Both Voltage- and Ca2+-Dependent Inactivation of Cav1.2 Ca2+ Channels are Suppresed in Myotubes, Likely Because of Triad Junction Proteins other than RyR1

L-type calcium channels (LTCCs) are critical elements of normal cardiac function, playing a major role in orchestrating cardiac electrical activity and initiating downstream signaling processes. LTCCs thus use feedback mechanisms to precisely control calcium (Ca2+) entry into cells. Of these, Ca2+-dependent inactivation (CDI) is significant because it shapes cardiac action potential duration and is essential for normal cardiac rhythm. This important form of regulation is mediated by a resident Ca2+ sensor, calmodulin (CaM), which is comprised of two lobes that are each capable of responding to spatially distinct Ca2+ sources. Disruption of CaM-mediated CDI leads to severe forms of long-QT syndrome (LQTS) and life-threatening arrhythmias. Thus, a model capable of capturing the nuances of CaM-mediated CDI would facilitate increased understanding of cardiac (patho)physiology. However, one critical barrier to achieving a detailed kinetic model of CDI has been the lack of quantitative data characterizing CDI as a function of Ca2+. This data deficit stems from the experimental challenge of uncoupling the effect of channel gating on Ca2+ entry. To overcome this obstacle, we use photo-uncaging of Ca2+ to deliver a measurable Ca2+ input to CaM/LTCCs, while simultaneously recording CDI. Moreover, we use engineered CaMs with Ca2+ binding restricted to a single lobe, to isolate the kinetic response of each lobe. These high-resolution measurements enable us to build mathematical models for each lobe of CaM, which we use as building blocks for a full-scale bilobal model of CDI. Finally, we use this model to probe the pathogenesis of LQTS associated with mutations in CaM (calmodulinopathies). Each of these models accurately recapitulates the kinetics and steady-state properties of CDI in both physiological and pathological states, thus offering powerful new insights into the mechanistic alterations underlying cardiac arrhythmias.

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Voltage- and Calcium-Dependent Inactivation of Calcium Channels in Lymnaea Neurons

The Journal of General Physiology ◽

10.1085/jgp.114.4.535 ◽

1999 ◽

Vol 114 (4) ◽

pp. 535-550 ◽

Cited By ~ 6

Author(s):

Shalini Gera ◽

Lou Byerly

Keyword(s):

Cytochalasin B ◽

Lymnaea Stagnalis ◽

Slow Component ◽

Freshwater Snail ◽

High Concentration ◽

Calcium Dependent ◽

Channel Inactivation ◽

Dependent Inactivation ◽

Lymnaea Neurons ◽

Ca2 Channels

Ca2+ channel inactivation in the neurons of the freshwater snail, Lymnaea stagnalis, was studied using patch-clamp techniques. In the presence of a high concentration of intracellular Ca2+ buffer (5 mM EGTA), the inactivation of these Ca2+ channels is entirely voltage dependent; it is not influenced by the identity of the permeant divalent ions or the amount of extracellular Ca2+ influx, or reduced by higher levels of intracellular Ca2+ buffering. Inactivation measured under these conditions, despite being independent of Ca2+ influx, has a bell-shaped voltage dependence, which has often been considered a hallmark of Ca2+-dependent inactivation. Ca2+-dependent inactivation does occur in Lymnaea neurons, when the concentration of the intracellular Ca2+ buffer is lowered to 0.1 mM EGTA. However, the magnitude of Ca2+-dependent inactivation does not increase linearly with Ca2+ influx, but saturates for relatively small amounts of Ca2+ influx. Recovery from inactivation at negative potentials is biexponential and has the same time constants in the presence of different intracellular concentrations of EGTA. However, the amplitude of the slow component is selectively enhanced by a decrease in intracellular EGTA, thus slowing the overall rate of recovery. The ability of 5 mM EGTA to completely suppress Ca2+-dependent inactivation suggests that the Ca2+ binding site is at some distance from the channel protein itself. No evidence was found of a role for serine/threonine phosphorylation in Ca2+ channel inactivation. Cytochalasin B, a microfilament disrupter, was found to greatly enhance the amount of Ca2+ channel inactivation, but the involvement of actin filaments in this effect of cytochalasin B on Ca2+ channel inactivation could not be verified using other pharmacological compounds. Thus, the mechanism of Ca2+-dependent inactivation in these neurons remains unknown, but appears to differ from those proposed for mammalian L-type Ca2+ channels.

