A-type Currents Research Articles

Caffeine modulates potassium currents in Drosophila neurons

We investigated the effects of caffeine on the delayed-rectifier potassium current (IKDR) which is important in repolarizing the membrane potential, and the transient A-type potassium current (IKA) which regulates neuronal firing threshold and the rate of repetitive action potentials. The whole-cell patch-clamp technique was used to measure the currents from cultured Drosophila neurons derived from embryonic neuroblasts. The currents were measured from neurons before and after the application of 1mM caffeine to the external saline of the same neuron. IKDR measured in the caffeine-containing solution (470±36pA, n=18), was smaller than that measured in the control 6K/0Ca Tris solution (745±51pA, n=18). IKA measured in the caffeine-containing solution (17±2pA, n=16) was smaller than that measured in the control 6K/0Ca Tris solution (35±4pA, n=16).These results indicate that caffeine reduces IKDR and IKA amplitudes and possibly leads to increased action potential frequency and enhanced neuronal excitability.

Seeking a mechanism of action for the novel anticonvulsant lacosamide

Lacosamide (LCM) is anticonvulsant in animal models and is in phase 3 assessment for epilepsy and neuropathic pain. Here we seek to identify cellular actions for the new drug and effects on recognised target sites for anticonvulsant drugs. Radioligand binding and electrophysiology were used to study the effects of LCM at well-established mammalian targets for clinical anticonvulsants. 10 μM LCM did not bind with high affinity to a plethora of rodent, guinea pig or human receptor sites including: AMPA; Kainate; NMDA (glycine/PCP/MK801); GABA A (muscimol/benzodiazepine); GABA B; adenosine A 1,2,3; α 1, α 2; β 1, β 2; M 1,2,3,4,5; H 1,2,3; CB 1,2; D 1,2,3,4,5; 5HT 1A,1B,2A,2C,3,5A,6,7 and K ATP. Weak displacement (25%) was evident at batrachotoxin site 2 on voltage gated Na + channels. LCM did not inhibit neurotransmitter transport mechanisms for norepinephrine, dopamine, 5-HT or GABA, nor did it inhibit GABA transaminase. LCM at 100 μM produced a significant reduction in the incidence of excitatory postsynaptic currents (EPSC's) and inhibitory postsynaptic currents (IPSC's) in cultured cortical cells and blocked spontaneous action potentials (EC 50 61 μM). LCM did not alter resting membrane potential or passive membrane properties following application of voltage ramps between −70 to +20 mV. The voltage-gated sodium channel (VGSC) blocker phenytoin potently blocked sustained repetitive firing (SRF) but, in contrast, 100 μM LCM failed to block SRF. No effect was observed on voltage-clamped Ca 2+ channels (T-, L-, N- or P-type). Delayed-rectifier or A-type potassium currents were not modulated by LCM (100 μM). LCM did not mimic the effects of diazepam as an allosteric modulator of GABA A receptor currents, nor did it significantly modulate evoked excitatory neurotransmission mediated by NMDA or AMPA receptors ( n ≥ 5). Evidently LCM perturbs excitability in primary cortical cultures but does not appear to do so via a high-affinity interaction with an acknowledged recognition site on a target for existing antiepileptic drugs.

The Kv4.2 Potassium Channel Subunit Is Required for Pain Plasticity

A-type potassium currents are important determinants of neuronal excitability. In spinal cord dorsal horn neurons, A-type currents are modulated by extracellular signal-regulated kinases (ERKs), which mediate central sensitization during inflammatory pain. Here, we report that Kv4.2 mediates the majority of A-type current in dorsal horn neurons and is a critical site for modulation of neuronal excitability and nociceptive behaviors. Genetic elimination of Kv4.2 reduces A-type currents and increases excitability of dorsal horn neurons, resulting in enhanced sensitivity to tactile and thermal stimuli. Furthermore, ERK-mediated modulation of excitability in dorsal horn neurons and ERK-dependent forms of pain hypersensitivity are absent in Kv4.2(-/-) mice compared to wild-type littermates. Finally, mutational analysis of Kv4.2 indicates that S616 is the functionally relevant ERK phosphorylation site for modulation of Kv4.2-mediated currents in neurons. These results show that Kv4.2 is a downstream target of ERK in spinal cord and plays a crucial role in pain plasticity.

