Single-Channel Properties and Pharmacological Characteristics of KATP Channels in Primary Afferent Neurons

ATPsensitive potassium (KATP) channels first discovered in cardiac myocytes in 1983 (Noma, 1983), and then found in many other metabolically active tissues, including central nervous systems (Babenko et al., 1998). KATP channels are inhibited by physiological concentration of intracellular ATP, and are activated when the intracellular ADP/ATP ratio increases secondary to hypoxia, ischemia, or metabolic stress (Babenko et al., 1998; Miki & Seino, 2005; Nichols, 2006). The activation of KATP channels results in an enhanced outward repolarizing flow of K+ and cell membrane hyperpolarization, and thus they regulate cell excitability and mediate cellular responses that determine cell survival during metabolic stress (Miki & Seino, 2005).


Introduction
ATP-sensitive potassium (K ATP ) channels first discovered in cardiac myocytes in 1983 (Noma, 1983), and then found in many other metabolically active tissues, including central nervous systems (Babenko et al., 1998).K ATP channels are inhibited by physiological concentration of intracellular ATP, and are activated when the intracellular ADP/ATP ratio increases secondary to hypoxia, ischemia, or metabolic stress (Babenko et al., 1998;Miki & Seino, 2005;Nichols, 2006).The activation of K ATP channels results in an enhanced outward repolarizing flow of K + and cell membrane hyperpolarization, and thus they regulate cell excitability and mediate cellular responses that determine cell survival during metabolic stress (Miki & Seino, 2005).
In central nervous systems, K ATP channels are predominantly expressed in basal ganglia, thalamus, hippocampus, and cerebral cortex (Mourre et al., 1989).Although detailed functional roles of K ATP cahnnel in central nervous systems still remain to be clarified, their activation results in K + efflux, leading to membrane hyperpolarization, decreased excitability, attenuation of transmitter release, and protection from cell death (Yamada et al., 2001).
In addition to central nervous systems, we have recently reported that functional K ATP channels are expressed in rat sensory neurons (Kawano et al., 2009a(Kawano et al., , 2009b(Kawano et al., , 2009c)).The primary sensory neuron is known to be an important site of pathogenesis for neuropathic pain, which is a common clinical condition that is difficult to treat by current methods (Amir et al., 2005;Gold, 2000;Zimmermann, 2001).Since our observations also reveal that currents through K ATP channels are significantly decreased by painful nerve injury (Sarantopoulos et al., 2003;Kawano et al., 2009a, 2009b, 2009c, Zoga et al., 2010), identification of their roles in sensory neurons, particularly regarding excitability, may reveal a contribution to the genesis of neuropathic pain.These researches may lead to development of novel therapies for neuropathic pain.Thus, the overall objective of this chapter is to discuss the characteristics and role of K ATP channels in rat sensory neurons.

