Pyramidal neurons in the medial prefrontal cortical layer 2/3 are an essential contributor to the cellular basis of working memory; thus, changes in their intrinsic excitability critically affect medial prefrontal cortex (mPFC) functional properties. Transient Receptor Potential Melastatin 4 (TRPM4), a calcium-activated nonselective cation channel (CAN), regulates the membrane potential in a calcium-dependent manner. In this study, we uncovered the role of TRPM4 in regulating the intrinsic excitability plasticity of pyramidal neurons in the mouse mPFC layer of 2/3 using a combination of conventional and nystatin perforated whole-cell recordings. Interestingly, we found that TRPM4 is open at resting membrane potential, and its inhibition increases input resistance and hyperpolarizes membrane potential. After high-frequency stimulation, pyramidal neurons increase a calcium-activated non-selective cation current, increase the action potential firing, and the amplitude of the afterdepolarization, these effects depend on intracellular calcium. Furthermore, pharmacological inhibition or genetic silencing of TRPM4 reduces the firing rate and the afterdepolarization after high frequency stimulation. Together, these results show that TRPM4 plays a significant role in the excitability of mPFC layer 2/3 pyramidal neurons by modulating neuronal excitability in a calcium-dependent manner.

Cell migration is critical for several physiological and pathophysiological processes. It depends on the coordinated action of kinases, phosphatases, Rho-GTPases proteins, and Ca²⁺ signaling. Interestingly, ubiquitination events have emerged as regulatory elements of migration. Thus, the role of proteins involved in ubiquitination processes could be relevant to a complete understanding of pro-migratory mechanisms. KCTD5 is a member of Potassium Channel Tetramerization Domain (KCTD) proteins that have been proposed as a putative adaptor for Cullin3-E3 ubiquitin ligase and a novel regulatory protein of TRPM4 channels. Here, we study whether KCTD5 participates in cell migration-associated mechanisms, such as focal adhesion dynamics and cellular spreading. Our results show that KCTD5 CRISPR/Cas9- and shRNA-based depletion in B16-F10 cells promoted an increase in cell migration and cell spreading, and a decrease in the focal adhesion area, consistent with an increased focal adhesion disassembly rate. The expression of a dominant-negative mutant of Rho-GTPases Rac1 precluded the KCTD5 depletion-induced increase in cell spreading. Additionally, KCTD5 silencing decreased the serum-induced Ca²⁺ response, and the reversion of this with ionomycin abolished the KCTD5 knockdown-induced decrease in focal adhesion size. Together, these data suggest that KCTD5 acts as a regulator of cell migration by modulating cell spreading and focal adhesion dynamics through Rac1 activity and Ca²⁺ signaling, respectively.

Neurons undergo a series of perturbations that alter their firing rate and synaptic transmission; however, they can adapt to keep a target level of electrical activity in the long term. Muscarinic receptor (mAChR) transmission modulates intrinsic excitability and allows for fast changes through phasic transmission and long-term effects through volume transmission. Earlier studies on mAChR transmission have primarily focused on the effects of long-term mAChR stimulation on excitability; however, the impact of long-term inhibition is still unknown. In this study, we used a combination of patch-clamp and immunofluorescence techniques to examine the effects short-term (3 h) and long-term (0–10 days) muscarinic or nicotinic (nAChR) receptor inhibition on the intrinsic excitability of cortical pyramidal neurons in culture. We found that short term mAChR or nAChR inhibition has no effect either in AIS or in neuronal excitability, however, prolonged mAChR, but not nAChR blockade, increases the AIS length with no change in its position. Moreover, prolonged mAChR blockade increases firing frequency and intrinsic excitability, through a reduction in the action potential duration that is the result of an increase in a 4-AP sensitivity K⁺ current in cortical pyramidal neurons in culture. Together, our work demonstrates that prolonged mAChR, but not nAChR, blockade induces structural and functional changes to compensate for the lack of mAChR signaling and to sustain a target level of electrical activity.

TRPM4 is a calcium activated non-selective cation channel, impermeable to Ca²⁺, in neurons it has been implicated in the regulation of the excitability and in the persistent firing. Cholinergic stimulation is also implicated in changes in excitability that leads neurons to an increased firing frequency, however it is not clear whether TRPM4 is involved in the cholinergic-induced increase in firing frequency. Here using a combination of patch clamp electrophysiology, Ca²⁺ imaging, immunofluorescence, fluorescence recovery after photobleaching (FRAP) and pharmacological approach, we demonstrate that carbachol (Cch) increases firing frequency, intracellular Ca²⁺ and that TRPM4 inhibition using 9-Ph and CBA reduces firing frequency and decreases the peak in intracellular Ca²⁺ induced by Cch in cortical pyramidal neurons in culture. Moreover, we determined that cholinergic stimulation reduces TRPM4 recycling and stabilizes TRPM4 in the plasma membrane. Together our results indicate that cholinergic stimulation increases firing in a TRPM4 dependent manner, and also increases the TRPM4 stability in the membrane, suggesting that TRPM4 is locked in microdomains in the membrane, possibly signaling or cytoskeleton proteins complexes.

Background/Objectives: P2X receptor channels are widely expressed in the CNS, where they have multiple functions in health and disease. The rat P2X2a (rP2X2a) receptor channel is modulated by copper, an essential trace element that plays important roles in synaptic modulation and neurodegenerative disorders. Although essential extracellular amino acids that coordinate copper have been identified, the exact mechanism of copper-induced modulation has not been yet elucidated. Methods: We used HEK293T cells expressing rP2X2a channel(s) and performed outside-out single-channel and whole-cell recordings to explore copper’s effects on rP2X2 currents and determine whether this metal can increase the cooperative gating of rP2X2a channel. Results: In whole-cell recordings and in patches containing 2 or 3 rP2X2a channels, copper enhanced the ATP-induced currents, significantly reducing the ATP EC₅₀ and increasing the Hill coefficient. Moreover, copper increased the apparent P_o in patches containing two or three channels. By contrast, in patches containing only one rP2X2a channel, we did not observe any significant changes in ATP EC₅₀, the Hill coefficient, or P_o. Conclusions: Copper modulates the gating of rP2X2a channels, enhancing interchannel cooperativity without altering single-channel conductance or P_o. This novel regulatory mechanism could be relevant for understanding the role of P2X2 channels in physiological and pathological processes.