Transient Calcium Channel Research Articles

Panama: An Open-Source Educational App for Ion Channel Biophysics Simulation.

This article describes an open-source educational software, called Panama, developed using R, that simulates the biophysics of voltage-gated ion channels. It is made publicly available as an R package called Panama and as a web app at http://www.neuronsimulator.com. A need for such a tool was observed after surveying available software packages. Available packages are either not robust enough to simulate multiple ion channels, too complicated, usable only as desktop software, not optimized for mobile devices, not interactive, lack intuitive graphical controls, or not appropriate for educational purposes. This app simulates the physiology of voltage-gated sodium, potassium, and chlorine channels; A channel; M channel; AHP channel; calcium-activated potassium channel; transient-calcium channel; and leak-calcium channel, under current-clamp or voltage-clamp conditions. As the input values on the app are changed, the output can be instantaneously visualized on the web browser and downloaded as a data table to be further analyzed in a spreadsheet program. This app is a first-of-its-kind, mobile-friendly, and touchscreen-friendly online tool that can be used as an installable R package. It has intuitive touch-optimized controls, instantaneous graphical output, and yet is pedagogically robust for educational purposes.

Potential Combination Drug Therapy to Prevent Redox Stress and Mitophagy Dysregulation in Retinal Müller Cells under High Glucose Conditions: Implications for Diabetic Retinopathy.

Chronic hyperglycemia-induced thioredoxin-interacting protein (TXNIP) expression, associated oxidative/nitrosative stress (ROS/RNS), and mitochondrial dysfunction play critical roles in the etiology of diabetic retinopathy (DR). However, there is no effective drug treatment to prevent or slow down the progression of DR. The purpose of this study is to examine if a combination drug treatment targeting TXNIP and the mitochondria-lysosome pathway prevents high glucose-induced mitochondrial stress and mitophagic flux in retinal Müller glial cells in culture, relevant to DR. We show that diabetes induces TXNIP expression, redox stress, and Müller glia activation (gliosis) in rat retinas when compared to non-diabetic rat retinas. Furthermore, high glucose (HG, 25 mM versus low glucose, LG 5.5 mM) also induces TXNIP expression and mitochondrial stress in a rat retinal Müller cell line, rMC1, in in vitro cultures. Additionally, we develop a mitochondria-targeted mCherry and EGFP probe tagged with two tandem COX8a mitochondrial target sequences (adenovirus-CMV-2×mt8a-CG) to examine mitophagic flux in rMC1. A triple drug combination treatment was applied using TXNIP-IN1 (which inhibits TXNIP interaction with thioredoxin), Mito-Tempo (mitochondrial anti-oxidant), and ML-SA1 (lysosome targeted activator of transient calcium channel MCOLN1/TRPML1 and of transcription factor TFEB) to study the mitochondrial–lysosomal axis dysregulation. We found that HG induces TXNIP expression, redox stress, and mitophagic flux in rMC1 versus LG. Treatment with the triple drug combination prevents mitophagic flux and restores transcription factor TFEB and PGC1α nuclear localization under HG, which is critical for lysosome biosynthesis and mitogenesis, respectively. Our results demonstrate that 2×mt8a-CG is a suitable probe for monitoring mitophagic flux, both in live and fixed cells in in vitro experiments, which may also be applicable to in vivo animal studies, and that the triple drug combination treatment has the potential for preventing retinal injury and disease progression in diabetes.

Modification by solvents of the action of nifedipine on calcium channel currents in neuroblastoma cells.

The effect of nifedipine dissolved in different solvents on the two types of calcium channel currents in neuroblastoma cells was investigated using the whole cell version of the patch clamp technique. Nifedipine dissolved in dimethylsulfoxide (nifedipine/DMSO) decreased the transient calcium channel (T channel) current by 50% at a concentration of 10 microM. This inhibitory effect was concentration-dependent and reversible. In contrast, T channel currents were not inhibited by nifedipine at a similar concentration dissolved in acetone or ethanol. Further experiments were carried out with dried nifedipine/DMSO. Dried nifedipine/DMSO powder re-dissolved in acetone or ethanol at a concentration of 10 microM decreased the T channel current by 32% and 37%, respectively. In addition, within the concentration range of 10 nM to 100 microM nifedipine/DMSO inhibited the long-lasting calcium channel (L channel) current more effectively than did nifedipine dissolved in acetone. The concentration of solvent (DMSO, ethanol, acetone) in the bath was fixed at 0.3% to reach different final concentrations of nifedipine. Solvents alone at a final concentration of 0.3% did not show any effect on T or L channel currents. UV absorbance measurements indicated that the combination of nifedipine, solvent and bath solution did not result in precipitation of the dihydropyridine during the experimental protocol. It is concluded that when DMSO is used as the solvent, nifedipine is not only a more effective L channel antagonist but also a T channel antagonist in neuroblastoma cells.

Post‐natal disappearance of transient calcium channels in mouse skeletal muscle: effects of denervation and culture.

