Biology subjects

Cantero, M. d. R.

Publications and source records attributed to Cantero, M. d. R..

7 recordsLinked to original sources

bioRxiv2025

Differences in Brain Microtubule Electrical Activity of the Hippocampus and Neocortex from the Adult Rat

Microtubules (MTs) are essential cytoskeletal structures in neurons that generate electrical oscillations in the frequency range of mammalian brain waves. However, the role of these MT oscillations in brain function remains largely unknown. Here, we sought to gain insight into MT electrical oscillatory activity from different brain regions with specific functions, the hippocampus and the neocortex from the adult rat brain. We obtained local field potentials (LFP) from the frozen brain regions under non-depolarized (high external NaCl) and depolarized (high external KCl) saline solutions, observing spontaneous oscillations under both conditions. The electrical oscillations of the brain tissue had different amplitudes in the absence (0 mV) or presence (100 mV) of holding potential and were inhibited by the MT stabilizer paclitaxel. A frequency domain spectral analysis of the time records revealed the presence of two major peaks at approximately [~]38 Hz and [~]93 Hz in both preparations. However, the energy contribution of each peak was different in the hippocampus compared to neocortex. Coupled with our electron microscopy observations, these data suggest that rat brain MTs produce electrical oscillations with specific properties in the various regions of the mammalian brain, which could be partially related as their intra-axonal distributions. MT oscillations may be implicated in the wave coherence of brain activity, supporting their contribution to the concept of a brain central oscillator that drives its function.

biophysics↗

bioRxiv2025

Dehydration Adaptation of the Adult Rat Modifies Brain Microtubule Electrical Activity of the Osmosensitive Supraoptic Nucleus

Brain microtubules (MTs) are important cytoskeletal structures in neurons that generate electrical oscillations in the frequency range of mammalian brain waves. However, the functional role of MT oscillations in the physiology of brain function remains unknown. Acute dehydration (DH) induced by water restriction results in changes in osmolality that require the modification of osmosensitive neuronal activity in the regions of the CNS involved in osmoregulation, in particular magnocellular neurons from the hypothalamic supraoptic nucleus (SON), responsible for the release of vasopressin and implicate osmosensor channels, associated with an intricate mesh of MTs. In the present study, we evaluated the effect of 24 h water deprivation (DH, dehydrated group) on the MT-based cytoskeleton of adult Wistar rats, as compared to a control group with free access to water (Control, euhydrated group). We obtained local field potentials (LFP) from isolated SON and cortex (CTX) brain tissue, observing spontaneous oscillations under both conditions. The electrical oscillations of the brain tissue from DH animals had different amplitudes and frequency peaks as compared to controls. A frequency domain spectral analysis of the time records indicated specific, SON-associated MT energy contributions of the frequency peaks in the challenged group, most particularly a dramatic increase in the 10-20 Hz range, and a statistically significant reduction in the high frequency 83-100 Hz region, not observed in the CTX samples. The data indicate that brain MTs respond to dehydration to produce electrical oscillations with distinct properties in the areas of the mammalian brain that specifically contribute to the homeostatic response. The present study provides the first evidence for a novel physiological mechanism associated with the electrical activity of the neuronal cytoskeleton and points to its possible involvement in the brain osmoregulatory mechanism.

biophysics↗

bioRxiv2025

Electrical Oscillations in Microtubules

Environmental perturbations and local changes in cellular electric potential can stimulate cytoskeletal filaments to transmit ionic currents along their surface. Advanced models and accurate experiments may provide a molecular understanding of these processes and reveal their role in cell electrical activities. This article introduces a multi-scale electrokinetic model incorporating atomistic protein details and biological environments to characterize electrical impulses along microtubules. We consider that condensed ionic layers on microtubule surfaces form two coupled asymmetric nonlinear electrical transmission lines. The model accounts for tubulin-tubulin interactions, dissipation, and a nanopore coupling between inner and outer surfaces, enabling luminal currents, energy transfer, amplification, and oscillatory dynamics that resemble the experimentally observed transistor properties of microtubules. The approach has been used to analyze how different electrolyte conditions and voltage stimuli affect electrical impulses shape, attenuation, oscillation, and propagation velocity along microtubules. Integrating transistor-like properties in the microtubules model has profound implications for intracellular communication and bioelectronic applications.

biophysics↗

bioRxiv2022

High Calcium Transport by Polycystin-2 (TRPP2) Induces Channel Clustering and Coordinated Oscillatory Behavior

