Theodore R. Cummins - US grants

Voltage-gated sodium channels are critical determinants of neuronal and muscle cellular excitability. These channels may also play a crucial role in chronic pain, epilepsy and other neurological disorders. However, investigations into the precise functional role that specific sodium channel isoforms play in normal and abnormal cellular excitability is lacking. A main objective of our research is to identify molecular mechanism(s) underlying alterations in the electrical excitability of sensory neurons. Experimental and clinical studies have clearly shown that the peripheral nerve fibers, and the neuronal cell bodies that give rise to them, can become hyperexcitable after injury and that this hyperexcitability contributes to neuropathic pain. Changes in sodium currents are likely to alter the excitability of sensory neurons, and could contribute to the reduced threshold for repetitive firing and increased level of spontaneous firing that has been observed in injured and inflamed sensory neurons. Subthreshold sodium currents, currents that are active at membrane potentials negative to the threshold for action potential generation, can play crucial roles in regulating electrogenesis in neurons. The present proposal focuses on tetrodotoxin-sensitive subthreshold sodium currents in sensory neurons and their role in chronic pain mechanisms. This project will address the hypothesis that altered sodium currents play a crucial role in the development of enhanced excitability associated with chronic pain with the following specific aims: 1. Characterize the properties of sodium currents in cutaneous afferent dorsal root ganglion neurons acutely isolated from normal adult rats, after chronic peripheral inflammation and after peripheral nerve injury. 2. Determine how specific sodium channel isoforms contribute to sodium currents in control and sensitized neurons. 3. Examine the effect of sodium channel mutations that cause the inherited painful neuropathy primary erythermalgia in humans on Nav1.7 sodium channel properties and excitability in sensory neurons. Understanding the changes that occur in the sodium currents of sensory neurons following inflammation and/or nerve injury and how specific sodium channel isoforms contribute to these changes should enhance our understanding of the normal and abnormal physiology of sensory neurons and should aid the development of new therapeutic strategies for the treatment of pain.

[unreadable] DESCRIPTION (provided by applicant): Voltage-gated sodium channels are critical determinants of neuronal excitability. Experimental and clinical evidence clearly demonstrates that sodium channels and changes in the properties of these channels can play crucial roles in chronic pain and other neurological disorders. However, at least six different sodium channel isoforms are expressed in peripheral sensory neurons, and investigating the role of specific voltage- gated sodium channel isoforms in normal and abnormal electrical activity of sensory neurons has been hindered by the lack of isoform specific neuronal sodium channel blockers. The goal of this R21 grant proposal is to develop selective sodium channel blockers based on biological peptide toxins that can be used to investigate the role(s) of specific sodium channel isoforms in disorders of excitability such as pain. Biological toxins have been invaluable in researching the role of ion channels in disease and some toxins that target ion channels are important therapeutics. Our preliminary data show that a toxin in the venom from the tarantula Ornithoctonus huwena inhibits TTX-sensitive neuronal sodium channels but not TTX- resistant neuronal sodium channels, cardiac sodium channels or skeletal muscle sodium channels. Furthermore, these data indicate that this toxin is more potent at blocking Nav1.6 and Nav1.7 TTX-sensitive channels than Nav1.1, 1.2 and 1.3 TTX-sensitive sodium channels. This toxin is an excellent candidate molecule on which to base the development of isoform specific sensory neuronal sodium channel blockers. This project has two related specific aims: 1. Identify the isoform specific determinants of sodium channel sensitivity to tarantula toxins. 2. Develop toxin analogs with enhanced selectivity for specific voltage-gated sodium channel isoforms. Whole-cell patch-clamp electrophysiology, mutagenesis of recombinant ion channels and peptide synthesis will be used to investigate toxin-channel interactions. The experiments proposed here will determine the feasibility of using tarantula toxins as the basis for developing novel tools to determine the roles of specific sodium channels in normal and abnormal physiology of sensory neurons. The long-term goal of this research is to develop sodium channel blockers that are useful as novel therapeutics for the treatment of neurological disorders of hyperexcitability such as chronic pain. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Pain is significant national health problem, costing the American public more than $100 billion each year. Voltage-gated sodium currents are essential to the generation of action potentials and contribute to the hyperexcitability of nociceptive neurons. However, although sodium channel blockers are useful for preventing acute pain, they are often associated with undesirable cardiac and CNS side effects due to their lack of isoform selectivity, limiting their therapeutic window and their effectiveness in treating chronic and neuropathic pain. Several voltage-gated sodium channels are preferentially expressed in nociceptive neurons (Nav1.7, Nav1.8 and Nav1.8). Nav1.7 in particular has been shown to play an absolutely crucial role in pain, including chronic neuropathic pain. Developing drugs that specifically target these nociceptive sodium channels could substantially increase the therapeutic armamentarium for pain. The majority of sodium channel inhibitors and modulators that have been identified interact with the sodium channel pore - because the amino acid residues lining the pore are highly conserved among the nine sodium channel genes, it has been difficult to identify pore blockers with isoform specificity. By contrast, the voltage-sensors of sodium channels show greater divergence and therefore it should be possible to develop voltage gating modifiers that target specific sodium channel isoforms. We are proposing to harness gating-pore currents to monitor sodium channel voltage-sensor function and enhance our ability to screen for voltage- gating modifiers. These gating-pore currents are currents that selectively flow through the voltage sensor domains of ion channels and provide a direct read-out of the voltage-sensor position. Importantly, they do not reflect pore activity. Two specific aims are proposed: AIM I will determine if gating-pore currents can be used to monitor the activity of voltage-sensor modulators of voltage-gated sodium channels. AIM II will determine if isolated voltage sensors or chimeric bacterial sodium channels containing a single voltage sensor of Nav1.7 can generate gating-pore currents that are sensitive to specific voltage-sensor modulators. Although we initially target Nav1.7 channels because of their importance in pain, this approach should be readily adaptable for identification and characterization of voltage-sensor modulators of other voltage-gated channels and therefore could be used to help identify isoform specific voltage-gated channel modulators that can be used in research and for the development of better therapeutics to treat a multitude of disorders of excitability such as pain, epilepsy and cardiac arrhythmias.