Vitamins C and E Modulate Neuronal Potassium Currents

We investigated the effects of vitamins C and E on the delayed-rectifier potassium current (IK(DR)), which is important in repolarizing the membrane potential, and on the transient A-type potassium current (IK(A)), which regulates neuronal firing frequency. The whole-cell patch-clamp technique was used to measure the currents from cultured Drosophila neurons derived from embryonic neuroblasts. The membrane potential was stepped to different voltages between -40 and +60 mV from a holding potential of -80 mV. IK(DR) and IK(A) measured in the vitamin C-containing solution (IK(DR) 305 +/- 16 pA, IK(A) 11 +/- 2 pA) were smaller than those measured in the control solution (488 +/- 21 pA, IK(A )28 +/- 3 pA). By contrast, IK(DR) and IK(A) measured in the vitamin E-containing solution (IK(DR) 561 +/- 21 pA, IK(A )31 +/- 3 pA) were greater than those measured in the control solution (422 +/- 15 pA, 17 +/- 2 pA). These results indicate that vitamins C and E can modulate potassium current amplitudes and possibly lead to altered neuronal excitability.

A potassium channel (Kv4) cloned from the heart of the tunicateCiona intestinalisand its modulation by a KChIP subunit

Voltage-gated ion channels of the Kv4 subfamily produce A-type currents whose properties are tuned by accessory subunits termed KChIPs, which are a family of Ca2+ sensor proteins. By modifying expression levels and the intrinsic biophysical properties of Kv4 channels, KChIPs modulate the excitability properties of neurons and myocytes. We studied how a Kv4 channel from a tunicate, the first branching clade of the chordates, is modulated by endogenous KChIP subunits. BLAST searches in the genome of Ciona intestinalis identified a single Kv4 gene and a single KChIP gene, implying that the diversification of both genes occurred during early vertebrate evolution, since the corresponding mammalian gene families are formed by several paralogues. In this study we describe the cloning and characterization of a tunicate Kv4 channel, CionaKv4, and a tunicate KChIP subunit, CionaKChIP. We demonstrate that CionaKChIP strongly modulates CionaKv4 by producing larger currents that inactivate more slowly than in the absence of the KChIP subunit. Furthermore, CionaKChIP shifted the midpoints of activation and inactivation and slowed deactivation and recovery from inactivation of CionaKv4. Modulation by CionaKChIP requires the presence of the intact N terminus of CionaKv4 because, except for a minor effect on inactivation, CionaKChIP did not modulate CionaKv4 channels that lacked amino acids 2-32. In summary, our results suggest that modulation of Kv4 channels by KChIP subunits is an ancient mechanism for modulating electrical excitability.

Functional properties and oxidative modulation of A‐type K+ currents in hippocampal granule cells of control and chronically epileptic rats

A-type K+ channels are crucial determinants of neuronal firing. For example, reducing the amplitude of A-type currents (I(A)) increases seizure susceptibility. We have therefore examined the functional and molecular properties of I(A) in dentate granule neurons following pilocarpine-induced status epilepticus (SE). We found that the levels of various A-type channel subunit mRNAs are unaltered following SE. Furthermore, current density and biophysical properties of I(A) recorded in outside-out and cell-attached patches from dentate granule cells are not modified by SE. However, I(A) in both control and epileptic rats was powerfully regulated by the cellular redox state. I(A) was recorded in outside-out patches with the recording pipette containing either reduced (GSH) or oxidized (GSSG) glutathione. In both control and epileptic rats, the presence of GSSG caused a similar, marked acceleration of recovery from inactivation. Additionally, GSSG produced a small but significant reduction of I(A) amplitudes only in control rats. The inactivation time course of I(A) during depolarizing voltage steps was not modified by GSH or GSSG. Cell-attached recordings, in which the intracellular milieu is conserved, revealed a slow time course of recovery more comparable to that with GSH. In summary, epileptic activity does not produce chronic changes in the molecular and functional properties of the somatic I(A) of dentate granule cells. However, I(A) is powerfully modulated by oxidation in both control and epileptic rats. This finding suggests that the availability of I(A) may be strongly regulated by changes in the GSH/GSSG ratio occurring during prolonged seizure activity or hypoxia.

