gadolinium was applied.

The other channel we could characterize was insensitive to extracellular application of 100 μM Gd³⁺ and had a smaller conductance of 76 pS. Interestingly, the channel with lower conductivity and, therefore, smaller channel width was insensitive to Gd³⁺, for the action of this substance is sometimes observed as mainly obstructing the pore. In a simple model, it would be easier to obstruct a small pore than a wide pore. Hence, not only the diameter but also the geometry or electrostatic properties of the pore may have an influence on the attachment and blocking effect of Gd³⁺. The insensitive channel shared properties of a 54 pS stretch-activated channel in colon sensory neurons (Su et al., 2000), which was also unaffected by 100 μM Gd³⁺, but in contrast to this channel, the MS channels of nodose neurons were not influenced by 40 mM TEA either on the extracellular side of the membrane.

Although gadolinium is considered a general inhibitor, several studies have shown that not all cellular mechano-transduction depended on Gd³-sensitive channels, so a classical Gd³ blockade of stretch-activated or MS cation channels failed to inhibit a mechanically evoked response (Sakamoto et al., 2010; Malek and Izumo, 1996; Steffensen et al., 1991).

Hence, gadolinium can help—as in our study—to distinguish between MS cation channels. The exact meaning of this finding is not yet understood.

Stretch-inactivated channels that were described in other preparations have not been observed at any patch on nodose neurons with cardiac afferents (Matsumoto et al., 2011). The existence of a mechanical notch filter that limits the range of the cell potential by two antagonizing channel types can, therefore, be assumed to be rather unlikely (Sackin, 1995a; Murthy, 2023).

The channels were predominantly selective for potassium and sodium and can, therefore, be classified as cation channels. In our measurements, the relative permeability of potassium versus sodium was 3.4 in Gd³⁺-sensitive channels and 3.3 in Gd³⁺-insensitive channels. Taking the Goldman equation into account with typical ion concentrations ([Na]_ec/[Na]_ic = [K]_ic/[K]_ec = 140 mM/5 mM), the reversal potential of the channels could depolarize the neurons to −33 mV, where voltage-sensitive sodium conductances are already activated.

Chloride or calcium conductances could not be detected in the two types of channels in our experiments. The calcium gradient used did not shift the reversal potential significantly from 0 mV. Therefore, the eventual permeability for this ion must be very low. Sharma et al. (1995) detected Gd³⁺-sensitive increases in intracellular Ca²⁺ of approximately 500 nM using the fura-2 technique when nodose neurons were mechanically stimulated. However, until now, it has not been possible to determine special membrane channels responsible for this increase.

When symmetrical potassium solutions were used, the two channel types showed the same reversal potential (0 mV) and a linear I–V relationship. The open probabilities (Bennett et al., 2000) were independent of voltage, suggesting that no voltage sensor is present in the channel proteins. So, they are unlikely to be influenced during the generation of action potentials in the neurons. Voltage insensitivity is a common feature of many MS ion channels in different preparations (Su et al., 2000; Sackin, 1995a).

The number of active MS channels in a patch decreased when extracellular calcium was buffered with EGTA. This suggests a new gating mechanism for neuronal MS channels as calcium normally acts from the intracellular side of the membrane on channel proteins, as observed in calcium-activated potassium channels. Extracellular Ca²⁺-dependent inhibition is known from the hair bundles in vertebrate hair cells, which are involved in the process of hearing. Here, mechanotransduction is abolished when reduced Ca²⁺ disrupts the tip links (Hamill and Martinac, 2001; Bennett et al., 2000). Calcium may modulate the structure of the channel protein, rendering it stretch-sensitive or inducing an active conformational state. On the other hand, EGTA (Kra et al., 1998) might be involved in the interaction itself.

The activity of MS ion channels in cardiac nodose neurons increased when the patch-clamp mode was changed from cell-attached to inside-out. Activation by the force of pipette retraction is not likely because microscopy analysis of patches showed purely fixed membranes (Hamill and Martinac, 2001; Gil et al., 1999). There is more evidence that membrane–cytoskeleton interactions are involved in the activation, as other authors have reported (Kra et al., 1998; Cunningham et al., 1997). Here, cytochalasin D and colchicine, which disrupted filaments of the cytoskeleton, are potent enough to increase channel activity in cell-attached patches (Lesage et al., 2000; Maingret et al., 1999). So, these channels may be inactivated by intracellular components in the parent cell.

