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6. 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 (+).

Author

Lopin, Kyle V., Obejero-Paz, Carlos A., and Jones, Stephen W.

Subjects
CALCIUM channels, ELECTROLYSIS, ELECTRONS, SOLUTION (Chemistry), CALCIUM, ION channels
Abstract

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. [ABSTRACT FROM AUTHOR]

Published
2010
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7. Fe2+Block and Permeation of CaV3.1 (α1G) T-Type Calcium Channels: Candidate Mechanism for Non–Transferrin-Mediated Fe2+Influx

Author

Lopin, Kyle V., Gray, I. Patrick, Obejero-Paz, Carlos A., Thévenod, Frank, and Jones, Stephen W.

Abstract

Iron is a biologically essential metal, but excess iron can cause damage to the cardiovascular and nervous systems. We examined the effects of extracellular Fe2+on permeation and gating of CaV3.1 channels stably transfected in HEK293 cells, by using whole-cell recording. Precautions were taken to maintain iron in the Fe2+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 Fe2+rapidly blocked currents with 2 mM extracellular Ca2+in a voltage-dependent manner, as described by a Woodhull model with KD= 2.5 mM at 0 mV and apparent electrical distance δ = 0.17. Extracellular Fe2+also shifted activation to more-depolarized voltages (by ∼10 mV with 1.8 mM extracellular Fe2+) somewhat more strongly than did extracellular Ca2+or Mg2+, which is consistent with a Gouy-Chapman-Stern model with surface charge density σ = 1 e−/98 Å2and KFe= 4.5 M−1for extracellular Fe2+. In the absence of extracellular Ca2+(and with extracellular Na+replaced by TEA), Fe2+carried detectable, whole-cell, inward currents at millimolar concentrations (73 ± 7 pA at −60 mV with 10 mM extracellular Fe2+). With a two-site/three-barrier Eyring model for permeation of CaV3.1 channels, we estimated a transport rate for Fe2+of ∼20 ions/s for each open channel at −60 mV and pH 7.2, with 1 μM extracellular Fe2+(with 2 mM extracellular Ca2+). Because CaV3.1 channels exhibit a significant “window current” at that voltage (open probability, ∼1%), CaV3.1 channels represent a likely pathway for Fe2+entry into cells with clinically relevant concentrations of extracellular Fe2+.

Published
2012
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8. Divalent Effects on Permeatin and Gating of T-type calcium channels

Author

Lopin, Kyle V.

Subjects
Biophysics, Physiology, T-Type, Calcium Channels, iron, cadmium, Erying rate theory, Permeation
Abstract

Cells carefully regulate their intracellular ion composition and their membrane potential. Voltage gated calcium channels change both of these properties by allowing Ca2+ to enter the cell, causing membrane depolarization and the entry of Ca2+ which acts as important signaling molecule. Much research over the last few decades has gone into discovering the different types of voltage gated calcium channels and their permeation and gating properties. Many models have been proposed to explain how ions permeate through calcium channels. The second chapter of this thesis uses a 2 binding site 3 barrier model of permeation to fit a wide range of permeation data previously collected from a1G T-type calcium channels. Using this model of permeation we were able to fit permeation and block data well over a wide range of concentrations and voltages for four different ions (Ca2+, Ba2+, Mg2+, Na+).T-type calcium channels are expressed in a wide range of cells in a diverse set of tissue types. In the third chapter of this thesis, electrophysiological measurements were made of the effects of block and permeation of Fe2+ on a1G channels and the model of permeation was extended to this physiological ion to examine the role that calcium channels can play in iron overload condition and in cadmium toxicity. Fe2+ entry through calcium channels has potentially deleterious consequences as this entry is unregulated and in iron overload condition where cells have an impaired iron efflux mechanism, calcium channels may play a role in increasing the labile iron pool inside a cell. This labile iron pool is unbound iron that is redox active and can cause oxidative damage to proteins and lipids. We observed currents through a1G calcium channels carried by Fe2+ showing that a1G calcium channels can allows ferrous iron (Fe2+) to enter into cells. Rates of Fe2+ entry in physiological concentrations of Fe2+ using the extended model of permeation showed that a1G calcium channels can be a mechanism for non transferrin bound iron entry.The fourth chapter of this thesis focuses on the permeation and blocking properties of Cd2+ on a1G channels. Cd2+ is known to have cytotoxic effects and while the exact mechanism of Cd2+ toxicity is still being elucidated, cadmium is increasingly contaminating the environment leading to increased exposure to humans. Electrophysiology experiments show that Cd2+ permeates extremely well through the T-type calcium channel and only incompletely block the channel. The model of permeation was extended again for a1G channels to include Cd2+, and it was able to fit the data collected in Cd2+ and to predict the transport rate of Cd2+ through the channels in Cd2+ concentrations seen in populations exposed to environmental containment. The calculated rates of Cd2+ entry showed that in Cd2+ exposure, a1G calcium channels can be a mechanism for Cd2+ entry into cells.

Published
2013

9. Cd²⁺ block and permeation of CaV3.1 (α1G) T-type calcium channels: candidate mechanism for Cd²⁺ influx.

Author

Lopin KV, Thévenod F, Page JC, and Jones SW

Subjects
Calcium metabolism, Cell Line, HEK293 Cells, Humans, Ion Channel Gating drug effects, Membrane Potentials drug effects, Patch-Clamp Techniques methods, Cadmium metabolism, Cadmium pharmacology, Calcium Channels, T-Type metabolism
Abstract

Cd²⁺ is an industrial pollutant that can cause cytotoxicity in multiple organs. We examined the effects of extracellular Cd²⁺ 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, Cd²⁺ rapidly blocked currents with 2 mM Ca²⁺ in a voltage-dependent manner. The block caused by Cd²⁺ was relieved at more-hyperpolarized potentials, which suggests that Cd²⁺ can permeate through the selectivity filter of the channel into the cytosol. In the absence of other permeant ions (Ca²⁺ and Na⁺ replaced by N-methyl-d-glucamine), Cd²⁺ carried sizable inward currents through Ca(v)3.1 channels (210 ± 20 pA at -60 mV with 2 mM Cd²⁺). Ca(v)3.1 channels have a significant "window current" at that voltage (open probability, ∼1%), which makes them a candidate pathway for Cd²⁺ entry into cells during Cd²⁺ exposure. Incubation with radiolabeled ¹⁰⁹Cd²⁺ confirmed uptake of Cd²⁺ into cells with Ca(v)3.1 channels.

Published
2012
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