Tissue-specific moleculer structure of K ATP channel
K ATP channels are a widely distributed family of potassium-selective ion channels, whose structure consists of an inwardly rectifying, pore-forming, K + channel (Kir6.x)subunit, each coupled to a regulatory sulfonylurea receptor (SUR) subunit (Babenko et al., 1998;Miki et al., 1999;Yokoshiki et al., 1998).The Kir 6.x subunits belong to the Kir family, and determine the inward rectification, ATP-sensitivity, and unitary single-channel conductance of K ATP channel (Babenko et al., 1998).On the other hand, SUR subunits belong to the ATP-binding cassette superfamily, and confer responsiveness to K ATP channel openers and sulfonylureas (Miki et al., 1999).The functional K ATP channel is assembled as an octamer with a 4:4 stoichiometry of Kir6.x and SUR subunit (Fig. 1 and 2).Fig. 1.Molecular structure of the K ATP channel.Schematic representation of the transmembrane topology of a single sulfonylurea receptor (SUR) or inwardly rectifying K + channel (Kir6.x)subunit.SUR is an ABC protein bearing transmembrane domains and two nucleotide-binding domains (NBD1 and NBD2).The Kir6.x subunits presumably form the pore of the channel and determine the sensitivity of the channel to inhibition by ATP (Nichols, 2006).
Native K ATP channels in different tissues show distinct single channel properties, modulating potency of nucleotides, and varying sensitivity to drugs that act as channel openers (such as diazoxide, pinacidil, nicorandil, etc) or blockers (sulphonylureas, like glibenclamide, tolbutamide, etc).These tissue-specific biophysical and pharmacological properties of K ATP channels (summarized in Table 1) are thought to be endowed by their different molecular composition of Kir6.0 and SUR subunits (Yokoshiki et al., 1998).For instance, affinity for sulfonylureas is high for SUR1 but low for SUR2A and SUR2B subunits.Similarly, there are differences in response to openers, with the SUR1 and SUR2B channels responding more potently to diazoxide in contrast to the response of SUR2A channels.Fig. 2. Schematic representation of the octameric K ATP channel complex viewed in cross section.Four Kir6.x subunits come together to form the K + channel pore, and each is associated with a regulatory SURx subunit.Several subtypes have been identified based on subunit combinations; co-expressing SUR1 and Kir6.2 forms the pancreatic -cell and neuronal type K ATP channel, SUR2A and Kir6.2 form the cardiac type K ATP channel, and SUR2B and Kir6.1 form the vascular smooth muscle type K ATP channel (Babenko et al., 1998;Miki et al., 1999;Yokoshiki et al., 1998).
Table 1.Tissue-specific properties of K ATP channels.
In addition to neuronal somata, image analysis results of immunostaining show that K ATP channels are present in glial satellite and Schwann cells (Zoga et al., 2010), which are known to express K + currents (Chiu et al., 1984).K ATP channels in these sites are thought to convey glial cell-mediated clearance of extracellular K + , often termed "K + spatial buffering" (Kofuji et al., 2002).

Cell isolation and plating
The L5 and L4 dorsal root ganglia (DRG) was harvested after normal adult Sprague-Dawley rats were decapitated under isoflurane anesthesia.DRG neurons were enzymatically dissociated in a solution containing 0.25 ml Liberalize Blendzyme 2 (0.05%) and 0.25 ml Dulbecco's modified Eagle's medium (DMEM) for 30 min at 37 °C.
Fig. 3. Isolated DRG neurons.Neurons were viewed using Hoffman modulation optic systems under an inverted microscope (Nikon Diaphot 300).DRG neuronal somata observed after isolation varied in size about 15-50 µm in diameter, and were stratified by diameter into either large ( 40 m) or small (<30 m) neurons.These sizes correlates roughly with electrophysiological characteristics corresponding to either A or C fibers, respectively (Lawson, 2002;Harper & Lawson, 1985).Arrowhead (1) and ( 2) point to small (24 m in diameter) and large (43 m in diameter) neuron, respectively.
After centrifugation and removal of the supernatant, a second incubation at 37 °C followed for another 30 min in 0.2 ml trypsin (0.0625%) and deoxyribonuclease 1 (0.0125%) in 0.25 ml DMEM.Cells were then isolated by centrifugation (600 rpm for 5 min) after adding 0.25 ml www.intechopen.comtrypsin inhibitor (0.1%), and re-suspended in a medium consisting of 0.5 mM glutamine, 0.02 mg/ml gentamicin, 100 ng/ml nerve growth factor 7 S , 2% B27 supplement, and 98% neural basal medium A. Neurons were plated onto poly-L-lysine-coated 12-mm glass coverslips, and kept in a humidified incubator at 37 °C with 95% air and 5% CO 2 .Patchclamp experiments were performed within 3-8 h after the cell dissociation (Fig. 3).