Enhanced excitability of rat trigeminal root ganglion neurons via decrease in A-type potassium currents following temporomandibular joint inflammation

The aim of the present study was to investigate the effect of temporomandibular joint inflammation on the excitability of trigeminal root ganglion neurons innervating the temporomandibular joint using a perforated patch-clamp technique. Inflammation was induced by injection of complete Freund’s adjuvant into the rat temporomandibular joint. The threshold for escape from mechanical stimulation in the temporomandibular joint-inflamed rats was significantly lower than that in control rats. Fluorogold labeling was used to identify the trigeminal root ganglion neurons innervating the site of inflammation. When voltage-clamp (V h=−60 mV) conditions were applied to these Fluorogold-labeled small diameter trigeminal root ganglion neurons (<30 μm), voltage-dependent transient K + current densities were significantly reduced in the inflamed rats compared with controls. In addition, the voltage-dependence of inactivation of the voltage-dependent transient K + current was negatively shifted in the labeled temporomandibular joint-inflamed trigeminal root ganglion neurons. Furthermore, temporomandibular joint inflammation significantly reduced the threshold current and significantly increased action potential firings evoked at two-fold threshold in the Fluorogold-labeled small trigeminal root ganglion neurons. Application of 4-aminopyridine (0.5mM) to control trigeminal root ganglion neurons mimicked the changes in the firing properties observed after complete Freund’s adjuvant treatment. Together, these results suggest that temporomandibular joint inflammation increases the excitability of trigeminal root ganglion neurons innervating temporomandibular joint by suppressing voltage-dependent transient K + current via a leftward shift in the inactivation curve. These changes may contribute to trigeminal inflammatory allodynia in temporomandibular joint disorder.

Impaired Inflammatory Pain and Thermal Hyperalgesia in Mice Expressing Neuron-Specific Dominant Negative Mitogen Activated Protein Kinase Kinase (MEK)

BackgroundNumerous studies have implicated spinal extracellular signal-regulated kinases (ERKs) as mediators of nociceptive plasticity. These studies have utilized pharmacological inhibition of MEK to demonstrate a role for ERK signaling in pain, but this approach cannot distinguish between effects of ERK in neuronal and non-neuronal cells. The present studies were undertaken to test the specific role of neuronal ERK in formalin-induced inflammatory pain. Dominant negative MEK (DN MEK) mutant mice in which MEK function is suppressed exclusively in neurons were tested in the formalin model of inflammatory pain.ResultsFormalin-induced second phase spontaneous pain behaviors as well as thermal hyperalgesia measured 1 – 3 hours post-formalin were significantly reduced in the DN MEK mice when compared to their wild type littermate controls. In addition, spinal ERK phosphorylation following formalin injection was significantly reduced in the DN MEK mice. This was not due to a reduction of the number of unmyelinated fibers in the periphery, since these were almost double the number observed in wild type controls. Further examination of the effects of suppression of MEK function on a downstream target of ERK phosphorylation, the A-type potassium channel, showed that the ERK-dependent modulation of the A-type currents is significantly reduced in neurons from DN MEK mice compared to littermate wild type controls.ConclusionOur results demonstrate that the neuronal MEK-ERK pathway is indeed an important intracellular cascade that is associated with formalin-induced inflammatory pain and thermal hyperalgesia.

A-Type and T-Type Currents Interact to Produce a Novel Spike Latency-Voltage Relationship in Cerebellar Stellate Cells

The modification of first-spike latencies by low-threshold and inactivating K+ currents (IA) have important implications in neuronal coding and synaptic integration. To date, cells in which first-spike latency characteristics have been analyzed have shown that increased hyperpolarization results in longer first-spike latencies, producing a monotonic relationship between first-spike latency and membrane voltage. Previous work has established that cerebellar stellate cells express members of the Kv4 potassium channel subfamily, which underlie IA in many central neurons. Spike timing in stellate cells could be particularly important to cerebellar output, because the discharge of even single spikes can significantly delay spike discharge in postsynaptic Purkinje cells. In the present work, we studied the first-spike latency characteristics of stellate cells. We show that first-spike latency is nonmonotonic, such that intermediate levels of prehyperpolarization produce the longest spike latencies, whereas greater hyperpolarization or depolarization reduces spike latency. Moreover, the range of first-spike latency values can be substantial in spanning 20-128 ms with preceding membrane shifts of <10 mV. Using patch clamp and modeling, we illustrate that spike latency characteristics are the product of an interplay between IA and low-threshold calcium current (IT) that requires a steady-state difference in the inactivation parameters of the currents. Furthermore, we show that the unique first-spike latency characteristics of stellate cells have important implications for the integration of coincident IPSPs and EPSPs, such that inhibition can shift first-spike latency to differentially modulate the probability of firing.

