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Patch-clamp Studies (patch-clamp + studies)
Selected AbstractsExercise training attenuates ageing-induced BKCa channel downregulation in rat coronary arteriesEXPERIMENTAL PHYSIOLOGY, Issue 6 2010Sulayma Albarwani Physical inactivity and ageing are widely recognized as risk factors for development of coronary artery disease. One of the characteristic changes that occurs in aged coronary artery is downregulation of their large-conductance voltage- and calcium-activated K+ (BKCa) channels. In this study, we investigated the effects of moderate exercise training (ET) on the activity of BKCa channels in coronary arteries of aged rats. Old Fischer 344 rats (23,26 months old) were randomly assigned to sedentary (O-SED, n= 24) or exercise-trained groups (O-ET, n= 28). The O-ET rats underwent a progressive treadmill exercise-training programme for 60 min day,1, 5 days week,1 for 12 weeks. Young animals were used for comparison. Coronary arteries were mounted on a wire myograph, and contractions in response to 1, 10, 30, 50 and 100 nmol l,1 iberiotoxin were compared. Iberiotoxin (100 nmol l,1) contracted coronary arteries of young, O-SED and O-ET rats by 115 ± 14, 36 ± 5.6 and 61 ± 5% of 5-hydroxytryptamine-induced contractions, respectively. Patch-clamp studies revealed a larger magnitude of BKCa current in young (104 ± 15.6 pA pF,1) compared with O-ET (44 ± 9 pA pF,1) and least in O-SED coronary smooth muscle cells (8.6 ± 2 pA pF,1). Western immunoblotting was performed to study expression levels of BKCa channel proteins. The , and ,1 subunits of the BKCa channel were reduced by 40 ± 3.5 and 30 ± 2.6%, respectively, in coronary arteries of old compared with young rats, and ET attenuated this reduction in expression level to 28 ± 2 and 12 ± 4%, respectively. Our results showed that ageing was associated with a reduction in BKCa channels, and ET partly reversed this reduction. We conclude that low-intensity ET may be beneficial in restoring age-related decline in coronary vasodilatory properties mediated by BKCa channels. [source] The triakontatetraneuropeptide TTN increases [Ca2+]i in rat astrocytes through activation of peripheral-type benzodiazepine receptorsGLIA, Issue 2 2001Pierrick Gandolfo Abstract Astrocytes synthesize a series of regulatory peptides called endozepines, which act as endogenous ligands of benzodiazepine receptors. We have recently shown that one of these endozepines, the triakontatetraneuropeptide TTN, stimulates DNA synthesis in astroglial cells. The purpose of the present study was to determine the mechanism of action of TTN on cultured rat astrocytes. Binding of the peripheral-type benzodiazepine receptor ligand [3H]Ro5-4864 to intact astrocytes was displaced by TTN, whereas its C-terminal fragment (TTN[17,34], the octadecaneuropeptide ODN) did not compete for [3H]Ro5-4864 binding. Microfluorimetric measurement of cytosolic calcium concentrations ([Ca2+]i) with the fluorescent probe indo-1 showed that TTN (10,10 to 10,6 M) provokes a concentration-dependent increase in [Ca2+]i in cultured astrocytes. Simultaneous administration of TTN (10,8 M) and Ro5-4864 (10,5 M) induced an increase in [Ca2+]i similar to that obtained with Ro5-4864 alone. In contrast, the effects of TTN (10,8 M) and ODN (10,8 M) on [Ca2+]i were strictly additive. Chelation of extracellular Ca2+ by EGTA (6 mM) or blockage of Ca2+ channels with Ni2+ (2 mM) abrogated the stimulatory effect of TTN. The calcium influx evoked by TTN (10,7 M) or by Ro5-4864 (10,5 M) was not affected by the N- and T-type calcium channel blockers ,-conotoxin (10,6 M) and mibefradil (10,6 M), but was significantly reduced by the L-type calcium channel blocker nifedipine (10,7 M). Patch-clamp studies showed that, at negative potentials, TTN (10,7 M) induced a sustained depolarization. Reduction of the chloride concentration in the extracellular solution shifted the reversal potential from 0 mV to a positive potential. These data show that TTN, acting through peripheral-type benzodiazepine receptors, provokes chloride efflux, which in turn induces calcium influx via L-type calcium channels in rat astrocytes. GLIA 35:90,100, 2001. © 2001 Wiley-Liss, Inc. [source] Effect of Testosterone on Potassium Channel Opening in Human Corporal Smooth Muscle CellsTHE JOURNAL OF SEXUAL MEDICINE, Issue 4 2008Deok Hyun Han MD ABSTRACT Introduction., In humans, the role of testosterone in sexual functions, including sexual desire, nocturnal penile erections, and ejaculatory volume, has been relatively well established. However, the effects of testosterone on intrapenile structure in humans remains controversial. Aim., We assessed the direct effects of testosterone on potassium channels in human corporal smooth muscle cells, in an effort to understand the mechanisms inherent to the testosterone-induced relaxation of corporal smooth muscle cells at the cellular and molecular levels. Methods., We conducted electrophysiologic studies using cultured human corporal smooth muscle cells. We evaluated the effects of testosterone on potassium channels,BKCa and KATP channels,by determining the whole-cell currents and single-channel