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Faculty Opinions recommendation of Cav1.4alpha1 subunits can form slowly inactivating dihydropyridine-sensitive L-type Ca2+ channels lacking Ca2+-dependent inactivation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1014332.193319 ◽

2003 ◽

Author(s):

Annette Dolphin

Keyword(s):

Dependent Inactivation ◽

Ca2 Channels

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Molecular moieties masking Ca2+-dependent facilitation of voltage-gated Cav2.2 Ca2+ channels

The Journal of General Physiology ◽

10.1085/jgp.201711841 ◽

2017 ◽

Vol 150 (1) ◽

pp. 83-94 ◽

Cited By ~ 2

Author(s):

Jessica R. Thomas ◽

Jussara Hagen ◽

Daniel Soh ◽

Amy Lee

Keyword(s):

Functional Divergence ◽

Short Term ◽

Voltage Gated ◽

Regulatory Domains ◽

Dependent Inactivation ◽

Weak Binding ◽

Ef Hand ◽

Molecular Determinants ◽

Ca2 Channels ◽

Α1 Subunit

Voltage-gated Cav2.1 (P/Q-type) Ca2+ channels undergo Ca2+-dependent inactivation (CDI) and facilitation (CDF), both of which contribute to short-term synaptic plasticity. Both CDI and CDF are mediated by calmodulin (CaM) binding to sites in the C-terminal domain of the Cav2.1 α1 subunit, most notably to a consensus CaM-binding IQ-like (IQ) domain. Closely related Cav2.2 (N-type) channels display CDI but not CDF, despite overall conservation of the IQ and additional sites (pre-IQ, EF-hand–like [EF] domain, and CaM-binding domain) that regulate CDF of Cav2.1. Here we investigate the molecular determinants that prevent Cav2.2 channels from undergoing CDF. Although alternative splicing of C-terminal exons regulates CDF of Cav2.1, the splicing of analogous exons in Cav2.2 does not reveal CDF. Transfer of sequences encoding the Cav2.1 EF, pre-IQ, and IQ together (EF-pre-IQ-IQ), but not individually, are sufficient to support CDF in chimeric Cav2.2 channels; Cav2.1 chimeras containing the corresponding domains of Cav2.2, either alone or together, fail to undergo CDF. In contrast to the weak binding of CaM to just the pre-IQ and IQ of Cav2.2, CaM binds to the EF-pre-IQ-IQ of Cav2.2 as well as to the corresponding domains of Cav2.1. Therefore, the lack of CDF in Cav2.2 likely arises from an inability of its EF-pre-IQ-IQ to transduce the effects of CaM rather than weak binding to CaM per se. Our results reveal a functional divergence in the CDF regulatory domains of Cav2 channels, which may help to diversify the modes by which Cav2.1 and Cav2.2 can modify synaptic transmission.

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Multiple calcium currents in a thyroid C-cell line: biophysical properties and pharmacology

AJP Cell Physiology ◽

10.1152/ajpcell.1991.260.6.c1253 ◽

1991 ◽

Vol 260 (6) ◽

pp. C1253-C1263 ◽

Cited By ~ 8

Author(s):

B. A. Biagi ◽

J. J. Enyeart

Keyword(s):

Cell Line ◽

Time Constant ◽

Calcium Currents ◽

C Cells ◽

Patch Clamp Technique ◽

C Cell ◽

Dependent Inactivation ◽

Voltage Dependent ◽

Rat Thyroid ◽

Ca2 Channels

The whole cell version of the patch-clamp technique was used to characterize voltage-gated Ca2+ channels in the calcitonin-secreting rat thyroid C-cell line 6-23 (clone 6). Three types of Ca2+ channels could be distinguished based on differences in voltage dependence, kinetics, and pharmacological sensitivity. T-type current was half-maximal at -31 mV, showed steady-state voltage-dependent inactivation that was half-maximal at -57 mV, inactivated with a voltage-dependent time constant that reached a minimum of 20 ms at potentials positive to -20 mV, and deactivated with a single time constant of approximately 2 ms at -80 mV. Reactivation of inactivated channels occurred with a time constant of 1.26 s at -90 mV. T current was selectively blocked by Ni2+ at concentrations between 5 and 50 microM. La3+ and Y3+ blocked the T current at 10- to 20-fold lower concentrations. Dihydropyridine-sensitive L-type current was half-maximal at a test potential of -3 mV and was approximately doubled in size when Ba2+ replaced Ca2+ as the charge carrier. Unlike L-type Ca2+ current in many cells, this current in C-cells displayed little Ca(2+)-dependent inactivation. N-type current was composed of inactivating and sustained components that were inhibited by omega-conotoxin. The inactivating component was half-maximal at +9 mV and could be fitted by two exponentials with time constants of 22 and 142 ms. A slow inactivation of N current with a time constant of 24.9 s was observed upon switching the holding potential from -80 to -40 mV. These results demonstrate that, similar to other neural crest derived cells, thyroid C-cells express multiple Ca2+ channels, including one previously observed only in neurons.