The regulation by Ca2+ of Ca2+-permeable ion channels represents an important mechanism in the control of cell function. Polycystin-2 (PC2, TRPP2), a member of the TRP channel family (Transient Potential Receptor), is a Ca2+ permeable non-selective cation channel. Previous studies from our laboratory demonstrated that physiological concentrations of Ca2+ do not regulate in vitro translated PC2 (PC2iv) channel activity. However, the issue as to PC2s Ca2+ permeability and regulation remain ill-defined. In this study, we assessed Ca2+ transport by PC2iv, in a lipid bilayer reconstitution system in the presence of a high Ca2+ gradient (CaCl2 100 mM cis, CaCl2 10 mM trans). PC2iv channel reconstitution was conducted in the presence of either 3:7 or 7:3 1-palmitoyl-2-oleoyl-choline (POPC) and ethanolamine (POPE) lipid mixtures. Reconstituted PC2iv showed spontaneous Ca2+ currents, in both lipid mixtures with a maximum conductance of 63 {+/-} 13 pS (n = 19) and 105 pS {+/-} 9.8 (n = 9), respectively. In both cases, experimental data were best fitted with the Goldman-Hodgkin-Katz equation, showing a reversal potential (Vrev ~ -27 mV) consistent with strict Ca2+ selectivity. The R742X mutated PC2 (PC2R742X), lacking the carboxy terminal domain of the channel showed no differences with wild type PC2. Interestingly, spontaneous Ca2+ current oscillations were observed whenever PC2-containing samples were reconstituted in the 3:7, but not 7:3 POPC:POPE lipid mixture. The amplitude and frequency of the oscillations were highly dependent on the applied voltage, the imposed Ca2+ gradient, and the presence of high Ca2+, which induced PC2 channel clustering as observed by atomic force microscopy (AFM). We also used the QuB suite to kinetically model the PC2 channel Ca2+ oscillations based on the presence of subconductance states in the channel. The encompassed data provide new evidence to support a high Ca2+ permeability by PC2, and a novel regulatory feedback mechanism dependent on the presence of Ca2+ and phospholipids on its function. Statement of SignificanceThe regulation by Ca2+ of Ca2+-permeable ion channels represents an important mechanism in the control of cell function. The Transient Potential Receptor channel Polycystin-2 (TRPP2, PC2), is a Ca2+ permeable non-selective cation channel. Ca2+ transport by PC2 has largely been inferred by changes in reversal potential. This study provides experimental evidence on the Ca2+-transporting capabilities of PC2 in high Ca2+ that is modulated by lipids and generates a novel phenomenon of oscillatory currents by channel clustering and multiple subconductance behavior. PC2 can be self-regulated by feedback mechanisms, which are independent of external regulatory proteins. This oscillatory behavior, previously unknown for a single channel species, depend on the presence of Ca2+ interaction sites as have been postulated for the channel protein.

biophysics↗

bioRxiv2020

Ca2+-Dependent Regulation by the Cyclic AMP Pathway of Primary Cilium Length in LLC-PK1 Renal Epithelial Cells

The primary cilium is a sensory organelle projecting from the apical surface of renal epithelial cells. Dysfunctional cilia have been linked to a number of genetic diseases known as ciliopathies, which include autosomal dominant polycystic kidney disease (ADPKD). Previous studies have determined that renal epithelial primary cilia express both the polycystin-2 (PC2, TRPP2) channel and the type-2 vasopressin receptor (V2R), coupled to local cAMP production. However, little is known as to how Ca2+ and cAMP signals lead to changes in the length of the primary cilium. Here, we explored how cAMP signals regulate the length of the primary cilium in wild type LLC-PK1 renal epithelial cells. Primary cilia length was determined by immunocytochemical labeling of the ciliary axoneme. Treatment of cells with the cAMP analog 8-Br-cAMP (1 mM) in normal external Ca2+ (1.2 mM) produced a 25.3% increase (p < 0.0001) in the length of the primary cilium, a phenomenon also observed in cells exposed to high external Ca2+ (6.2 mM). However, exposure of cells to vasopressin (AVP, 10 M), which also increases cAMP in primary cilia of LLC-PK1 cells, mimicked the effect of 8-Br-cAMP in normal, but not in high Ca2+. Further, specific gene silencing of PC2 expression further increased primary cilium length after 8-Br-cAMP treatment, in normal, but not high Ca2+. The encompassed data indicate a crosstalk between the cAMP and Ca2+ signals to modulate the length of the primary cilium, in a phenomenon that implicates the expression of PC2. Significance StatementMorphological changes in primary cilia have been linked to genetic disorders, including autosomal dominant polycystic kidney disease (ADPKD), a major cause of kidney disease. Both cAMP and Ca2+ are universal second messengers that regulate polycystin-2 (PC2, TRPP2), a Ca2+ permeable non-selective cation channel implicated in ADPKD, and expressed in the primary cilium of renal epithelial cells. Despite current interest, little is known as to how second messenger systems and how aberrant regulation of PC2 may link primary cilium structure with cyst formation in ADPKD. Here we determined that both the cAMP analog 8-Br-cAMP and vasopressin increase the length of the primary cilium in renal epithelial cells. However, this phenomenon depends of external Ca2+ and PKD2 gene silencing. Proper cAMP signaling may be essential in the control of the primary cilium of renal epithelial cells, and the onset of cyst formation in ADPKD.