1. The whole-cell voltage clamp technique was used to record Ba2+ currents in voltage-sensitive Ca2+ channels in mouse flexor digitorum brevis muscles developing in situ from day 1 to 30 after birth. Effects of denervation and tissue culture on the Ca2+ channel currents were also studied. 2. The muscle fibres in newborn mice showed two distinct types of Ca2+ channel currents, a low-threshold transient current and a high-threshold sustained current. 3. The specific amplitude of the transient current was 2.7 +/- 1.7 (S.D.) A/F in response to -30 mV test pulses in medium containing 30 mM-Ba2+ on day 1 after birth. The transient current decreased progressively in the post-natal days and became undetectable by day 17. In contrast, the specific amplitude of the sustained current in response to +20 mV test pulses increased 4-fold from 6.9 A/F on day 1 to 27.7 A/F on day 30. 4. The disappearance of the transient current could not be accounted for by either shifts in voltage dependence of activation and inactivation or changes in activation and inactivation times of the two types of current during development. 5. Denervating muscle fibres on day 8 after birth did not prevent the disappearance of the transient current. Denervating them on day 17 did not allow reappearance of the transient current. However, the increase of the sustained current was suppressed by the denervation either on day 8 or day 17. 6. In muscle fibres isolated on day 8 after birth and cultured thereafter, the transient current did not disappear until day 19 in culture (27 days after birth), while the sustained current was maintained at the level on day 8. 7. In muscle fibres isolated on day 17, when the transient current had become undetectable, and cultured thereafter, the transient current did not reappear until day 15 in culture (32 days after birth), while the sustained current was maintained at a level similar to that on day 17. 8. We conclude that innervation has little influence on the developmental disappearance of the transient Ca2+ channel current in mouse muscle fibres, and suggest that some influencing factors from surroundings other than the nerve may be required for the disappearance of the functional transient channels.

Currents carried by sodium ions through transient calcium channels in clonal GH3 pituitary cells

The transient or T-type Ca channel currents of voltage-clamped clonal (GH3) pituitary cells were studied after blockade of Na and K channels. Removal of Ca ions from the bathing medium abolished the Ca currents and allowed Na ions to convey current through the transient channels. These Na currents (I Na,t) were activated at threshold potentials of ca. -85 mV, reached peak amplitudes (up to 2,000 pA) at -60 mV, and were 50% inactivated at holding membrane potentials of -110 to -120 mV. The time constant for inactivation of the maximal I Na,t was 12 +/- 2.4 msec (compared with 22.1 =/- 0.85 msec for maximal I Ca,t). The I Na,t was insensitive to tetrodotoxin (1 micron) and to nimodipine (200 nM) but was abolished by 100 micron external [Ca2+]. It is suggested that high affinity Ca binding sites in the channel modulate the ion selectivity and the current flow through T-type Ca channels.

Calcium and Sodium Channels in Spontaneously Contracting Vascular Muscle Cells

Electrophysiological recordings of inward currents from whole cells showed that vascular muscle cells have one type of sodium channel and two types of calcium channels. One of the calcium channels, the transient calcium channel, was activated by small depolarizations but then rapidly inactivated. It was equally permeable to calcium and barium and was blocked by cadmium, but not by tetrodotoxin. The other type, the sustained calcium channel, was activated by larger depolarizations, but inactivated very little; it was more permeable to barium than calcium. The sustained calcium channel was more sensitive to block by cadmium than the transient channel, but also was not blocked by tetrodotoxin. The sodium channel inactivated 15 times more rapidly than the transient calcium channel and at more negative voltages. This sodium channel, which is unusual because it is only blocked by a very high (60 microM) tetrodotoxin concentration but not by cadmium, is the first to be characterized in vascular muscle, and together with the two calcium channels, provides a basis for different patterns of excitation in vascular muscles.

Voltage-sensitive calcium flux promoted by vesicles in an isolated cardiac sarcolemma preparation.

The effect of membrane potential on the vesicular uptake of calcium in an isolated cardiac sarcolemma preparation from canine ventricle was evaluated. Membrane potentials were developed by the establishment of potassium gradients across the vesicular membranes. In the presence of valinomycin, the fluorescence changes of the voltage sensitive dye, diS-C3-(5) were consistent with the development of potassium equilibrium potentials. Using EGTA to remove endogenous calcium from the preparation and to maintain a low intravesicular calcium concentration over time, the uptake of calcium was linear from 5 to 100 sec, in the absence of sodium, at both -98 and -1 mV. The rate of calcium uptake (calcium influx) was approximately twofold greater at -1 mV than at -98 mV, and prepolarization of the membrane potential to -98 mV did not enhance calcium influx upon subsequent depolarization to -1 mV. Hence, calcium influx was voltage-sensitive but not depolarization-induced and did not inactivate with time. Furthermore, the calcium influx was not inhibited by the organic calcium antagonists, which suggests that this flux did not occur via the transient calcium channel. Evaluation of calcium influx over a wide range of membrane potentials produced a profile consistent with the hypothesis that calcium entered the vesicles through the pathway responsible for the persistent inward current observed in voltage-clamped isolated myocytes. A model was proposed to account for these results.