Single DRG K ATP channel currents from cell-attached patches
Biophysical and pharmacological characteristics of DRG K ATP channels were first studied in cell-attached recordings at a sampling frequency of 5 kHz with 1 kHz low-pass filter.Cellattached patch configuration is a non-invasive approach which is used to describe the endogenous properties of ion channels without disturbance of the intracellular milieu (Fig 4).
Both bath and pipette (extracellular) solutions were composed of the following (in mM): 140 KCl, 10 HEPES, 10 D-glucose, and 0.5 EGTA.The pH of all solutions was adjusted to 7.4 with KOH.Patch micropipettes were made from borosilicate glass capillaries using a Flaming/Brown micropipette puller, model P-97 (Sutter, San Rafael, CA) and flame polished with a microforge polisher (Narishige, Tokyo, Japan) prior to use.Their resistance ranged between 3 and 6 MΩ when filled with the internal solution, and placed into the recording solutions.In cell-attached patches (Fig 5), infrequent but significant spontaneous channel activity was recorded.These basal channel activities were observed in cell-size independent manner.However, bath application of the uncoupler of mitochondrial ATP synthesis, 2,4-Dinitrophenol (DNP, 100 µM), gradually activated these baseline currents.
Subsequent bath application of glibenclamide 1 µM, a specific K ATP channel inhibitor, completely blocked DNP-induced currents in both groups, indicating that these currents are conveyed via K ATP channels. www.intechopen.com

Single DRG K ATP channel currents from excised inside-out patches
In order to investigate the relative contribution of the intracellular milieu regulation of the intrinsic channel properties in DRG neurons, the K ATP channel behavior was next examined in excised inside-out membrane patches.Inside-out patches were made by pulling the membrane patch off the cell into the bath solution.
The bath (intracellular) solution contained 140 mM KCl, 1.2 mM MgCl 2 , 10 mM HEPES, 1.5 mM EGTA and 5.5 mM dextrose.The pipette (extracellular) solution was composed of the following: 140 mM KCl, 10 mM HEPES, 5.5 mM dextrose, and 1 mM EGTA.The pH of all solutions was adjusted to 7.4 with KOH.Osmolality was adjusted approximately to 300 mOsm/l by adding sucrose if necessary.When inside-out patch recordings at a holding potential of -60 mV were obtained in ATPfree solution, intense channel activity was observed in all patches without any differences between groups of neurons classified by size.The current-voltage relationship showed weak inward rectification with single channel conductance of 70-80 pS without any differences between neuron sizes.This channel activity was reversibly blocked by 1 mM of ATP.In the www.intechopen.comsame recordings, subsequent superfusion with glibenclamide (1 µM) eliminated channel activity in ATP-free solution.Channel activation by ATP-free solution, as well as the inhibition by ATP and glibenclamide, rapidly occurred within a few seconds (Fig. 6).
4. Functional roles of K ATP channel in primary afferent neurons

Basal K ATP channel activity contributes to the resting membrane potential
To test the functional importance of K ATP channel currents, their effect on resting membrane potential (RMP) in DRG neurons was examined using current-clamp recordings from escin perforated whole-cell patches.
RMP was recorded at baseline for at least 1 min.After stability was confirmed, glibenclamide (1 µM) was superfused in the bath by a gravity dependent flow system, which depolarized the RMP in DRG neurons.These results imply that basal K ATP channel opening physiologically regulates the RMP in DRG neurons (Fig 7).