The openings of nodose MS channels exhibited a flickering behavior, indicating an extremely low open-state time constant that could not be resolved by standard patch-clamp amplifiers. Based on amplifier specifications, the time constant must be faster than 0.1 ms. This limitation impacted both the accuracy of current measurements and the precise calculation of channel conductance. The flickering states were integrated into longer-lasting bursts on the millisecond timescale. In MS channels of chicken muscle cells, Guharay and Sachs (1984) showed that only this state was influenced by the cytoskeleton. Using cytochalasin, all other states remained unchanged (Sackin, 1995a; Sackin, 1995b).

Recent high-resolution electrophysiological studies have confirmed the presence of such rapid gating events in MS ion channels, emphasizing the need for advanced methods such as high-speed patch-clamp and fluorescence-based imaging to resolve these fast transitions (Cox et al., 2018; Wu et al., 2017; Jiang et al., 2021). The flickering states are typically integrated into longer bursts lasting several milliseconds.

In line with earlier findings, recent research confirms that cytoskeletal elements selectively influence particular gating states of MS channels. In particular, actin filament disruption using agents such as cytochalasin D alters prolonged open states, while rapid flicker transitions remain largely unaffected. These observations support a model in which mechanical force is transduced not only via membrane tension but also through tethering to the cytoskeletal network, particularly actin filaments (Jiang et al., 2021; Ingber, 2006).

In summary, the Gd-sensitive and -insensitive potassium channel types of cardiac nodose ganglion neurons shared many equivalent properties, with the exception of the conductivity. Further work is needed to elucidate the extent to which crosstalk between these channels and voltage-gated sodium channels influences the sensitivity and activity of cardiac afferent pathways in both healthy and diseased conditions.

Beyond the focus of this investigation, a large variety of stretch-activated channels (Webster et al., 2025) can be found in the nodose ganglion in general: Piezo1/2 were identified as directly stretch-activated cation channels essential for baroreceptor function, and their deletion abolished reflex-mediated activity (Zeng et al., 2018). In hypertensive rats, activation of Piezo1 by Yoda1 lowered the blood pressure through increased afferent firing, an effect prevented by GsMTx4, while SERCA2 inhibition attenuated these currents, indicating Ca²⁺-dependent gating (Cui et al., 2024; Zhao et al., 2025). TTN3 contributed to dynamic pressure sensing and baroreflex maintenance (Lu et al., 2020). Among TRP channels, TRPV4/TRPA1 were diet-sensitive, TRPC1/3 localized to low-threshold complexes, and TRPA1 and TRPV1 mediated visceral and gastro-esophageal mechanosensitivity, respectively. ENaC/ASIC2 deficits impaired baroreflex integration, while HCN channels, nitric oxide, and cytoskeleton-dependent stretch conductances further shaped mechanosensory excitability (Sharma et al., 1995; Kra et al., 1998; Li et al., 2016; Lu et al., 2009; Doan et al., 2004; Li et al., 1998). In addition to Piezo and TRP channels, members of the K2P family, such as TREK-1, have also been described in nodose ganglion neurons as MS potassium channels (Park et al., 2023). Although our data cannot exclude their contribution, the biophysical properties we observed (unitary conductance, Na⁺ permeability, and Gd³⁺ sensitivity) suggest that at least two distinct populations of MS K⁺ channels coexist. Future studies will be required to determine whether TREK1 or related K2P channels account for one of the populations described here.

A more definitive classification of the channels described in this publication would ideally involve genetic or structural analyses. Such experiments, however, were beyond the scope and feasibility of the present study. Instead, we adopted a functional classification approach based on comparisons with established mechanotransducers. Although this provides valuable insights into their properties, future studies employing genetic or structural methods will be required to confirm their molecular identity and further refine their mechanistic understanding.