Overexpressed Cavβ3 Inhibits N-type (Cav2.2) Calcium Channel Currents through a Hyperpolarizing Shift of “Ultra-slow” and “Closed-state” Inactivation (original) (raw)
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The Journal of General Physiology, 2004
It has been shown that  auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a novel inhibitory effect of  3 subunit on macroscopic Ba 2 ϩ currents through recombinant N-and R-type calcium channels expressed in Xenopus oocytes. Overexpressed  3 (12.5 ng/ cell cRNA) significantly suppressed N-and R-type, but not L-type, calcium channel currents at "physiological" holding potentials (HPs) of Ϫ 60 and Ϫ 80 mV. At a HP of Ϫ 80 mV, coinjection of various concentrations (0-12.5 ng) of the  3 with Ca v 2.2 ␣ 1 and ␣ 2 ␦ enhanced the maximum conductance of expressed channels at lower  3 concentrations but at higher concentrations ( Ͼ 2.5 ng/cell) caused a marked inhibition. The  3-induced current suppression was reversed at a HP of Ϫ 120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba 2 ϩ (40 mM) as a charge carrier also largely diminished the effect of  3 at Ϫ 80 mV. Therefore, experimental conditions (HP, divalent cation concentration, and  3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel  3 effect. Steady-state inactivation curves revealed that N-type channels exhibited "closed-state" inactivation without  3, and that  3 caused an ف 40-mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately Ϫ 85 mV. The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of  3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at Ϫ 80 mV. Similar "ultra-slow" inactivation was observed for N-type channels without  3. Thus,  3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by causing a hyperpolarizing shift of the inactivation without affecting "ultra-slow" and "closed-state" inactivation properties.
Ca2+- and voltage-dependent inactivation of the expressed L-type Cav1.2 calcium channel
Archives of Biochemistry and Biophysics, 2005
Ca 2+-dependent regulation of the ion current through the a 1C b 2a a 2 d-1 (L-type) calcium channel transiently expressed in HEK 293 cells was investigated using whole cell patch clamp method. Ca 2+ or Na + ions were used as a charge carrier. Intracellular Ca 2+ was either buffered by 10 mM EGTA or 200 lM Ca 2+ was added into non-buffered intracellular solution. Free intracellular Ca 2+ inactivated permanently about 80% of the L-type calcium current. The L-type calcium channel inactivated during a depolarizing pulse with two time constants, s fast and s slow. Free intracellular calcium accelerated both time constants. Effect on the s slow was more pronounced. About 80% of the channel inactivation during brief depolarizing pulse could be attributed to a Ca 2+-dependent mechanism and 20% to a voltage-dependent mechanism. When Na + ions were used as a charge carrier, the L-type current still inactivated with two time constants that were 10 times slower and were virtually voltage-independent. Ca 2+ ions stabilized the inactivated state of the channel in a concentration-dependent manner.
Proceedings of the National Academy of Sciences, 1997
Human epithelial kidney cells (HEK) were prepared to coexpress α1A, α2δ with different β calcium channel subunits and green fluorescence protein. To compare the calcium currents observed in these cells with the native neuronal currents, electrophysiological and pharmacological tools were used conjointly. Whole-cell current recordings of human epithelial kidney α1A-transfected cells showed small inactivating currents in 80 mM Ba 2+ that were relatively insensitive to calcium blockers. Coexpression of α1A, βIb, and α2δ produced a robust inactivating current detected in 10 mM Ba 2+ , reversibly blockable with low concentration of ω-agatoxin IVA (ω-Aga IVA) or synthetic funnel-web spider toxin (sFTX). Barium currents were also supported by α1A, β2a, α2δ subunits, which demonstrated the slowest inactivation and were relatively insensitive to ω-Aga IVA and sFTX. Coexpression of β3 with the same combination as above produced inactivating currents also insensitive to low concentration of ω-...
Modulation of gating currents of the Cav3.1 calcium channel by α2δ2a and γ5 subunits
Archives of Biochemistry and Biophysics, 2004
Modulatory effects of auxiliary c 5 and a 2 d 2a subunits on intramembrane charge movement originating from the expressed Ca v 3.1 calcium channel were investigated. Inward current was blocked by 1 mM La 3þ. Voltage dependences of Q on and Q off , kinetics of ON-and OFF-charge movement, and I max =Q max ratio were measured in the absence and the presence of an auxiliary subunit. The a 2 d 2a subunit accelerated significantly both ON-and OFF-charge movement. I max =Q max ratio and Q on-V, Q off-V relations were not affected. Coexpression of the a 2 d 2a subunit may accelerate channel transitions between individual closed states, but not the transition from the last closed channel state into an open state. Coexpression of the c 5 subunit accelerated the decay of the ON-charge transient and enhanced I max =Q max ratio. These effects suggest improvement of the coupling between the charge movement and the channel opening due to facilitation of transitions between individual closed states and the transition between the last closed state and an open state.