ERK/MAPK regulates the Kv4.2 potassium channel by direct phosphorylation of the pore-forming subunit

Kv4.2 is the primary pore-forming subunit encoding A-type currents in many neurons throughout the nervous system, and it also contributes to the transient outward currents of cardiac myocytes. A-type currents in the dendrites of hippocampal CA1 pyramidal neurons are regulated by activation of ERK/MAPK, and Kv4.2 is the likely pore-forming subunit of that current. We showed previously that Kv4.2 is directly phosphorylated at three sites by ERK/MAPK (T602, T607, and S616). In this study we determined whether direct phosphorylation of Kv4.2 by ERK/MAPK is responsible for the regulation of the A-type current observed in neurons. We made site-directed mutants, changing the phosphosite serine (S) or threonine (T) to aspartate (D) to mimic phosphorylation. We found that the T607D mutation mimicked the electrophysiological changes elicited by ERK/MAPK activation in neurons: a rightward shift of the activation curve and an overall reduction in current compared with wild type (WT). Surprisingly, the S616D mutation caused the opposite effect, a leftward shift in the activation voltage. K(+) channel-interacting protein (KChIP)3 ancillary subunit coexpression with Kv4.2 was necessary for the T607D effect, as the T607D mutant when expressed in the absence of KChIP3 was not different from WT Kv4.2. These data suggest that direct phosphorylation of Kv4.2 at T607 is involved in the dynamic regulation of the channel function by ERK/MAPK and an interaction of the primary subunit with KChIP is also necessary for this effect. Overall these studies provide new insights into the structure-function relationships for MAPK regulation of membrane ion channels.

ANEPIII, a new recombinant neurotoxic polypeptide derived from scorpion peptide, inhibits delayed rectifier, but not A-type potassium currents in rat primary cultured hippocampal and cortical neurons

A new recombinant neurotoxic polypeptide ANEPIII (BmK ANEPIII) derived from Scorpion peptide, which was demonstrated with antineuroexcitation properties in animal models, was examined for its action on K + currents in primary cultured rat hippocampal and cortical neurons using the patch clamp technique in the whole-cell configuration. The delayed rectifier K + current ( I k) was inhibited by externally applied recombinant BmK ANEPIII, while the transient A-current ( I A) remained virtually unaffected. BmK ANEPIII 3 μM, reduced the delayed rectifier current by 28.2% and 23.6% in cultured rat hippocampal and cortical neurons, respectively. The concentration of half-maximal block was 155.1 nM for hippocampal neurons and 227.2 nM for cortical neurons, respectively. These results suggest that BmK ANEPIII affect K + currents, which may lead to a reduction in neuronal excitability.

Effects of copper on A-type potassium currents in acutely dissociated rat hippocampal CA1 neurons

The effects of copper on voltage-gated A-type potassium currents were investigated in acutely dissociated rat hippocampal CA1 neurons using the whole-cell patch-clamp technique. Extracellular application of various concentrations of copper (1-1000 microM) reversibly reduced the amplitude of voltage-gated A-type potassium currents in a dose-dependent manner with a 50% inhibitory concentration value of 130 microM. Copper (300 microM) increased the V1/2 of the activation curve and state-inactivation curve by 17.2 and 9.0 mV, respectively. Thus, copper slowed down the activation and inactivation process of voltage-gated A-type potassium currents. This study indicated that copper reversibly inhibits the hippocampal CA1 neuronal voltage-gated A-type potassium current in a dose-dependent and voltage-dependent manner, and such actions are likely involved in the regulation of the neuronal excitability and the pathophysiology of Wilson's disease.