K ATP channel activity regulates neurotransmitter release
K ATP channel is known to modulate neurotransmission (Stefani & Gold, 2001;Steinkamp et al., 2007).To examine whether K ATP channel in DRG neuron also modulate neurotransmitter www.intechopen.com Single-Channel Properties and Pharmacological Characteristics of K ATP Channels in Primary Afferent Neurons 81 release, carbon-fiber amperometry was used to detect exocytosis from single DRG neuron in real time (Kawagoe, et al., 1993).
Amperometry provides high resolution to detect molecules released from single secretory vesicles.Amperometric measurements are normally limited to cells that package and secrete an endogenous oxidizable molecule such as catecholamines or serotonin; however, in some cases oxidizable molecules can be introduced artificially (Smith et al., 1995;Zhou & Misler, 1996).So, amperometric analysis from DRG neurons was conducted by measured release of the pseudo-transmitter dopamine that had been loaded in DRG neurons (Fig. 8).with NaOH at 37 ○ C. Recordings were performed at room temperature in amine-free external Tyrode's solution.To record amperometric events, 5 µm carbon fiber electrodes were backfilled with 3 M KCl.A carbon fiber electrode connected to a patch clamp amplifier was attached to the plasma membrane of the cell held at +800 mV for all experiments.The tip of the carbon fiber was manipulated onto the cell surface without disturbing cellular morphology.If infrequent response to high K + (70 mM KCl) was observed, these data results were discarded.Currents were low-pass filtered at 5 kHz and sampled at 1 kHz by a Digidata 1440 Series interface (Axon Instruments).

Neuroprtection
Increased neuronal survival through activation of K ATP channels has been demonstrated in association with membrane hyperpolarization, and reduction of excitability in response to hypoxia, ischemia or metabolic stress (Amoroso et al., 1990;Ballanyi, 2004;Sun et al., 2007;Yamada & Inagaki, 2005).K ATP channels also act as transducers and effectors of neuronal preconditioning (Blondeau et al., 2000;Heurteaux et al., 1995).Pharmacological induced preconditioning with the K ATP channel opener diazoxide may offer effective neuroprotection during hypothermic circulatory arrest (Shake et al., 2001).In addition, opening of K ATP channels in the hippocampus or neocortex stabilizes the resting potential against anoxic stress, and protects neurons (Sun et al., 2006(Sun et al., , 2007)).In whole animals, the absence of Kir6.2 was associated with dramatically increased damage following ischemia induced by middle cerebral artery occlusion (Sun et al., 2006).

Pathophysiology of neuropathic pain
The specific cellular and molecular mechanisms underlying neuropathic pain remains largely unknown, but membrane hyperexcitability in those neurons that have lost their normal synaptic, physiological or electrical patterns is a common feature of most conditions leading to neuropathic pain (Gold, 2000;Woolf, 2010;Zimmermann, 2001).
Following nerve injury, damaged peripheral nerves become more excitable, with regards to their capacity to generate action potentials, leading to spontaneous, ongoing, ectopic electrical activity (Chung et al., 2002;Raouf et al., 2010;Woolf, 2010).In addition to the injury site, neuronal somata in the dorsal root ganglia are recognized as an important focus of ectopic electrical activity (Devor, 2009;Sapunar et al., 2005).This increase in primary afferent traffic to the dorsal horn is thought to induce raw pain signal (Devor, 2009) as well as central sensitization (Woolf, 2010).Substantial evidence indicates that altered expressions of ion channels on peripheral afferent neuronal somata contribute to abnormal sensory function following nerve injury (Raouf et al., 2010).Specifically, important changes have been noted in sodium (Amir et al., 2006), calcium (Gemes et al., 2011;McCallum et al., 2006) and potassium channels (Abdulla & Smith, 2001). www.intechopen.com

Animal models of neuropathic pain
Examination of the pathogenesis of neuropathic pain has been aided by the development of increasingly sophisticated rodent models of nerve injury that produce behavior indicative of on-going and evoked pain (Hogan, 2002).
A complete section of a nerve produces spontaneous pain, whereas they also lead to an anesthetic limb.On the other hand, partial injury retains a subset of afferent fibers and results in altered sensory function.Therefore, the latter is currently widely used for the study of neuropathic pain.These models involve: chronic constriction injury by loose ligation of the sciatic nerve (CCI) model (Bennett & Xie, 1988), tight ligation of the partial sciatic nerve (PSL) model (Shir & Seltzer, 1990), and tight ligation of spinal nerves (SNL) model (Kim & Chung, 1992).

CCI surgery
The right common sciatic nerve is exposed at the level of the middle of the thigh.Four loose ligatures of 4-0 chromic gut are placed around the sciatic nerve , and are loosely tied such that the diameter of the nerve was barely constricted.

