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Effect of ciprofloxacin and biphenylacetic acid on voltage-gated ion channels in cultured rat hippocampal neurons
Abstracts / Neuroscience Research 58S (2007) S1–S244
3 Increased Ca2+ channel currents in cerebellar Purkinje P3-b0
S189
6 Effect of ciprofloxacin and biphenylacetic acid on P3-b0
cells of the ataxic Groggy rat
voltage-gated ion channels in cultured rat hippocampal neurons
Shuji Kaneko 1 , Kenta Tanaka 1 , Hisashi Shirakawa 1 , Satoko Tokuda 2 , Tadao Serikawa 2 1 Kyoto University Graduate School of Pharmaceutical Sciences, Japan; 2 Kyoto University Graduate School of Medicine, Laboratory Animal Center, Japan
Ryoko Hirakawa, Parto Khansari, Leanne Coyne, Robert F. Halliwell TJL School of Pharmacy and Health Science, University of the Pacific, Stockton, CA, USA
The groggy rat (strain name; GRY) is an autosomal recessive neurological mutant found in a closed colony of Slc:Wistar rats, whose phenotype is characterized by movement disorder, ataxia and unstable gait first observed at around postnatal day 15. Recently, genetic analysis has identified the missense (M251K) mutation in the coding region of Cav 2.1 ␣1 subunit in GRY rat. Here, we have evaluated the functional properties of Ca2+ channel currents in the cerebellar Purkinje cells acutely isolated from GRY rats. Our results demonstrate that high-voltage-activated (HVA) currents in Purkinje cells of GRY rats are distinctly upregulated with respect to the current density, voltage-dependence of activation and inactivation, whereas those of low-voltage-activated (LVA) currents remain unchanged. These results suggest that increased HVA Ca2+ channel function underlies the cerebellar dysfunction and ataxic phenotype of GRY rats.
4 Effect of RIM 1 on inactivation of neuronal voltageP3-b0 dependent calcium channels
Binding and electrophysiological studies have shown a novel antagonist interaction between ciprofloxacin and biphenylacetic acid (BPAA) at GABAA receptors. In the present study, the effect of these two drugs on voltage-gated K+ , Na+ and Ca2+ channels in the cultured rat hippocampal neurons was investigated using the whole-cell patch clamp techniques. BPAA (100 M), ciprofloxacin (100 M) and the combination of BPAA and ciprofloxacin (100 + 10 M) had little or no effect on the K+ or Na+ currents. While ciprofloxacin had no effect, BPAA and the combination of BPAA plus ciprofloxacin did increase the amplitude of Ca2+ currents. Voltage-dependentance of activation was not however changed by BPAA remaining at approximately V1/2 = −15 ± 1.4 mV in control and in the presence of the drug. These data show that ciprofloxacin and BPAA have little or no effects at voltage-gated K+ and Na+ channels but BPAA can enhance Ca2+ currents in hippocampal neurons.
7 Identification of the Ginsenoside interaction site in P3-b0 human Kv1.4 channels
Minoru Yoshitsugu Takafumi Shigeki Kiyonaka 2 , Yasuo Mori 2 1 Dept Oral Biol, Tohoku Univ, Sendai, Japan; 2 Dept Synth & Biol Chem, Kyoto Univ, Kyoto, Japan
Jun-Ho Lee 1 , Byung-Hwan Lee 1 , Sun-Hye Choi 1 , In-Soo Yoon 1 , Joon-Hee Lee 1 , Mi Kyung Pyo 1 , Sang Min Jeong 1 , Woo-Sung Choi 1 , Sang-Mok Lee 1 , Hyewhon Rhim 2 , Seung-Yeol Nah 1 1 Department of Physiology, College of Vet. Medicine, Konkuk University, Seoul, Korea; 2 Biomedical Res. Center, KIST, Seoul, Korea
Active zones (AZs) are highly specialized sites for release of neurotransmitter in presynaptic nerve terminals. The spacing between voltage-dependent calcium channels (VDCCs) and synaptic vesicles at AZs is thought to influence the dynamic properties of synaptic transmission. Recently we have demonstrated a novel molecular interaction between an AZ scaffolding protein, RIM1, and VDCCs. To elucidate the functional significance of this direct coupling, we characterized wholecell Ba2+ currents through recombinant VDCCs. The RIM1 induced a pronounced deceleration of inactivation rate and a depolarizing shift of the inactivation curve. Currents evoked by trains of action potential waveforms, a more physiological voltage-clamp protocol used particularly to reveal closed-state inactivation, further support profound suppression of voltage-dependent inactivation by the RIM1. Thus, the RIM1 coordinates calcium signaling and spatial organization of molecular constituents in presynaptic AZ.