activities. For the electrophysiologic recordings, whole-cell and cell-attached configuration patch-clamp techniques were utilized. Main Outcome Measures., Changes in whole-cell currents and channel activities of BKCa and KATP channels by testosterone. Results., Testosterone (200 nM) significantly increased the single-channel activity of calcium-activated potassium (BKCa) channels and whole-cell K+ currents by 443.4 ± 83.4% (at +60 mV; N = 11, P < 0.05), and this effect was abolished by tetraethylammonium (TEA) (1 mM), a BKCa channel blocker. The whole-cell inward K+ currents of the KATP channels were also increased by 226.5 ± 49.3% (at ,100 mV; N = 7, P < 0.05). In the presence of a combination of vardenafil (10 nM) and testosterone (200 nM), the BKCa channel was activated to a significantly higher degree than was induced by testosterone alone. Conclusions., The results of patch-clamp studies provided direct molecular evidence that testosterone stimulates the activity of BKCa channels and KATP channels. An understanding of the signaling mechanisms that couple testosterone receptor activation to potassium channel stimulation will provide us with an insight into the cellular processes underlying the vasorelaxant effects of testosterone. Han DH, Chae MR, Jung JH, So I, Park JK, and Lee SW. Effect of testosterone on potassium channel opening in human corporal smooth muscle cells. J Sex Med 2008;5:822,832. [source] A novel Nav1.7 mutation producing carbamazepine-responsive erythromelalgia,ANNALS OF NEUROLOGY, Issue 6 2009Tanya Z. Fischer MD Objective Human and animal studies have shown that Nav1.7 sodium channels, which are preferentially expressed within nociceptors and sympathetic neurons, play a major role in inflammatory and neuropathic pain. Inherited erythromelalgia (IEM) has been linked to gain-of-function mutations of Nav1.7. We now report a novel mutation (V400M) in a three-generation Canadian family in which pain is relieved by carbamazepine (CBZ). Methods We extracted genomic DNA from blood samples of eight members of the family, and the sequence of SCN9A coding exons was compared with the reference Nav1.7 complementary DNA. Wild-type Nav1.7 and V400M cell lines were then analyzed using whole-cell patch-clamp recording for changes in activation, deactivation, steady-state inactivation, and ramp currents. Results Whole-cell patch-clamp studies of V400M demonstrate changes in activation, deactivation, steady-state inactivation, and ramp currents that can produce dorsal root ganglia neuron hyperexcitability that underlies pain in these patients. We show that CBZ, at concentrations in the human therapeutic range, normalizes the voltage dependence of activation and inactivation of this inherited erythromelalgia mutation in Nav1.7 but does not affect these parameters in wild-type Nav1.7. Interpretation Our results demonstrate a normalizing effect of CBZ on mutant Nav1.7 channels in this kindred with CBZ-responsive inherited erythromelalgia. The selective effect of CBZ on the mutant Nav1.7 channel appears to explain the ameliorative response to treatment in this kindred. Our results suggest that functional expression and pharmacological studies may provide mechanistic insights into hereditary painful disorders. Ann Neurol 2009;65:733,741 [source] Effect of K+ and Rb+ on the action of verapamil on a voltage-gated K+ channel, hKv1.3: implications for a second open state?BRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2009Z Kuras Background and purpose:, Verapamil blocks current through the voltage-gated K+ channel Kv1.3 in the open and inactivated state of the channel but not the closed state. The binding site for verapamil was proposed to be close to the selectivity filter and the occupancy of the selectivity filter might therefore influence verapamil affinity. Experimental approach:, We investigated the influence of intra- and extracellular K+ and Rb+ on the effect of verapamil by patch-clamp studies, in COS-7 cells transfected with hKv1.3 channels. Key results:, Verapamil affinity was highest in high intracellular K+ concentrations ([K+]i) and lowest in low [Rb+]i, indicating an influence of intracellular cations on verapamil affinity. Experiments with a mutant channel (H399T), exhibiting a strongly reduced C-type inactivated state, demonstrated that part of this changed verapamil affinity in wild-type channels could be caused by altered C-type inactivation. External K+ and Rb+ could influence verapamil affinity by a voltage-dependent entry into the channel thereby modifying the verapamil off-rate and in addition causing a voltage-dependent verapamil off-rate. Conclusions and implications:, Recovery from verapamil block was mainly due to the voltage-dependent closing of channels (state-dependent block), implying a second open state of the channel. This hypothesis was confirmed by the dependency of the tail current time course on duration of the prepulse. We conclude that the wild-type hKv1.3 channel undergoes at least two different conformational changes before finally closing with a low verapamil affinity in one open state and a high verapamil affinity in the other open state. [source] | |||||||||||||