Lack of diversity in neuroscience graduate programs and advanced positions is a substantial problem. The Neuroscience Experience and Undergraduate Research Opportunities Program (NEUROP) is designed to increase the diversity of prepared neuroscience scholars at the predoctoral, postdoctoral, and (ultimately) faculty levels. To address this problem the NEUROP objectives are to expand the exposure of undergraduate underrepresented minorities (URMs) to neuroscience research, and to enhance the exposure of graduate URMs to cutting-edge research methodologies and professional skills training with the goal of fostering the next generation of URM scientists. To meet the objectives and long-term goals we propose a multipronged approach. We will enhance URM undergraduate neuroscience students' exposure to neuroscience themes and research with NEUROP elements across all four undergraduate years. The 1st year Neuroscience Learning Community will expose freshmen to the excitement of neuroscience research and the overall neuroscience community. The 2nd year Neuroscience Research Skills Course will expose students to state-of-the art techniques and additional faculty mentors. The Neuroscience Research Topics Course will bring together students from multiple years in order to continue to build excitement and a knowledge base of neuroscience and a sense of community using a journal club format, involving appropriate role models and potential faculty mentors. After the 2nd year, summer and academic research internships will provide hands-on exposure to neuroscience research, culminating in a 4th year capstone research experience to prepare undergraduate students for the transition to graduate school. By increasing opportunities and desire to join the neuroscience research community, providing enhanced mentoring, and maximizing authentic research experiences, we will grow the pool of trained URM students entering neuroscience graduate programs. In addition to undergraduates, graduate student NEUROP scholars will be selected. The NEUROP will allow for increased impact of research, intensified sense of community, and enhanced professional skills. We propose that this will enhance graduate student preparedness for postdoctoral training and intensify their desire for an academic faculty position in neurosciences research. Overall, we are committed to decreasing attrition at the undergraduate level, increasing transitions to graduate school, and enhancing the graduate student to postdoc/faculty transition by engaging and supporting neuroscience students at multiple stages of their burgeoning careers.