Voltage-gated K+ channels play critical roles in a variety of physiological processes in nervous systems and heart. We found that Rg3-induced inhibition of hKv1.4 channel currents was concentration-dependent and reversible. Raising extracellular [K+]o induced K+ activation by enhancing outward K+ currents and abolished the effect of Rg3 on hKv1.4 channels. We examined whether the K531 residue (K+ activation and external TEA binding site) is also involved in Rg3 interactions. Mutation of K531 to K531Y abolished the effects of Rg3 and rendered channels sensitive to TEA. Examinations of the effect of TEA on Rg3-induced regulation of wild-type channels and K531Y mutant channels revealed that the K531 is an overlapping interaction site for Rg3 and TEA. These results indicate that Rg3 is a novel hKv1.4 channel regulator that functions by interacting with the K531 residue.
Wakamori 1,2 ,
Uriu 2 ,
Miki 2 ,
Research funds: Korean Research Foundation, KRF-2004-015-E00234 and KRF-2006-005-J03403
5 Activity-dependent relief of G-protein-dependent inhibiP3-b0 tion of presynaptic P/Q-type Ca2+ channels Masahiro Kaneko, Tomoyuki Takahashi Department of Neurophysics, University Tokyo Grad. Sch. Med., Tokyo, Japan Activation of GPCRs reduces Ca2+ influx via G␥-subunits binding with voltage-gated Ca2+ channels, thereby decreasing neuro transmitter release. Though the depolarization-induced relief of G-protein-dependent inhibition is well-known phenomenon, it is not known whether action potential can relief the inhibition. To address this issue, we recorded Nand P/Q-type Ca2+ currents directly from calyces of Held nerve terminals. The G-protein-dependent inhibition of Ca2+ channels was elicited by intracellular application of GTP␥-S or extracellular application of the GABAB receptor agonist baclofen. The duration and potential of depolarizing pulse required for disinhibition was shorter and lower in P/Q- than N-type channels, suggesting the higher affinity of G␥-subunit for N-type channels. The high frequency trains of action potential waveform can relieve the inhibition in P/Q-type channels. We conclude that the developmental switch from N- to P/Q-type channels endows presynaptic terminals with the property of the activity dependent relief of G-protein-dependent inhibition. Research funds: JSPS Research Fellowships
8 Expression and localization of TREK-1 channel in P3-b0 Schwann cells Dawon Kang, Eun-Jin Kim, Gyu-Tae Kim, Chang-Gi Hur, Jae-Yong Park, Seong-Geun Hong, Jaehee Han Department of Physiology, Gyeongsang Nati. University, Jinju, South Korea The Schwann cell has widely studied for repair lesioned peripheral nerve and spinal cord. In the peripheral nervous system, the neuron-glial signaling is mediated by axon electrical activity through ion channels. TWIK-related K+ channels (TREKs; TREK-1 and TREK-2) are functionally expressed in dorsal root ganglion (DRG) neurons. Whether TREKs are expressed in Schwann cells has not yet been reported. Here we report the expression and localization of TREK-1 and TRPV1 in Schwann celllike glial (SCLG) cells obtained from rat DRG. Glial cells obtained from DRG demonstrated positive staining with antibodies of Schwann cell markers (S-100 and GFAP). Immunostaining data showed that TREK-1 was highly expressed in SCLG cells, but was localized only in nucleus. However, TREK-1-like channel was functionally expressed and activated by arachidonic acid, low pH, and stretch as judged by single channel recording. These results suggest that TREK-1 may serve for background K+ conductance in SCLG cells. Research funds: The Basic Research Program of the KOSEF (R13-2005012-01002-0)
3 Increased Ca2+ channel currents in cerebellar Purkinje P3-b0
S189
6 Effect of ciprofloxacin and biphenylacetic acid on P3-b0
cells of the ataxic Groggy rat
voltage-gated ion channels in cultured rat hippocampal neurons