A-Type Potassium Current in Retinal Arteriolar Smooth Muscle Cells

By their control of membrane potential and intracellular free Ca(2+) ([Ca(2+)](i)), K(+) currents are pivotal in the regulation of arterial smooth muscle tone. The goal of the present study was to identify and characterize the A-type K(+) current in retinal microvascular smooth muscle (MVSM) and to examine its role in modulating membrane potential and cellular contractility. Whole-cell perforated patch-clamp recordings were made from MVSM cells within intact isolated arteriolar segments. Before patch-clamping, retinal arterioles were anchored in the physiological recording bath and perfused with an enzyme cocktail to remove surface basal lamina and to uncouple electrically the endothelial cells from the overlying MVSM cells. K(+) currents were activated by depolarizing steps from -80 to +100 mV in 20-mV increments. A dominant, noninactivating current was elicited by depolarization to potentials positive of -50 mV. Inhibition of this current by 100 nM of the Ca(2+)-activated K(+) channel blocker, Penitrem A, revealed a rapidly inactivating K(+) current that resembled an A-type current. The A-type current was insensitive to tetraethylammonium (TEA) at 1 mM, but was partially suppressed by higher concentrations (10 mM). 4-Aminopyridine (10 mM; 4-AP) completely blocked the A-type current. The 4-AP-sensitive transient current was activated at a potential of -60 mV with peak current densities averaging 29.7 +/- 5.68 pA/pF at +60 mV. The voltage of half-inactivation was -28.3 +/- 1.9 mV, and the time constant for recovery from inactivation at +60 mV was 118.7 +/- 7.9 ms. Under current-clamp conditions 4-AP depolarized the membrane potential by approximately 3 to 4 mV and triggered small contractions and relaxations of individual MVSM cells within the walls of the arterioles. A-type current is the major voltage-dependent K(+) current in retinal MVSM and appears to play a physiological role in suppressing cell excitability and contractility.

A Novel Mechanism for the Suppression of a Voltage-gated Potassium Channel by Glucose-dependent Insulinotropic Polypeptide: PROTEIN KINASE A-DEPENDENT ENDOCYTOSIS

The mechanisms involved in glucose regulation of insulin secretion by ATP-sensitive (K(ATP)) and calcium-activated (K(CA)) potassium channels have been extensively studied, but less is known about the role of voltage-gated (K(V)) potassium channels in pancreatic beta-cells. The incretin hormone, glucose-dependent insulinotropic polypeptide (GIP) stimulates insulin secretion by potentiating events underlying membrane depolarization and exerting direct effects on exocytosis. In the present study, we identified a novel role for GIP in regulating K(V)1.4 channel endocytosis. In GIP receptor-expressing HEK293 cells, GIP reduced A-type peak ionic current amplitude of K(V)1.4 via activation of protein kinase A (PKA). Using mutant forms of K(V)1.4 with Ala-Ser/Thr substitutions in a potential PKA phosphorylation site, C-terminal phosphorylation was shown to be linked to GIP-mediated current amplitude decreases. Proteinase K digestion and immunocytochemical studies on mutant K(V)1.4 localization following GIP stimulation demonstrated phosphorylation-dependent rapid endocytosis of K(V)1.4. Expression of K(V)1.4 protein was also demonstrated in human beta-cells; GIP treatment resulting in similar decreases in A-type potassium current peak amplitude to those in HEK293 cells. Transient overexpression in INS-1 beta-cells (clone 832/13) of wild-type (WT) K(V)1.4, or a T601A mutant form resistant to PKA phosphorylation, resulted in reduced glucose-stimulated insulin secretion; WT K(V)1.4 overexpression potentiated GIP-induced insulin secretion, whereas this response was absent in T601A cells. These results strongly support an important novel role for GIP in regulating K(V)1.4 cell surface expression and modulation of A-type potassium currents, which is likely to be critically important for its insulinotropic action.

Chloride concentration affects Kv channel voltage-gating kinetics: Importance of experimental anion concentrations

