Browsing by Subject "Beta subunit"

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    Increased Resurgent Sodium Currents in Nav1.8 Contribute to Nociceptive Sensory Neuron Hyperexcitability Associated with Peripheral Neuropathies
    (Society for Neuroscience, 2019-02-20) Xiao, Yucheng; Barbosa, Cindy; Pei, Zifan; Xie, Wenrui; Strong, Judith A.; Zhang, Jun-Ming; Cummins, Theodore R.; Biology, School of Science
    Neuropathic pain is a significant public health challenge, yet the underlying mechanisms remain poorly understood. Painful small fiber neuropathy (SFN) may be caused by gain-of-function mutations in Nav1.8, a sodium channel subtype predominantly expressed in peripheral nociceptive neurons. However, it is not clear how Nav1.8 disease mutations induce sensory neuron hyperexcitability. Here we studied two mutations in Nav1.8 associated with hypersensitive sensory neurons: G1662S reported in painful SFN; and T790A, which underlies increased pain behaviors in the Possum transgenic mouse strain. We show that, in male DRG neurons, these mutations, which impair inactivation, significantly increase TTX-resistant resurgent sodium currents mediated by Nav1.8. The G1662S mutation doubled resurgent currents, and the T790A mutation increased them fourfold. These unusual currents are typically evoked during the repolarization phase of action potentials. We show that the T790A mutation greatly enhances DRG neuron excitability by reducing current threshold and increasing firing frequency. Interestingly, the mutation endows DRG neurons with multiple early afterdepolarizations and leads to substantial prolongation of action potential duration. In DRG neurons, siRNA knockdown of sodium channel β4 subunits fails to significantly alter T790A current density but reduces TTX-resistant resurgent currents by 56%. Furthermore, DRG neurons expressing T790A channels exhibited reduced excitability with fewer early afterdepolarizations and narrower action potentials after β4 knockdown. Together, our data demonstrate that open-channel block of TTX-resistant currents, enhanced by gain-of-function mutations in Nav1.8, can make major contributions to the hyperexcitability of nociceptive neurons, likely leading to altered sensory phenotypes including neuropathic pain in SFN.SIGNIFICANCE STATEMENT This work demonstrates that two disease mutations in the voltage-gated sodium channel Nav1.8 that induce nociceptor hyperexcitability increase resurgent currents. Nav1.8 is crucial for pain sensations. Because resurgent currents are evoked during action potential repolarization, they can be crucial regulators of action potential activity. Our data indicate that increased Nav1.8 resurgent currents in DRG neurons greatly prolong action potential duration and enhance repetitive firing. We propose that Nav1.8 open-channel block is a major factor in Nav1.8-associated pain mechanisms and that targeting the molecular mechanism underlying these unique resurgent currents represents a novel therapeutic target for the treatment of aberrant pain sensations.