PSL surgery
The right sciatic nerve was exposed near the trochanter.An 8-0 silk suture was inserted in the middle of the nerve, trapping in a tight ligation.

SNL surgery
The right paravertebral region was exposed via a lumbar incision, and the L6 transverse process was removed.The L5 and L6 spinal nerves were tightly ligated with a 6-0 silk suture and transected distal to the ligature.

Sensory testing
The purpose of behavioral sensory testing is to identify rats in which nerve injury has successfully produced behavior consistent with neuropathic pain.Operated rats are being tested for mechanical hyperalgesia, which provides a more consistent feature of the SNL model.Testing includes preoperative familiarization and acclimatization to testing environment, and subsequent repeated testing sessions.
Recently, Hogan et al. demonstrated the novel sensory testing that identifies hyperalgesia after SNL with high specificity (Hogan et al., 2004).Briefly, the plantar surface of each hind paw was touched with the tip of a Quincke 22-gauge spinal needle, which was applied with pressure adequate to indent but not penetrate the skin.Five needle applications were delivered in random order to each paw and repeated 5 min later for a total of 10 applications per session.These mechanical stimuli produced either a normal brief reflexive withdrawal or a hyperalgesia-type response that included sustained (> 1 s) paw lifting, shaking, and grooming (Fig. 9).The latter response occurs only after true SNL, and thus this may be accepted as an indication of a neuropathic pain.The intensity of hyperalgesia was assessed by the probability (%) of hyperalgesia-type responses out of ten trials of needle stimulation.
www.intechopen.comFig. 9. Behavioral testing using a Quincke 22-gauge spinal needle.When a pin is applied to rat planter, the response is either a brief reflex withdrawal or a hyperalgesic reaction characterized by sustained lifting, shaking, and licking of the paw.

K ATP channel activity in cell-attached patches depends on nerve injury status
To tested whether K ATP channels in primary afferent neuron contribute to the pathogenesis of neuropathic pain, basal K ATP channel openings in either control (non-surgery) or SNL neurons were measured at -60 mV membrane holding potential using cell-attached patch clamp configurations.In these recordings, basal channel opening was observed in both control and SNL neurons.
In SNL neurons, however, basal K ATP channel activity was diminished compared to controls (Fig. 10).NPo values in SNL neuron were also significantly reduced compared to control neurons.Analysis of single channel kinetics indicated that mean open time was shorter in SNL group compared to control group.Furthermore, basal K ATP channel NPo correlated inversely with the probability of the donor animal responding to punctuate mechanical stimulus with a sustained, complex hyperalgesia-type behavior (Fig. 11.) These results suggest that loss of current through these channels contributes to the pathogenesis of neuropathic pain.This hypothesis is further supported by a previous report that non-specific K + channel blockade evokes spontaneous firing in large A fibers after SNL (Liu et al., 2001).In addition, other study reported that hyperexcitability following peripheral nerve injury is mediated by loss of various K + currents (Chung & Chung, 2002).

K ATP channel activity in inside-out patches are not altered by nerve injury
To examine whether unitary K ATP channel currents is altered by axotomy, single channel properties in cell-free patches in either control (non-surgery) or SNL neurons were measured at -60 mV membrane holding potential.
In these recordings, marked current activity was observed in inside-out patches excised from either control or SNL neurons into ATP-free solution with symmetrical 140 mM K + condition (Fig. 12).In both groups, inside-out patches showed only one type of K + channel current, especially at negative potentials.In addition, single-channel conductance was the same in control and SNL neurons (70-80 pS).Furthermore, sensitivity to ATP and diazoxide, a selective SUR-1 containing K ATP channel opener, also did not differ between groups (Fig. 12).
www.intechopen.comThese results suggest that SNL does not affect the K ATP channel per se or any associated membrane-resident regulatory proteins.Our findings further imply that the molecular composition of the K ATP channels is not affected by axotomy.
Therefore, the suppressed K ATP channel activity observed from cell attached recordings may be attributed to alterations in the cytosolic signaling following painful nerve injury.