cell biology↗

bioRxiv2020

Electrical Oscillations of Brain Microtubules

Microtubules (MTs) are important cytoskeletal structures engaged in a number of specific cellular activities, including vesicular traffic and motility, cell division, and information transfer within neuronal processes. MTs also are highly charged polyelectrolytes. Recent in vitro electrophysiological studies indicate that different brain MT structures, including two-dimensional (2D) sheets (MT sheets) and bundles, generate highly synchronous electrical oscillations. However, no information has been heretofore available as to whether isolated MTs also engage in electrical oscillations, despite the fact that taxol-stabilized isolated MTs are capable of amplifying electrical signals. Herein we tested the effect of voltage clamping on the electrical properties of isolated non-taxol stabilized brain MTs. Electrical oscillations were observed on application of holding potentials between {+/-}200 mV that responded accordingly with changes in amplitude and polarity. Frequency domain spectral analysis of time records from isolated MTs disclosed a richer oscillatory response as compared to that observed in voltage clamped MT sheets from the same preparation. The data indicate that isolated brain MTs are electrical oscillators that behave as "ionic-based" transistors whose activity may be synchronized in higher MT structures. The ability of MTs to generate, propagate, and amplify electrical signals may have important implications in neuronal computational capabilities. Significance StatementMicrotubules (MTs) are important cytoskeletal structures engaged in a number of specific cellular activities. Recent in vitro electrophysiological studies indicate that different brain MT structures generate highly synchronous electrical oscillations. However, no information is available as to whether isolated MTs also engage in electrical oscillations. In the present study, we provide evidence that non-taxol stabilized isolated MTs generated electrical oscillations with richer frequency spectrum as compared to MT sheets. Thus, structured MT complexes may render more coherent responses at given oscillatory frequencies, suggesting entrainment in combined MT structures. The present study provides to our knowledge the first experimental evidence for electrical oscillations of single brain MTs.

biophysics↗

bioRxiv2020

Polycystin-2 (TRPP2) Regulates Primary Cilium Length in LLC-PK1 Renal Epithelial Cells

Polycystin-2 (PC2, TRPP2) is a Ca2+ permeable non-selective cation channel whose dysfunction generates autosomal dominant polycystic kidney disease (ADPKD). PC2 is present in different cell locations, including the primary cilium of renal epithelial cells. Little is known, however, as to whether PC2 contributes to the structure of the primary cilium. Here, we explored the effect(s) of external Ca2+, PC2 channel blockers, and PKD2 gene silencing on the length of primary cilia in wild type LLC-PK1 renal epithelial cells. To identify primary cilia and measure their length, confluent cell monolayers were fixed and immuno-labeled with an anti-acetylated -tubulin antibody. Although primary cilia length measurements did not follow a Normal distribution, data were normalized by Box-Cox transformation rendering statistical difference under all experimental conditions. Cells exposed to high external Ca2+ (6.2 mM) decreased a 13.5% (p < 0.001) primary cilia length as compared to controls (1.2 mM Ca2+). In contrast, the PC2 inhibitors amiloride (200 M) and LiCl (10 mM), both increased primary ciliary length by 33.2% (p < 0.001), and 17.4% (p < 0.001), respectively. PKD2 gene silencing by siRNA also elicited a statistically significant, 10.3% (p < 0.001) increase in primary cilia length, as compared to their respective scrambled RNA transfected cells. The data indicate that maneuvers that either regulate PC2 function or gene expression, modify the length of primary cilia in renal epithelial cells. Proper regulation of PC2 function in the primary cilium may be essential in the onset of mechanisms that trigger cyst formation in ADPKD. Significance StatementPolycystin-2 (PC2, TRPP2) is a Ca2+ permeable non-selective cation channel causing the autosomal dominant polycystic kidney disease (ADPKD). The importance of intact cilia and of fully functional polycystins in the onset of ADPKD cyst formation, point to yet unknown signaling mechanisms occurring within this organelle. We determined that the extracellular Ca2+ concentration, PC2 channel blockers, and PKD2 gene silencing, all contribute to the length of primary cilia in wild type LLC-PK1 renal epithelial cells. The data indicate that proper regulation of PC2 function in the primary cilium may be essential in the onset of mechanisms that trigger cyst formation in ADPKD.

cell biology↗