M-current induced Bogdanov–Takens bifurcation and switching of neuron excitability class

In this work, we consider a general conductance-based neuron model with the inclusion of the acetycholine sensitive, M-current. We study bifurcations in the parameter space consisting of the applied current $I_{app}$ I a p p , the maximal conductance of the M-current $g_{M}$ g M and the conductance of the leak current $g_{L}$ g L . We give precise conditions for the model that ensure the existence of a Bogdanov–Takens (BT) point and show that such a point can occur by varying $I_{app}$ I a p p and $g_{M}$ g M . We discuss the case when the BT point becomes a Bogdanov–Takens–cusp (BTC) point and show that such a point can occur in the three-dimensional parameter space. The results of the bifurcation analysis are applied to different neuronal models and are verified and supplemented by numerical bifurcation diagrams generated using the package . We conclude that there is a transition in the neuronal excitability type organised by the BT point and the neuron switches from Class-I to Class-II as conductance of the M-current increases.

SLO2.1 and NALCN form a functional complex to modulate myometrial cell excitability

At the end of pregnancy, the uterus transitions from a quiescent state to an excitable, contractile state. These changes are linked to depolarization of the myometrial smooth muscle cell (MSMC) resting membrane potential. The membrane potential is primarily determined by the balance between an outward potassium (K+) leak current and an inward sodium (Na+) leak current. We recently described a Na+-activated K+ channel (SLO2.1) and a non-selective Na+ leak channel (NALCN) in human MSMCs. Here, we asked whether these channels function together. We show that SLO2.1 currents are activated by an inward NALCN-dependent Na+ leak current, leading to MSMC hyperpolarization. The regulation of the membrane potential by NALCN/SLO2.1 activity modulates both Ca2+ entry through VDCCs, and myometrial contractility. Finally, NALCN and SLO2.1 are in proximity to one another in human MSMCs. We conclude that SLO2.1 and NALCN function together to regulate human MSMC membrane potential and excitability.

A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer

Automated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to ground occurs whenever the seal between a pipette and cell (or internal solution and cell in high-throughput machines) is not perfectly insulated from the bath (extracellular) solution. Over 1 GΩ seal resistance between pipette and bath solutions is commonly used as a quality standard for manual patch work. With automated platforms it can be difficult to obtain such a high seal resistance between the intra- and extra-cellular solutions. One suggested method to alleviate this problem is using an F− containing internal solution together with a Ca2+ containing external solution — so that a CaF2 crystal forms when the two solutions meet which ‘plugs the holes’ to enhance the seal resistance. However, we observed an unexpected nonlinear-in-voltage and time-dependent current using these solutions on an automated patch-clamp platform. We performed manual patch-clamp experiments with the automated patch-clamp solutions, but no biological cell, and observed the same nonlinear time-dependent leak current. The current could be completely removed by washing out F− ions to leave a conventional leak current that was linear and not time-dependent. We therefore conclude fluoride ions interacting with the CaF2 crystal are the origin of the nonlinear time-dependent leak current. The consequences of such a nonlinear and time-dependent leak current polluting measurements should be considered carefully if it cannot be isolated and subtracted.

Progesterone and estrogen regulate NALCN expression in human myometrial smooth muscle cells

During pregnancy, the uterus transitions from a quiescent state to an excitable, highly contractile state to deliver the fetus. Two important contributors essential for this transition are hormones and ion channels, both of which modulate myometrial smooth muscle cell (MSMC) excitability. Recently, the sodium (Na+) leak channel, nonselective (NALCN), was shown to contribute to a Na+ leak current in human MSMCs, and mice lacking NALCN in the uterus had dysfunctional labor. Microarray data suggested that the proquiescent hormone progesterone (P4) and the procontractile hormone estrogen (E2) regulated this channel. Here, we sought to determine whether P4 and E2 directly regulate NALCN. In human MSMCs, we found that NALCN mRNA expression decreased by 2.3-fold in the presence of E2 and increased by 5.6-fold in the presence of P4. Similarly, E2 treatment decreased, and P4 treatment restored NALCN protein expression. Additionally, E2 significantly inhibited, and P4 significantly enhanced an NALCN-dependent leak current in MSMCs. Finally, we identified estrogen response and progesterone response elements (EREs and PREs) in the NALCN promoter. With the use of luciferase assays, we showed that the PREs, but not the ERE, contributed to regulation of NALCN expression. Our findings reveal a new mechanism by which NALCN is regulated in the myometrium and suggest a novel role for NALCN in pregnancy.