Chloride concentration has been shown to have a dramatic impact on protein folding and subsequent tertiary conformation [K.D. Collins, Ions from the Hofmeister series and osmolytes: effects on proteins in solution and in the crystallization process, Methods 34 (2004) 300–311; I. Jelesarov, E. Durr, R.M. Thomas, H.R. Bosshard, Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper), Biochemistry 37 (1998) 7539–7550]. As it is known that Kv channel gating is linked to the stability of the cytoplasmic T1 multimerization domain conformation [D.L. Minor, Y.F. Lin, B.C. Mobley, A. Avelar, Y.N. Jan, L.Y. Jan, J.M. Berger, The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel, Cell 102 (2000) 657–670; B.A. Yi, D.L. Minor Jr., Y.F. Lin, Y.N. Jan, L.Y. Jan, Controlling potassium channel activities: interplay between the membrane and intracellular factors, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 11016–11023] and that intracellular chloride concentration has been linked to Kv channel kinetics [L.K. Bekar, W. Walz, Intracellular chloride modulates A-type potassium currents in astrocytes, Glia 39 (2002) 207–216; W.B. Thoreson, S.L. Stella, Anion modulation of calcium current voltage dependence and amplitude in salamander rods, Biochim. Biophys. Acta 1464 (2000) 142–150], the objective of the present study was to address how chloride concentration changes affect Kv channel kinetics more closely in an isolated expression system. Initially, no significant chloride concentration-dependent effects on channel steady-state gating kinetics were observed. Only after disruption of the cytoskeleton with cytochalasin-D did we see significant chloride concentration-dependent shifts in gating kinetics. This suggests that the shift in gating kinetics is mediated through effects of intracellular chloride concentration on cytoplasmic domain tertiary conformation as cytoskeletal interaction appears to mask the effect. Furthermore, as cytoskeletal disruption only impacts channel gating kinetics at low physiological intracellular chloride concentrations, these studies highlight the importance of paying close attention to anion concentrations used under experimental conditions.

Expression and function of dipeptidyl‐aminopeptidase‐like protein 6 as a putative β‐subunit of human cardiac transient outward current encoded by Kv4.3

Dipeptidyl-aminopeptidase-like protein 6 (DPPX) was recently shown in the brain to modulate the kinetics of transient A-type currents by accelerating inactivation and recovery from inactivation. Since the kinetics of human cardiac transient outward current (I(to)) are not mimicked by coexpression of the alpha-subunit Kv4.3 with its known beta-subunit KChIP2, we have tested the hypothesis that DPPX may serve as an additional beta-subunit in the human heart. With quantitative real-time RT-PCR strong mRNA expression of DPPX was detected in human ventricles and was verified at the protein level in human but not in rat heart by a DPPX-specific antibody. Co-expression of DPPX with Kv4.3 in Chinese hamster ovary cells produced I(to)-like currents, but compared with expression of KChIP2a and Kv4.3, the time constant of inactivation was faster, the potential of half-maximum steady-state inactivation was more negative and recovery from inactivation was delayed. Co-expression of DPPX in addition to Kv4.3 and KChIP2a produced similar current kinetics as in human ventricular myocytes. We therefore propose that DPPX is an essential component of the native cardiac I(to) channel complex in human heart.

DPP10 Modulates Kv4-mediated A-type Potassium Channels

A new member of a family of proteins characterized by structural similarity to dipeptidyl peptidase (DPP) IV known as DPP10 was recently identified and linked to asthma susceptibility; however, the cellular functions of DPP10 are thus far unknown. DPP10 is highly homologous to subfamily member DPPX, which we previously reported as a modulator of Kv4-mediated A-type potassium channels (Nadal, M. S., Ozaita, A., Amarillo, Y., Vega-Saenz de Miera, E., Ma, Y., Mo, W., Goldberg, E. M., Misumi, Y., Ikehara, Y., Neubert, T. A., and Rudy, B. (2003) Neuron. 37, 449-461). We studied the ability of DPP10 protein to modulate the properties of Kv4.2 channels in heterologous expression systems. We found DPP10 activity to be nearly identical to DPPX activity and significantly different from DPPIV activity. DPPX and DPP10 facilitated Kv4.2 protein trafficking to the cell membrane, increased A-type current magnitude, and modified the voltage dependence and kinetic properties of the current such that they resembled the properties of A-type currents recorded in neurons in the central nervous system. Using in situ hybridization, we found DPP10 to be prominently expressed in brain neuronal populations that also express Kv4 subunits. Furthermore, DPP10 was detected in immunoprecipitated Kv4.2 channel complexes from rat brain membranes, confirming the association of DPP10 proteins with native Kv4.2 channels. These experiments suggest that DPP10 contributes to the molecular composition of A-type currents in the central nervous system. To dissect the structural determinants of these integral accessory proteins, we constructed chimeras of DPPX, DPP10, and DPPIV lacking the extracellular domain. Chimeras of DPPX and DPP10, but not DPPIV, were able to modulate the properties of Kv4.2 channels, highlighting the importance of the intracellular and transmembrane domains in this activity.