Inhibition of voltage-gated calcium channels by sequestration of β subunits
Biochemical and Biophysical Research Communications, 2003
The auxiliary Ca v b subunit is essential for functional expression of high-voltage activated Ca 2þ channels. Here, we describe a lure sequence designed to sequester the Ca v b subunits in transfected bovine chromaffin cells. This sequence is composed of the extracellular and transmembrane domains of the a chain of the human CD8, the I-II loop of Ca v 2.1 subunit, and EGFP. We showed that expressing the CD8-I-II-EGFP sequence in chromaffin cells led to a >50% decrease in overall Ca 2þ current density. Although this decrease involved all the Ca 2þ channel types (L, N, P/Q, R), the proportion of each type supporting the remaining current was altered. A similar effect was observed after transfection when measuring the functional role of Ca 2þ channels in catecholamine release by chromaffin cells: global decrease of release and change of balance between the different channel types supporting it. Possible explanations for this apparent discrepancy are further discussed.
Gating of the expressed T-type Cav3.1 calcium channels is modulated by Ca2+
Acta Physiologica, 2006
We have investigated the influence of Ca2+ ions on the basic biophysical properties of T-type calcium channels. The Cav3.1 calcium channel was transiently expressed in HEK 293 cells. Current was measured using the whole cell patch clamp technique. Ca2+ or Na+ ions were used as charge carriers. The intracellular Ca2+ was either decreased by the addition of 10 mm ethyleneglycoltetraacetic acid (EGTA) or increased by the addition of 200 microm Ca2+ into the non-buffered intracellular solution. Various combinations of extra- and intracellular solutions yielded high, intermediate or low intracellular Ca2+ levels. The amplitude of the calcium current was independent of intracellular Ca2+ concentrations. High levels of intracellular Ca2+ accelerated significantly both the inactivation and the activation time constants of the current. The replacement of extracellular Ca2+ by Na+ as charge carrier did not affect the absolute value of the activation and inactivation time constants, but significantly enhanced the slope factor of the voltage dependence of the inactivation time constant. Slope factors of voltage dependencies of channel activation and inactivation were significantly enhanced. The recovery from inactivation was faster when Ca2+ was a charge carrier. The number of available channels saturated for membrane voltages more negative than -100 mV for the Ca2+ current, but did not reach steady state even at -150 mV for the Na+ current. Ca2+ ions facilitate transitions of Cav3.1 channel from open into closed and inactivated states as well as backwards transition from inactivated into closed state, possibly by interacting with its voltage sensor.
Gating of the expressed Cav 3.1 calcium channel
FEBS Letters, 2002
Intramembrane charge movement originating from Ca v 3.1 (T-type) channel expressed in HEK 293 cells was investigated. Ion current was blocked by 1 mM La 3+. Charge movement was detectable for depolarizations above V V3 370 mV and saturated above +60 mV. The voltage dependence of charge movement followed a single Boltzmann function with half-maximal activation voltage +12.9 mV and +12.3 mV and with slopes of 22.4 mV and 18.1 mV for the ON-and OFF-charge movement, respectively. Inactivation of I Ca by prolonged depolarization pulse did not immobilize intramembrane charge movement in the Ca v 3.1 channel.
γ 1 Dependent Down-regulation of Recombinant Voltage-gated Ca 2+ Channels
Cellular and Molecular Neurobiology, 2007
(1) Voltage-gated Ca2+ (CaV) channels are multi-subunit membrane complexes that allow depolarization-induced Ca2+ influx into cells. The skeletal muscle L-type CaV channels consist of an ion-conducting CaV1.1 subunit and auxiliary α2δ−1, β1 and γ1 subunits. This complex serves both as a CaV channel and as a voltage sensor for excitation–contraction coupling. (2) Though much is known about the mechanisms by which the α2δ−1 and β1 subunits regulate CaV channel function, there is far less information on the γ1 subunit. Previously, we characterized the interaction of γ1 with the other components of the skeletal CaV channel complex, and showed that heterologous expression of this auxiliary subunit decreases Ca2+ current density in myotubes from γ1 null mice. (3) In the current report, using Western blotting we show that the expression of the CaV1.1 protein is significantly lower when it is heterologously co-expressed with γ1. Consistent with this, patch-clamp recordings showed that transient transfection of γ1 drastically inhibited macroscopic currents through recombinant N-type (CaV2.2/α2δ−1/β3) channels expressed in HEK-293 cells. (4) These findings provide evidence that co-expression of the auxiliary γ1 subunit results in a decreased expression of the ion-conducting subunit, which may help to explain the reduction in Ca2+ current density following γ1 transfection.
The Journal of General Physiology, 1998
In studies of gating currents of rabbit cardiac Ca channels expressed as α1C/β2aor α1C/β2a/α2δ subunit combinations in tsA201 cells, we found that long-lasting depolarization shifted the distribution of mobile charge to very negative potentials. The phenomenon has been termed charge interconversion in native skeletal muscle (Brum, G., and E. Ríos. 1987.J. Physiol. (Camb.).387:489–517) and cardiac Ca channels (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1992.J. Gen. Physiol.99:863–895). Charge 1 (voltage of half-maximal transfer, V1/2≃ 0 mV) gates noninactivated channels, while charge 2 (V1/2≃ −90 mV) is generated in inactivated channels. In α1C/β2acells, the available charge 1 decreased upon inactivating depolarization with a time constant τ ≃ 8, while the available charge 2 decreased upon recovery from inactivation (at −200 mV) with τ ≃ 0.3 s. These processes therefore are much slower than charge movement, which takes <50 ms. This separation between the time scale of meas...