Shuji Kaneko 1 , Kenta Tanaka 1 , Hisashi Shirakawa 1 , Satoko Tokuda 2 , Tadao Serikawa 2 1 Kyoto University Graduate School of Pharmaceutical Sciences, Japan; 2 Kyoto University Graduate School of Medicine, Laboratory Animal Center, Japan
Ryoko Hirakawa, Parto Khansari, Leanne Coyne, Robert F. Halliwell TJL School of Pharmacy and Health Science, University of the Pacific, Stockton, CA, USA
The groggy rat (strain name; GRY) is an autosomal recessive neurological mutant found in a closed colony of Slc:Wistar rats, whose phenotype is characterized by movement disorder, ataxia and unstable gait first observed at around postnatal day 15. Recently, genetic analysis has identified the missense (M251K) mutation in the coding region of Cav 2.1 ␣1 subunit in GRY rat. Here, we have evaluated the functional properties of Ca2+ channel currents in the cerebellar Purkinje cells acutely isolated from GRY rats. Our results demonstrate that high-voltage-activated (HVA) currents in Purkinje cells of GRY rats are distinctly upregulated with respect to the current density, voltage-dependence of activation and inactivation, whereas those of low-voltage-activated (LVA) currents remain unchanged. These results suggest that increased HVA Ca2+ channel function underlies the cerebellar dysfunction and ataxic phenotype of GRY rats.
4 Effect of RIM 1 on inactivation of neuronal voltageP3-b0 dependent calcium channels
Binding and electrophysiological studies have shown a novel antagonist interaction between ciprofloxacin and biphenylacetic acid (BPAA) at GABAA receptors. In the present study, the effect of these two drugs on voltage-gated K+ , Na+ and Ca2+ channels in the cultured rat hippocampal neurons was investigated using the whole-cell patch clamp techniques. BPAA (100 M), ciprofloxacin (100 M) and the combination of BPAA and ciprofloxacin (100 + 10 M) had little or no effect on the K+ or Na+ currents. While ciprofloxacin had no effect, BPAA and the combination of BPAA plus ciprofloxacin did increase the amplitude of Ca2+ currents. Voltage-dependentance of activation was not however changed by BPAA remaining at approximately V1/2 = −15 ± 1.4 mV in control and in the presence of the drug. These data show that ciprofloxacin and BPAA have little or no effects at voltage-gated K+ and Na+ channels but BPAA can enhance Ca2+ currents in hippocampal neurons.
7 Identification of the Ginsenoside interaction site in P3-b0 human Kv1.4 channels
Minoru Yoshitsugu Takafumi Shigeki Kiyonaka 2 , Yasuo Mori 2 1 Dept Oral Biol, Tohoku Univ, Sendai, Japan; 2 Dept Synth & Biol Chem, Kyoto Univ, Kyoto, Japan
Jun-Ho Lee 1 , Byung-Hwan Lee 1 , Sun-Hye Choi 1 , In-Soo Yoon 1 , Joon-Hee Lee 1 , Mi Kyung Pyo 1 , Sang Min Jeong 1 , Woo-Sung Choi 1 , Sang-Mok Lee 1 , Hyewhon Rhim 2 , Seung-Yeol Nah 1 1 Department of Physiology, College of Vet. Medicine, Konkuk University, Seoul, Korea; 2 Biomedical Res. Center, KIST, Seoul, Korea
Active zones (AZs) are highly specialized sites for release of neurotransmitter in presynaptic nerve terminals. The spacing between voltage-dependent calcium channels (VDCCs) and synaptic vesicles at AZs is thought to influence the dynamic properties of synaptic transmission. Recently we have demonstrated a novel molecular interaction between an AZ scaffolding protein, RIM1, and VDCCs. To elucidate the functional significance of this direct coupling, we characterized wholecell Ba2+ currents through recombinant VDCCs. The RIM1 induced a pronounced deceleration of inactivation rate and a depolarizing shift of the inactivation curve. Currents evoked by trains of action potential waveforms, a more physiological voltage-clamp protocol used particularly to reveal closed-state inactivation, further support profound suppression of voltage-dependent inactivation by the RIM1. Thus, the RIM1 coordinates calcium signaling and spatial organization of molecular constituents in presynaptic AZ.