Shal and Shaker Differential Contribution to the K+Currents in theDrosophilaMushroom Body Neurons

Shaker, a voltage-dependent K+ channel, is enriched in the mushroom bodies (MBs), the locus of olfactory learning in Drosophila. Mutations in the shaker locus are known to alter excitability, neurotransmitter release, synaptic plasticity, and olfactory learning. However, a direct link of Shaker channels to MB intrinsic neuron (MBN) physiology has not been documented. We found that transcripts for shab, shaw, shaker, and shal, among which only Shaker and Shal have been reported to code for A-type currents, are present in the MBs. The electrophysiological data showed that the absence of functional Shaker channels modifies the distribution of half-inactivation voltages (V(i1/2)) in the MBNs, indicating a segregation of Shaker channels to only a subset (approximately 28%) of their somata. In harmony with this notion, we found that approximately one-fifth of MBNs lacking functional Shaker channels displayed dramatically slowed-down outward current inactivation times and reduced peak-current amplitudes. Furthermore, whereas all MBNs were sensitive to 4-aminopyridine, a nonspecific A-type current blocker, a subset of neurons (approximately 24%) displayed little sensitivity to a Shal-specific toxin. This subset of neurons displaying toxin-insensitive outward currents had more depolarized V(i1/2) values attributable to Shaker channels. Our findings provide the first direct evidence that altered Shaker channel function disrupts MBN physiology in Drosophila. To our surprise, the experimental data also indicate that Shaker channels segregate to a minor fraction of MB neuronal somata (20-30%), and that Shal channels contribute the somatic A-type current in the majority of MBNs.

Frequency-dependent potentiation of voltage-activated responses only in the intact neurohypophysis of the rat

The loose-patch-clamp technique was used with multiple-pulse protocols to study the frequency dependence of currents from the surface of the intact rat neurohypophysis (NH) and hypothalamus. In the NH, but not in the corresponding supraoptic nucleus of the hypothalamus, an initial, single pulse of 3-8 ms duration (long pulse) potentiated a secondary pulse response starting 20-50 ms after the initial pulse. Potentiation was abolished by 4-aminopyridine (4-AP), but not by tetraethylammonium (TEA) chloride or tetrandrine, indicating the participation of A-type potassium currents. Potentiation was also abolished by CdCl2, CoCl2 or 1 microM nicardipine, indicating the participation of calcium currents. The potentiation was reduced significantly in the presence of 4-6 mM extracellular CaCl2, indicating that the potentiation is not due to calcium influx. An initial train with as few as two pulses, each of 0.3-0.7 ms duration (short pulses) at 64-1,100 Hz also potentiated the secondary short pulse response significantly. We conclude that voltage-gated channels underlie this potentiation, which is due to interstitial calcium and potassium homeostasis changes induced by action potential activity and occurs only in the intact NH. A model is proposed for the participation of calcium and potassium channels in the burst patterning that is optimal for secretion from the NH.

A-Type Voltage-Gated K+ Currents Influence Firing Properties of Isolectin B4-Positive But Not Isolectin B4-Negative Primary Sensory Neurons

Voltage-gated K+ channels (Kv) in primary sensory neurons are important for regulation of neuronal excitability. The dorsal root ganglion (DRG) neurons are heterogeneous, and the types of native Kv currents in different groups of nociceptive DRG neurons are not fully known. In this study, we determined the difference in the A-type Kv current and its influence on the firing properties between isolectin B4 (IB4)-positive and -negative DRG neurons. Whole cell voltage- and current-clamp recordings were performed on acutely dissociated small DRG neurons of rats. The total Kv current density was significantly higher in IB+-positive than that in IB(4)-negative neurons. Also, 4-aminopyridine (4-AP) produced a significantly greater reduction in Kv currents in IB4-positive than in IB4-negative neurons. In contrast, IB4-negative neurons exhibited a larger proportion of tetraethylammonium-sensitive Kv currents. Furthermore, IB4-positive neurons showed a longer latency of firing and required a significantly larger amount of current injection to evoke action potentials. 4-AP significantly decreased the latency of firing and increased the firing frequency in IB4-positive but not in IB4-negative neurons. Additionally, IB4-positive neurons are immunoreactive to Kv1.4 but not to Kv1.1 and Kv1.2 subunits. Collectively, this study provides new information that 4-AP-sensitive A-type Kv currents are mainly present in IB4-positive DRG neurons and preferentially dampen the initiation of action potentials of this subpopulation of nociceptors. The difference in the density of A-type Kv currents contributes to the distinct electrophysiological properties of IB4-positive and -negative DRG neurons.