Inhibitory Actions of HERG Currents by the Immunosuppressant Drug Cyclosporin A

The effect of cyclosporin A (CsA), an immunosuppressant, on human ether-a-go-go-related gene (HERG) channel as it is expressed in human embryonic kidney cells was studied using a whole-cell, patch-clamp technique. CsA inhibited the HERG channel in a concentration-dependent manner, with an IC50 value and a Hill coefficient of 3.17 microM and 0.89, respectively. Pretreatment with cypermethrine, a calcineurin inhibitor, had no effect on the CsA-induced inhibition of the HERG channel. The CsA-induced inhibition of HERG channels was voltage-dependent, with a steep increase over the voltage range of the channel opening. However, the inhibition exhibited voltage independence over the voltage range of fully activated channels. CsA blocked the HERG channels predominantly in the open and inactivated states rather than in the closed state. Results of the present study suggest that CsA acts directly on the HERG channel as an open-channel blocker, and it acts independently of its effect on calcineurin activity.

DAMGO, a micro-opioid agonist and cholecystokinin-octapeptide have dual modulatory effects on capsaicin-activated current in rat dorsal root ganglion neurons

Capsaicin, a pungent ingredient of hot pepper, elicits an intense burning pain when applied cutaneously and intradermally. Activation of capsaicin-gated channel in. C-type dorsal root ganglion (DRG) neurons produces nonselective cationic currents. Although electrophysiological and biochemical properties of capsaicin-activated current (ICAP) were studied, the regulatory mechanism and intracellular signaling pathway are still unclear. In the present study, we investigated the modulations of ICAP by DAMGO (micro-opioid agonist) and cholecystokinin octapeptide (CCK-8). In 18 out of 86 cells, the amplitude of ICAP was significantly increased by DAMGO and completely reversed after washout, while ICAP was decreased by DAMGO in 25 cells. In 43 cells, DAMGO had no effect on ICAP. Mean action potential duration was significantly different between 'increased-by-DAMGO' group and 'decreased-by-DAMGO' group. Mean amplitudes of IH were not significantly different between both groups. CCK-8 reversibly enhanced the amplitude of ICAP (5/13). DAMGO also increased ICAP amplitude significantly in the same cells. The amplitude of ICAP was increased in additive manner by combined applications of DAMGO and CCK-8 in these cells. These results suggest that DAMGO and CCK-8 can either increase or decrease ICAP presumably depending on the subtypes of DRG cells and classified by electrophysiological properties.

The effects of sera from amyotrophic lateral sclerosis patients on neuromuscular transmission and calcium channels in mice

Amyotrophic lateral sclerosis (ALS) is a degenerative neuromuscular disease of unknown etiology in which the upper and lower motor neurons are progressively destroyed. Recent evidences support the role of autoimmune mechanisms in the pathogenesis of ALS. This study investigated the effects of sera from ALS patients on neuromuscular transmission in phrenic nerve-hemidiaphragm preparations and on calcium currents of single isolated dorsal root ganglion (DRG) cells in mice. Mice were injected with either control sera from healthy adults or ALS sera from 18 patients with ALS of sporadic form, for three days. Miniature end plate potential (MEPP) and nerve-evoked end plate potential (EPP) were measured using intracellular recording technique and the quantal content was determined. Single isolated DRG cells were voltage-clamped with the whole-cell configuration and membrane currents were recorded. Sera from 14 of 18 ALS patients caused a significant increase in MEPP frequency in normal Ringer's solution (4.62+/-0.14 Hz) compared with the control (2.18+/-0.15 Hz). In a high Mg2+/low Ca2+ solution, sera from 13 of 18 ALS patients caused a significant increase in MEPP frequency, from 2.18+/-0.31 Hz to 6.09+/-0.38 Hz. Sera from 11 of 18 patients produced a significant increase of nerve-evoked EPP amplitude, from 0.92+/-0.05 mV to 1.30+/-0.04 mV, while the other seven ALS sera did not alter EPP amplitude. In the ALS group, EPP quantal content was also elevated by the sera of 14 patients (from 1.49+/-0.07 to 2.35+/-0.07). MEPP frequency and amplitude in wobbler mouse were 4.03+/-0.53 Hz and 1.37+/-0.18 mV, respectively, which were significantly higher than those of wobbler controls (wobblers without the symptoms of wobbler). Sera from ALS patients significantly reduced HVA calcium currents of DRG cells to 42.7% at -10 mV. Furthermore, the inactivation curve shifted to more negative potentials with its half-inactivation potential changed by 6.98 mV. There were, however, significant changes neither in the reversal potential of ICa nor in the I-V curve. From these results it was concluded that: 1) The serum factors of sporadic ALS patients increase neuromuscular transmission and can alter motor nerve terminal presynaptic function. This suggests that ALS serum factors may play an important role in the early stage of ALS, and 2) Calcium currents in DRG cells were reduced and rapidly inactivated by ALS sera, suggesting that in these cells, ALS serum factors may exert interaction with the calcium channel.

Characterization of voltage-gated potassium currents in dorsal root ganglion neurons of neonatal rats

Dorsal root ganglion (DRG) is composed of neuronal cell bodies of primary afferents with diverse functions. Various types of ion channels present on DRG neurons may reflect those functions. In the present study, voltage-gated potassium currents in DRG neurons of neonatal rats were characterized by whole-cell voltage clamp method. Two types of delayed rectifier and three types of transient potassium currents were identified according to their electrophysiological properties. The delayed rectifier currents were named IKe (early inactivating) and IKl (late inactivating). Steady state inactivation of IKe began from -100 mV lasting until -20 mV. IKl could be distinguished from IKe by its inactivation voltage range, from -70 mV to + 10 mV. Three transient currents were named IAf (fast inactivation), IAi (intermediate inactivation kinetics), and IAs (slow inactivation). IAf showed fast inactivation with time constant of 10.6 +/- 2.0 msec, IAi of 36.9 +/- 13.9 msec, and IAs of 60.6 +/- 2.9 msec at +30 mV, respectively. They also had distinct steady state inactivation range of each. Each cell expressed diverse combination of potassium-currents. The cells most frequently observed were those which expressed both IKl and IAf, and they had large diameters. The cells expressing IKe and expressing IKe, IAi, and IAs usually had small diameters. Judging from cell diameter, capsaicin sensitivity or action potential duration, candidates for nociceptor were the cells expressing IKe, expressing IKe and IAi, and expressing IKe and IAs. The types and distribution of potassium currents in neonatal rat DRG were similar to those of adult rat DRG (Gold et al, 1996b).