Voltage-gated K+ channels play critical roles in a variety of physiological processes in nervous systems and heart. We found that Rg3-induced inhibition of hKv1.4 channel currents was concentration-dependent and reversible. Raising extracellular [K+]o induced K+ activation by enhancing outward K+ currents and abolished the effect of Rg3 on hKv1.4 channels. We examined whether the K531 residue (K+ activation and external TEA binding site) is also involved in Rg3 interactions. Mutation of K531 to K531Y abolished the effects of Rg3 and rendered channels sensitive to TEA. Examinations of the effect of TEA on Rg3-induced regulation of wild-type channels and K531Y mutant channels revealed that the K531 is an overlapping interaction site for Rg3 and TEA. These results indicate that Rg3 is a novel hKv1.4 channel regulator that functions by interacting with the K531 residue.
Wakamori 1,2 ,
Uriu 2 ,
Miki 2 ,
Research funds: Korean Research Foundation, KRF-2004-015-E00234 and KRF-2006-005-J03403
5 Activity-dependent relief of G-protein-dependent inhibiP3-b0 tion of presynaptic P/Q-type Ca2+ channels Masahiro Kaneko, Tomoyuki Takahashi Department of Neurophysics, University Tokyo Grad. Sch. Med., Tokyo, Japan Activation of GPCRs reduces Ca2+ influx via G␥-subunits binding with voltage-gated Ca2+ channels, thereby decreasing neuro transmitter release. Though the depolarization-induced relief of G-protein-dependent inhibition is well-known phenomenon, it is not known whether action potential can relief the inhibition. To address this issue, we recorded Nand P/Q-type Ca2+ currents directly from calyces of Held nerve terminals. The G-protein-dependent inhibition of Ca2+ channels was elicited by intracellular application of GTP␥-S or extracellular application of the GABAB receptor agonist baclofen. The duration and potential of depolarizing pulse required for disinhibition was shorter and lower in P/Q- than N-type channels, suggesting the higher affinity of G␥-subunit for N-type channels. The high frequency trains of action potential waveform can relieve the inhibition in P/Q-type channels. We conclude that the developmental switch from N- to P/Q-type channels endows presynaptic terminals with the property of the activity dependent relief of G-protein-dependent inhibition. Research funds: JSPS Research Fellowships
8 Expression and localization of TREK-1 channel in P3-b0 Schwann cells Dawon Kang, Eun-Jin Kim, Gyu-Tae Kim, Chang-Gi Hur, Jae-Yong Park, Seong-Geun Hong, Jaehee Han Department of Physiology, Gyeongsang Nati. University, Jinju, South Korea The Schwann cell has widely studied for repair lesioned peripheral nerve and spinal cord. In the peripheral nervous system, the neuron-glial signaling is mediated by axon electrical activity through ion channels. TWIK-related K+ channels (TREKs; TREK-1 and TREK-2) are functionally expressed in dorsal root ganglion (DRG) neurons. Whether TREKs are expressed in Schwann cells has not yet been reported. Here we report the expression and localization of TREK-1 and TRPV1 in Schwann celllike glial (SCLG) cells obtained from rat DRG. Glial cells obtained from DRG demonstrated positive staining with antibodies of Schwann cell markers (S-100 and GFAP). Immunostaining data showed that TREK-1 was highly expressed in SCLG cells, but was localized only in nucleus. However, TREK-1-like channel was functionally expressed and activated by arachidonic acid, low pH, and stretch as judged by single channel recording. These results suggest that TREK-1 may serve for background K+ conductance in SCLG cells. Research funds: The Basic Research Program of the KOSEF (R13-2005012-01002-0)