Lab research interests:
The study of KATP channels in normal and injured sensory transduction.
My lab currently investigates the biophysical properties, pharmacology and roles of ATP-sensitive potassium (KATP) channels in peripheral sensory neurons, in the normal state and after painful nerve injury. My research is currently supported by a grant from the National Institute of Neurological Disorders and Stroke.
An estimated four to five million people in the US suffer from neuropathic pain, which can result from trauma or disease affecting the peripheral nerves. Injured nerves develop a series of structural and functional changes that lead to severe, intractable pain. In addition to traumatic injuries, other conditions associated with pain originating from the nerves include spine disease, diabetes, infections and cancer. Chronic neuropathic pain is persistent and resistant to most current treatments.
ATP-sensitive potassium channels in the heart, pancreas and brain, regulate the excitability of the cells, the release of transmitters, and protect the cells from death. The role of these channels in peripheral nerves remains unknown. Previous studies from my laboratory have shown the presence of these channels on peripheral nerves (Figures 1-3)
|
|
|
Figure 1: Effect of glibenclamide on potassium current in a control DRG neuron. Current changes were recorded in response to 10 mV voltage commands from –100 mV holding potential, in baseline external solution ("Baseline" traces) and in the presence of 1 mM glibenclamide ("After Glibenclamide" traces). The "Difference" current indicates the glibenclamide sensitive component, and was obtained after subtracting the traces in the presence of glibenclamide from the baseline traces. The corresponding I-V relationship is also shown. |
|
|
|
Figure 2: ATP dependence of glibenclamide effects on potassium currents recorded from normal DRG neurons. Currents were recorded before ("Baseline") and after ("Glibenclamide") the application of 1mM glibenclamide in the presence of high (2.5 mM), and low (0.5-1 mM) pipette [ATP]. Traces were compensated for capacitance, averaged, and plotted I-V curves are shown (means±SE), indicating the ATP-dependence of the glibenclamide effects, as well as the lower baseline currents in the presence of higher [ATP]. |
|
|
|
Figure 3: Effects of KATP channel openers (diazoxide, pinacidil) and blocker glibenclamide on potassium currents in DRG neurons dissociated from control rats. I-V curves indicating the diazoxide, pinacidil and glibenclamide-sensitive potassium current components (means ±SE) in DRG neurons dissociated from control rats. Curves were plotted from averaged, normalized for capacitance, subtracted current components. In the case of glibenclamide (KATP-channel blocker), curves were obtained by subtracting the drug-induced from control baseline traces. For the KATP channel openers (diazoxide and pinacidil) each curve was obtained by subtracting the baseline from the drug enhanced trace. |
|
|
|
Figure 4: Effects of KATP channel openers (diazoxide, pinacidil) and blocker glibenclamide on potassium currents in DRG neurons dissociated from rats with painful neuropathy after sciatic nerve chronic constriction injury (CCI). I-V curves indicating the diazoxide, pinacidil and glibenclamide-sensitive potassium current components (means ±SE) in DRG neurons dissociated from CCI rats. Curves were plotted from averaged, normalized for capacitance, subtracted current components. In the case of glibenclamide (blocker), curves were obtained by subtracting the drug-induced from control baseline traces. For the KATP channel openers (diazoxide and pinacidil) each curve was obtained by subtracting the baseline from the drug enhanced trace. (Compare with Figure 3 which shows KATP current I-V curves in neurons from control rats). |
and their loss after nerve injury (Figure 4).
Increased neuronal excitability, neurotransmitter release, and cell death are key features of neuropathic injury that results in pain. Because KATP channels are implicated in the regulation of these functions, they are potentially significant foci of investigation and therapeutic interventions with promising results.
My research explores the role and regulation of peripheral neuronal KATP channels employing primarily electrophysiological (whole-cell and single-channel recordings), molecular, and genetic techniques. Specifically, investigations include:
1.- The detailed biophysical and pharmacological characterization of KATP current in primary afferent neurons, exploring the hypothesis that these cells express KATP current conveyed via the Kir6.2/SUR1 channel. This is the predominant neuronal KATP channel subtype, and has a distinct pharmacological profile. My goal is to characterize the properties of this channel using pharmacological and biophysical tools.
