Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells.

Adrenal neuroendocrine chromaffin cells receive excitatory synaptic input from the sympathetic nervous system and secrete hormones into the peripheral circulation. Under basal sympathetic tone, modest amounts of freely soluble catecholamine are selectively released through a restricted fusion pore formed between the secretory granule and the plasma membrane. Upon activation of the sympathoadrenal stress reflex, elevated stimulation drives fusion pore expansion, resulting in increased catecholamine secretion and facilitating release of copackaged peptide hormones. Thus regulated expansion of the secretory fusion pore is a control point for differential hormone release of the sympathoadrenal stress response. Previous work has shown that syndapin 1 deletion alters transmitter release and that the dynamin 1-syndapin 1 interaction is necessary for coupled endocytosis in neurons. Dynamin has also been shown to be involved in regulation of fusion pore expansion in neuroendocrine chromaffin cells through an activity-dependent association with syndapin. However, it is not known which syndapin isoform(s) contributes to pore dynamics in neuroendocrine cells. Nor is it known at what stage of the secretion process dynamin and syndapin associate to modulate pore expansion. Here we investigate the expression and localization of syndapin isoforms and determine which are involved in mediating fusion pore expansion. We show that all syndapin isoforms are expressed in the adrenal medulla. Mutation of the SH3 dynamin-binding domain of all syndapin isoforms shows that fusion pore expansion and catecholamine release are limited specifically by mutation of syndapin 3. The mutation also disrupts targeting of syndapin 3 to the cell periphery. Syndapin 3 exists in a persistent colocalized state with dynamin 1.

Cd²âº block and permeation of CaV3.1 (α1G) T-type calcium channels: candidate mechanism for Cd²âº influx.

Cd²âº is an industrial pollutant that can cause cytotoxicity in multiple organs. We examined the effects of extracellular Cd²âº on permeation and gating of Ca(v)3.1 (α1G) channels stably transfected in HEK293 cells, by using whole-cell recording. With the use of instantaneous I-V currents (measured after strong depolarization) to isolate the effects on permeation, Cd²âº rapidly blocked currents with 2 mM Ca²âº in a voltage-dependent manner. The block caused by Cd²âº was relieved at more-hyperpolarized potentials, which suggests that Cd²âº can permeate through the selectivity filter of the channel into the cytosol. In the absence of other permeant ions (Ca²âº and Na⁺ replaced by N-methyl-d-glucamine), Cd²âº carried sizable inward currents through Ca(v)3.1 channels (210 ± 20 pA at -60 mV with 2 mM Cd²âº). Ca(v)3.1 channels have a significant "window current" at that voltage (open probability, ∼1%), which makes them a candidate pathway for Cd²âº entry into cells during Cd²âº exposure. Incubation with radiolabeled ¹°9Cd²âº confirmed uptake of Cd²âº into cells with Ca(v)3.1 channels.

Fe²âº block and permeation of CaV3.1 (α1G) T-type calcium channels: candidate mechanism for non-transferrin-mediated Fe²âº influx.

Iron is a biologically essential metal, but excess iron can cause damage to the cardiovascular and nervous systems. We examined the effects of extracellular Fe²âº on permeation and gating of Ca(V)3.1 channels stably transfected in HEK293 cells, by using whole-cell recording. Precautions were taken to maintain iron in the Fe²âº state (e.g., use of extracellular ascorbate). With the use of instantaneous I-V currents (measured after strong depolarization) to isolate the effects on permeation, extracellular Fe²âº rapidly blocked currents with 2 mM extracellular Ca²âº in a voltage-dependent manner, as described by a Woodhull model with K(D) = 2.5 mM at 0 mV and apparent electrical distance δ = 0.17. Extracellular Fe²âº also shifted activation to more-depolarized voltages (by ∼10 mV with 1.8 mM extracellular Fe²âº) somewhat more strongly than did extracellular Ca²âº or Mg²âº, which is consistent with a Gouy-Chapman-Stern model with surface charge density σ = 1 e(-)/98 Ų and K(Fe) = 4.5 M⁻¹ for extracellular Fe²âº. In the absence of extracellular Ca²âº (and with extracellular Na⁺ replaced by TEA), Fe²âº carried detectable, whole-cell, inward currents at millimolar concentrations (73 ± 7 pA at -60 mV with 10 mM extracellular Fe²âº). With a two-site/three-barrier Eyring model for permeation of Ca(V)3.1 channels, we estimated a transport rate for Fe²âº of ∼20 ions/s for each open channel at -60 mV and pH 7.2, with 1 µM extracellular Fe²âº (with 2 mM extracellular Ca²âº). Because Ca(V)3.1 channels exhibit a significant "window current" at that voltage (open probability, ∼1%), Ca(V)3.1 channels represent a likely pathway for Fe²âº entry into cells with clinically relevant concentrations of extracellular Fe²âº.

Evaluation of a two-site, three-barrier model for permeation in Ca(V)3.1 (alpha1G) T-type calcium channels: Ca (2+), Ba (2+), Mg (2+), and Na (+).

We explored the ability of a two-site, three-barrier (2S3B) Eyring model to describe recently reported data on current flow through open Ca(V)3.1 T-type calcium channels, varying Ca(2+) and Ba(2+) over a wide range (100 nM: -110 mM: ) while recording whole-cell currents over a wide voltage range (-150 mV to +100 mV) from channels stably expressed in HEK 293 cells. Effects on permeation were isolated using instantaneous current-voltage relationships (IIV) after strong, brief depolarizations to activate channels with minimal inactivation. Most experimental results were reproduced by a 2S3B model. The model described the IIV relationships, apparent affinities for permeation and block for Ca(2+) and Ba(2+), and shifts in reversal potential between Ca(2+) and Ba(2+). The fit to block by 1 mM Mg(2+)(i) was reasonable, but block by Mg(2+)(0) was described less well. Surprisingly, fits were comparable with strong ion-ion repulsion, with no repulsion, or with intermediate values. With weak repulsion, there was a single high-affinity site, with a low-affinity site near the cytoplasmic side of the pore. With strong repulsion, the net charge of ions in the pore was near +2 over a relatively wide range of concentration and voltage, suggesting a knockoff mechanism. With strong repulsion, Ba(2+) preferred the inner site, while Ca(2+) preferred the outer site, potentially explaining faster entry of Ni(2+) and other pore blockers when Ba(2+) is the charge carrier.

PSoC-Stat: A single chip open source potentiostat based on a Programmable System on a Chip.

In this paper we demonstrate a potentiostat built with a single commercially available integrated circuit (IC) that does not require any external electronic components to perform electrochemical experiments. This is done using the capabilities of the Programmable System on a Chip (PSoC®) by Cypress Semiconductor, which integrates all of the necessary electrical components. This is in contrast to other recent papers that have developed potentiostats but require technical skills or specialized equipment to produce. This eliminates the process of having to make a printed circuit board and soldering on electronic components. To control the device, a graphical user interface (GUI) was developed in the python programming language. Python is open source, with a style that makes it easy to read and write programs, making it an ideal choice for open source projects. As the developed device is open source and based on a PSoC, modification to implement other electrochemical techniques is straightforward and only requires modest programming skills, but no expensive equipment or difficult techniques. The potentiostat developed here adds to the growing amount of open source laboratory equipment. To demonstrate the PSoC potentiostat in a wide range of applications, we performed cyclic voltammetry (to measure vitamin C concentration in orange juice), amperometry (to measure glucose with a glucose strip), and stripping voltammetry experiments (to measure lead in water). The device was able to perform all experiments and could accurately measure Vitamin C, glucose, and lead.