Conclusion
K ATP channels couple cellular electrical activity to cytosolic metabolic status in various excitable tissues.These channels are widely expressed in central neurons, wherein they regulate membrane excitability and neurotransmitter release, and they provide neuroprotection.In addition to the functional K ATP channels in the central nervous system, we have identified these channels in rat primary afferent neurons, dissociated from the rat DRG. www.intechopen.com Single-Channel Properties and Pharmacological Characteristics of K ATP Channels in Primary Afferent Neurons 87 Altered sensory function contributes to the pathogenesis of neuropathic pain via hyperexcitability in injured axons and the corresponding somata in the DRG, increased synaptic transmission at the dorsal horns, and loss of DRG neurons.We have identified loss of K ATP currents in DRG somata from rats that demonstrated sustained hyperalgesia-type response to nociceptive stimulation after axotomy.Thus, reduced K ATP currents may be a factor in generating neuropathic pain through increased excitability, amplified excitatory neurotransmission, and enhanced susceptibility to neuronal cell death.In addition, intrinsic single-K ATP channel characteristics are preserved even after painful nerve injury.Therefore, intact biophysical and pharmacological properties provide opportunities for therapeutic targeting with K ATP channel openers against neuropathic pain.

Fig. 4 .
Fig. 4. Cell-attached patch clamp configuration.(a) Diagram illustrating the methods of making cell-attached patches.(b) Cell-attached pipette on a large DRG somata.

Fig. 5 .
Fig. 5. Single-channel characteristics of K ATP channel in DRG neurons from cell-attached patch clamp configuration.(a) Representative current trace of K ATP channels in isolated DRG neurons recorded in cell-attached configuration at a holding potential of -60mV.These patches typically showed one predominant channel-type at a holding potential of -60 mV, whereas a second channel-type was observed infrequently in 5-10% of all patches.Arrows indicate closed channel state.(b) Basal channel open probability (Po) in individual DRG neurons.Po was determined from the ratios of the area under the peaks in the amplitude histograms fitted by a Gaussian distribution.Channel activity was calculated as NPo, where N is the number of observed channels in the patch.

Fig. 6 .
Fig. 6.Single-channel characteristics of K ATP channel in DRG neurons from inside-out patch clamp configuration.(a) Representative trace of K ATP channel activity recorded in cell-free patches excised from DRG neurons under symmetrical 140 mM K + conditions.Membrane potential was clamped at -60 mV.Upon patch excision (vertical arrow) into an ATP-free bath marked channel activity ensued.Horizontal arrows indicate closed channel state.(b) Current amplitude-voltage relationships, showing weak inward rectification.Means ± SD are shown (n=10).

Fig. 7 .
Fig. 7. Changes in resting membrane potential (RMP) induced by glibenclamide in DRG neurons.Representative RMP traces recorded in the current-clamp whole-cell patch configuration during the bath application of glibenclamide (1 µM).

Fig. 10 .
Fig. 10.Basal K ATP channel activity from cell-attached patches in control and SNL neurons.Representative cell-attached recording traces in control (non surgery) and SNL neurons.Basal single channel currents were recorded at -60 mV.Horizontal arrows indicate closed channel state.

Fig. 11 .
Fig. 11.Correlation of basal K ATP channel NPo with each donor rat's probability of hyperalgesia, showing an inverse relationship.Simple linear regression curve and confidence intervals are shown.

Fig. 12 .
Fig. 12. Single-channel characteristics of K ATP channels from inside-out recordings from control or SNL neurons.Membrane potential was clamped at -60 mV.Upon patch excision (vertical arrow) into an ATP-free bath marked channel activity ensued.ATP (1 mM), diazoxide (100 µM), and glibenclamide (1 µM) was added to the intracellular (bath) solution as indicated by the horizontal solid bar.Horizontal arrows indicate closed channel state.