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The STIM1 CTID domain determines access of SARAF to SOAR to regulate Orai1 channel function

The Journal of Cell Biology ◽

10.1083/jcb.201301148 ◽

2013 ◽

Vol 202 (1) ◽

pp. 71-79 ◽

Cited By ~ 74

Author(s):

Archana Jha ◽

Malini Ahuja ◽

József Maléth ◽

Claudia M. Moreno ◽

Joseph P. Yuan ◽

...

Keyword(s):

Molecular Mechanism ◽

Cell Damage ◽

Cell Functions ◽

Dependent Inactivation ◽

Dependent Cell ◽

Inhibitory Domain ◽

Molecular Components ◽

Full Activation ◽

Ca2 Channels

Ca2+ influx by store-operated Ca2+ channels (SOCs) mediates all Ca2+-dependent cell functions, but excess Ca2+ influx is highly toxic. The molecular components of SOC are the pore-forming Orai1 channel and the endoplasmic reticulum Ca2+ sensor STIM1. Slow Ca2+-dependent inactivation (SCDI) of Orai1 guards against cell damage, but its molecular mechanism is unknown. Here, we used homology modeling to identify a conserved STIM1(448–530) C-terminal inhibitory domain (CTID), whose deletion resulted in spontaneous clustering of STIM1 and full activation of Orai1 in the absence of store depletion. CTID regulated SCDI by determining access to and interaction of the STIM1 inhibitor SARAF with STIM1 Orai1 activation region (SOAR), the STIM1 domain that activates Orai1. CTID had two lobes, STIM1(448–490) and STIM1(490–530), with distinct roles in mediating access of SARAF to SOAR. The STIM1(448–490) lobe restricted, whereas the STIM1(490–530) lobe directed, SARAF to SOAR. The two lobes cooperated to determine the features of SCDI. These findings highlight the central role of STIM1 in SCDI and provide a molecular mechanism for SCDI of Orai1.

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Molecular basis of calmodulin tethering and Ca2+-dependent inactivation of L-type Ca2+ channels.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)86775-1 ◽

2001 ◽

Vol 276 (39) ◽

pp. 36862

Author(s):

Geoffrey S. Pitt ◽

Roger D. Zühlke ◽

Andy Hudmon ◽

Howard Schulman ◽

Harald Reuter ◽

...

Keyword(s):

Molecular Basis ◽

Dependent Inactivation ◽

Ca2 Channels

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Ion-dependent Inactivation of Barium Current through L-type Calcium Channels

The Journal of General Physiology ◽

10.1085/jgp.109.4.449 ◽

1997 ◽

Vol 109 (4) ◽

pp. 449-461 ◽

Cited By ~ 86

Author(s):

Gonzalo Ferreira ◽

Jianxun Yi ◽

Eduardo Ríos ◽

Roman Shirokov

Keyword(s):

Ionic Current ◽

Reversal Potential ◽

Slow Phase ◽

Gating Currents ◽

Current Decay ◽

Gating Charge ◽

Long Duration ◽

Dependent Inactivation ◽

Voltage Dependent ◽

Ca2 Channels

It is widely believed that Ba2+ currents carried through L-type Ca2+ channels inactivate by a voltage- dependent mechanism similar to that described for other voltage-dependent channels. Studying ionic and gating currents of rabbit cardiac Ca2+ channels expressed in different subunit combinations in tsA201 cells, we found a phase of Ba2+ current decay with characteristics of ion-dependent inactivation. Upon a long duration (20 s) depolarizing pulse, IBa decayed as the sum of two exponentials. The slow phase (τ ≈ 6 s, 21°C) was parallel to a reduction of gating charge mobile at positive voltages, which was determined in the same cells. The fast phase of current decay (τ ≈ 600 ms), involving about 50% of total decay, was not accompanied by decrease of gating currents. Its amplitude depended on voltage with a characteristic U-shape, reflecting reduction of inactivation at positive voltages. When Na+ was used as the charge carrier, decay of ionic current followed a single exponential, of rate similar to that of the slow decay of Ba2+ current. The reduction of Ba2+ current during a depolarizing pulse was not due to changes in the concentration gradients driving ion movement, because Ba2+ entry during the pulse did not change the reversal potential for Ba2+. A simple model of Ca2+-dependent inactivation (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1993. J. Gen. Physiol. 102:1005–1030) robustly accounts for fast Ba2+ current decay assuming the affinity of the inactivation site on the α1 subunit to be 100 times lower for Ba2+ than Ca2+.

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