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    The Role of β4 Subunit in Epilepsy Susceptibility
    (2024-08) Fahim, Ahmed; Cummins, Theodore; Berbari, Nicolas F.; Mastracci, Teresa
    Seizure involves a sudden, uncontrolled electrical disturbance of the brain due to many different causes apart from epilepsy, for example, high fever, low level of blood sugar, alcohol withdrawal, and many more, including the infections in the brain. In fact, epilepsy is a group of chronic neurological disorders characterized by recurrent unprovoked sudden-onset seizures. It stands as one of the prevalent brain disorders globally, impacting over 70 million individuals. The origins of epilepsy are multifaceted, coming from a mix of genetic and environmental factors including genetic predispositions, brain-related conditions (like tumors or strokes), infectious diseases, and traumatic brain injuries. Seizures can be partly referred to the dysregulation of ion channels, including voltage-gated sodium channels which will impact the action potential (electrical impulses that are responsible for the communication that takes place between neurons in the brain). These voltage-gated sodium channels mediate the depolarization responsible for the generation and conduction of action potentials. They are crucial in the generation and continuous electrical signals of the tissues that respond rapidly, like the neurons, and thus forming part of their function. In epilepsy, therefore, it is relevant to that domain in which abnormal functions of these sodium channels come up. Any change or dysfunction of these channels affect the excitability of the neurons themselves, with the consequence that an increased probability occurs in which abnormal electrical activity can be generated, hence the convulsions. Voltage-gated sodium channels are made up of large transmembrane proteins, having a single alpha subunit and related beta subunits. The beta subunit is an auxiliary protein that modulates channel gating, kinetics, surface expression, and the unique resurgent current, thereby influencing neuronal excitability and signaling. Resurgent currents represent a kind of current that can develop during action potential repolarization. They are characterized by a resurgent sodium current, the current which follows the initial sodium inflow in depolarization. Resurgent sodium currents are characterized by a rebound increase in sodium current during the repolarization phase of the action potential. Unlike the classic transient sodium current that inactivates rapidly upon membrane depolarization, the resurgent current is facilitated by the partial block and unblock of the sodium channel pore by the β subunit or other intracellular molecules during the repolarization phase. This allows sodium ions to flow into the cell when this blockage removed before it goes to closed state. It is believed widely to be of keen importance in neuronal excitability. The role of resurgent currents in epilepsy is likely genetically influenced with some environmental influence. Genetic mutations and dysregulation of the gene code for voltage-gated sodium channels, especially those related to beta subunits, can be linked to some atypical resurgent current. This increases the chance of having a seizure, which could develop into epilepsy. Four beta subunits have been identified up to now. As such, my investigation will focus on the beta 4 subunit and its possible involvement in increased susceptibility to seizures. My study will involve a genetically modified mouse β4 knockout (K.O) of the voltage-gated sodium channel, which will be compared with a wild type (WT) mouse model. To facilitate this comparison, I will prepare cortical brain slices from both the genetically modified and WT mice using a (Leica VT1200s vibratome). These slices will then be analyzed with multi-electrode arrays to detect electrical activity and measure the neurons' electrical responses. Additionally, I use 4-Aminopyridine, a potassium channel blocker, to stimulate electrical activity in the neurons and brain slices. Using the methodology outlined above, I aimed to investigate the ability to induce and measure neuronal activity in the β4 K.O mouse model. This involved comparing the neuronal activity between the β4 K.O and WT mice in terms of frequency and amplitude. The analysis of the recorded data was performed using Spike2 software, in conjunction with the multi-electrode array recordings. Furthermore, I explored whether variations in temperature (body temp vs 40℃) affect neuronal activity differently in β4 K.O compared to WT mice. In conclusion, my observations revealed that neuronal activity could indeed be induced in the β4 K.O mice, with a noted decrease in the frequency of this activity compared to WT mice, but an increase in amplitude. These outcomes were consistent at both normal body temperature and at an elevated temperature of 40°C, as analyzed using Spike2 software. However, when conducting a statistical analysis using a two-way ANOVA to compare between the β4 K.O and WT mice, and between body temperature and 40°C conditions, no significant differences were observed. Despite this, it is a general observation and conclusion that β4 K.O mice exhibit altered neuronal activity compared to WT mice. To gain a deeper understanding of the role of the β4 subunit on the alpha subunit of the voltage-gated sodium channel, adopting alternative methods such as patch clamp techniques or in vivo studies with intracranial electrodes may be beneficial. This suggestion comes considering various challenges and limitations encountered during my study, such as maintaining the viability of the slices for extended periods and minimizing noise in multi-electrode array (MEA) recordings. Mutations of β-subunit-encoding genes have been associated with such a wide array of debilitating diseases that include epilepsy, cancer, neuropathic pain, and febrile seizures, to some of the most prevalent conditions in neurodegeneration. Further study will be needed to better understand the biology of these important proteins and their potential for use as new targets for several disease states. Even so, the role of β4 remains somewhat controversial.