MON-011 NALCN Expression Is Regulated by Progesterone and Estrogen in Human Myometrial Smooth Muscle Cells

During pregnancy, the uterus transitions from a quiescent state to a highly contractile, excitable state. Both ion channels and hormones are essential for this transition. We recently identified that the Na+ leak channel, non-selective (NALCN) contributes to a leak current in human MSMCs and mice lacking NALCN have prolonged and dysfunctional labor. Additionally, NALCN levels change throughout mouse pregnancy suggesting regulation by hormones of pregnancy, specifically estrogen and progesterone. Here, we tested the hypothesis that P4, a pro-quiescent hormone, and E2, a pro-contractile hormone, regulate NALCN expression and current in the myometrium. In a human immortalized myometrial cells (HM6ERMS2), using qPCR we measured a 2.3 fold decrease and a 5.6 fold increase in NALCN mRNA expression in the presence of E2 and P4, respectively. These findings were also confirmed when NALCN protein expression were measured by immunoblot. Conversely, treatment with the ER antagonist, ICI 182,780, significantly increased NALCN mRNA expression, while treatment with the PR antagonist RU486 significantly decreased NALCN mRNA expression suggesting E2 and P4 work through their respective receptors to regulate NALCN. P4 differentially regulates myometrial activity depending on which progesterone receptor is activated: PRA, promotes contractility, whereas PRB promotes quiescence. Thus to study the effect of each PR, we used a human myometrial cell line stably expressing PRA or PRB, and measured similar increases in NALCN mRNA expression in both cell lines treated with P4. To determine the functional consequences of E2 and P4, we measured NALCN-dependent leak current in MSMCs using whole cell patch clamping. We observed that E2 significantly inhibited while P4 significantly enhanced NALCN current. Finally, we identified estrogen response and progesterone response elements (ERE and PRE) in the NALCN promoter and showed that the PREs contributed to P4 regulation while the ERE did not contribute to the regulation of NALCN expression using luciferase based promoter assays. Overall, our findings show that NALCN is upregulated by P4, the pro-quiescent hormone, and downregulated by E2, the pro-contractile hormone. This data reveals a new mechanism by which NALCN is regulated in the myometrium and may suggest a novel role for NALCN during pregnancy. Further investigation into these novel roles can provide an insight into potential targets to modulate uterine quiescence and contractility.

Myotonia in a patient with a mutation in an S4 arginine residue associated with hypokalaemic periodic paralysis and a concomitant synonymous CLCN1 mutation

The sarcolemmal voltage gated sodium channel NaV1.4 conducts the key depolarizing current that drives the upstroke of the skeletal muscle action potential. It contains four voltage-sensing domains (VSDs) that regulate the opening of the pore domain and ensuing permeation of sodium ions. Mutations that lead to increased NaV1.4 currents are found in patients with myotonia or hyperkalaemic periodic paralysis (HyperPP). Myotonia is also caused by mutations in the CLCN1gene that result in loss-of-function of the skeletal muscle chloride channel ClC-1. Mutations affecting arginine residues in the fourth transmembrane helix (S4) of the NaV1.4 VSDs can result in a leak current through the VSD and hypokalemic periodic paralysis (HypoPP), but these have hitherto not been associated with myotonia. We report a patient with an Nav1.4 S4 arginine mutation, R222Q, presenting with severe myotonia without fulminant paralytic episodes. Other mutations affecting the same residue, R222W and R222G, have been found in patients with HypoPP. We show that R222Q channels have enhanced activation, consistent with myotonia, but also conduct a leak current. The patient carries a concomitant synonymous CLCN1 variant that likely worsens the myotonia and potentially contributes to the amelioration of muscle paralysis. Our data show phenotypic variability for different mutations affecting the same S4 arginine that have implications for clinical therapy.