2.- The investigation of the functional role of KATP currents in primary afferent neurons. Although KATP currents have been shown to control resting membrane potential, excitability and survival in other neuronal types, their role in DRG neurons is unknown. In order to test the hypothesis that these currents regulate neuronal excitability and enhance cell survival under conditions of metabolic stress, experiments designed to examine electrophysiological parameters defining neuronal excitability in primary afferent neuronal somata under current clamp conditions, at baseline and in the presence of KATP channel modulators are being conducted. Furthermore, neuronal ischemia and metabolic stress contribute to nerve dysfunction in a variety of conditions that share a common feature of sensory neuron cell loss. Thus, to identify the influence of KATP currents on the response to metabolic stress, other experiments aim to investigate the influence of KATP channel openers and blockers on rotenone-induced cell death of sensory neurons.
3.- Testing the effects of nerve injury on DRG neuronal KATP currents. Sensory neurons demonstrate elevated excitability in neuropathic pain states. My previous studies have shown loss of KATP current in neurons proximal to a distal sciatic injury. My current hypothesis is that decreased KATP current is a general feature of nerve injury producing neuropathic pain. For these studies, I am using the model of spinal nerve ligation (SNL) that allows clear identification between injured (by axotomy) and intact neurons, and investigate which neuronal group demonstrates KATP current loss. Current decrement may be due to either transcriptional down-regulation of channel protein, or post-translational regulatory decrease in channel conductance. In addition to current- and voltage-clamp recordings, the presence of channel protein and mRNAs encoding specific KATP channel subunits is investigated in control, axotomized and adjacent DRG neuronal somata using binding studies and RT-PCR.
4. Investigation of other channels that may control excitability in primary afferent neurons. In addition to KATP channels, investigation of other channels involved in regulation of neuronal excitability is complementary to my primary focus. Calcium-activated potassium channels (KCa) may also play a role, since they are co-expressed with KATP channels on primary afferents, and also link neuronal excitability to metabolism [Jonas P, et al. ATP-sensitive and Ca-activated K channels in vertebrate axons: novel links between metabolism and excitability. Pflugers Arch 418: 68-73, 1991]. We have previously shown alterations of ICa after neuropathic injury, so altered KCa currents can be expected. Preliminary results obtained from my lab show that all IK(Ca) subtypes (characterized by their sensitivity to apamin, clotrimazole, and iberiotoxin) are decreased by axotomy, but iberiotoxin-sensitive and clotrimazole-sensitive current densities are increased in adjacent to injury L4 neurons after SNL.
The translational research emanating from this project will be pertinent to improving the care of those who suffer from chronic pain and neurodegenerative conditions.
Pertinent published studies:
Q Hogan, JB McCallum, C Sarantopoulos, M Aason, M Mynlieff, WM Kwok, Z Bosnjak. Painful neuropathy decreases membrane calcium current in mammalian primary afferent neurons. Pain 86: 43-53, 2000.
C Sarantopoulos, JB McCallum, WM Kwok, Q Hogan. Gabapentin decreases membrane calcium currents in injured as well as in control mammalian primary afferent neurons. Regional Anesthesia and Pain Medicine 27: 47-57, 2002.
C Sarantopoulos, B. McCallum, D. Sapunar, WM Kwok, Q Hogan. ATP-sensitive potassium channels in rat primary afferent neurons: the effect of neuropathic injury and gabapentin. Neuroscience Letters 343: 185-189, 2003.
C Sarantopoulos, B McCallum, WM Kwok, Q Hogan. b-escin diminishes voltage-gated calcium channel rundown in perforated patch-clamp recordings from rat primary afferent neurons. Journal of Neuroscience Methods, 139: 61-68, 2004.
A Kanai A, C Sarantopoulos, JB McCallum, QH Hogan. Painful neuropathy alters the effect of gabapentin on sensory neuron excitability in rats. Acta Anaesthesiologica Scandinavica 48: 507-512, 2004.