Mutations of Nonconserved Residues within the Calcium Channel α1-interaction Domain Inhibit β-Subunit Potentiation

Voltage-dependent calcium channels consist of a pore-forming subunit (CaVα1) that includes all the molecular determinants of a voltage-gated channel, and several accessory subunits. The ancillary β-subunit (CaVβ) is a potent activator of voltage-dependent calcium channels, but the mechanisms and structural bases of this regulation remain elusive. CaVβ binds reversibly to a conserved consensus sequence in CaVα1, the α1-interaction domain (AID), which forms an α-helix when complexed with CaVβ. Conserved aromatic residues face to one side of the helix and strongly interact with a hydrophobic pocket on CaVβ. Here, we studied the effect of mutating residues located opposite to the AID-CaVβ contact surface in CaV1.2. Substitution of AID-exposed residues by the corresponding amino acids present in other CaVα1 subunits (E462R, K465N, D469S, and Q473K) hinders CaVβ's ability to increase ionic-current to charge-movement ratio (I/Q) without changing the apparent affinity for CaVβ. At the single channel level, these CaV1.2 mutants coexpressed with CaVβ2a visit high open probability mode less frequently than wild-type channels. On the other hand, CaV1.2 carrying either a mutation in the conserved tryptophan residue (W470S, which impairs CaVβ binding), or a deletion of the whole AID sequence, does not exhibit CaVβ-induced increase in I/Q. In addition, we observed a shift in the voltage dependence of activation by +12 mV in the AID-deleted channel in the absence of CaVβ, suggesting a direct participation of these residues in the modulation of channel activation. Our results show that CaVβ-dependent potentiation arises primarily from changes in the modal gating behavior. We envision that CaVβ spatially reorients AID residues that influence the channel gate. These findings provide a new framework for understanding modulation of VDCC gating by CaVβ.

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Effective gating charges per channel in voltage-dependent K+ and Ca2+ channels.

The Journal of General Physiology ◽

10.1085/jgp.108.3.143 ◽

1996 ◽

Vol 108 (3) ◽

pp. 143-155 ◽

Cited By ~ 86

Author(s):

F Noceti ◽

P Baldelli ◽

X Wei ◽

N Qin ◽

L Toro ◽

...

Keyword(s):

Ionic Current ◽

Variance Analysis ◽

Beta Subunit ◽

Charge Movement ◽

Gating Currents ◽

Open Probability ◽

K Channels ◽

Voltage Dependent ◽

Pore Opening ◽

Ca2 Channels

In voltage-dependent ion channels, the gating of the channels is determined by the movement of the voltage sensor. This movement reflects the rearrangement of the protein in response to a voltage stimulus, and it can be thought of as a net displacement of elementary charges (e0) through the membrane (z: effective number of elementary charges). In this paper, we measured z in Shaker IR (inactivation removed) K+ channels, neuronal alpha 1E and alpha 1A, and cardiac alpha 1C Ca2+ channels using two methods: (a) limiting slope analysis of the conductance-voltage relationship and (b) variance analysis, to evaluate the number of active channels in a patch, combined with the measurement of charge movement in the same patch. We found that in Shaker IR K+ channels the two methods agreed with a z congruent to 13. This suggests that all the channels that gate can open and that all the measured charge is coupled to pore opening in a strictly sequential kinetic model. For all Ca2+ channels the limiting slope method gave consistent results regardless of the presence or type of beta subunit tested (z = 8.6). However, as seen with alpha 1E, the variance analysis gave different results depending on the beta subunit used. alpha 1E and alpha 1E beta 1a gave higher z values (z = 14.77 and z = 15.13 respectively) than alpha 1E beta 2a (z = 9.50, which is similar to the limiting slope results). Both the beta 1a and beta 2a subunits, coexpressed with alpha 1E Ca2+ channels facilitated channel opening by shifting the activation curve to more negative potentials, but only the beta 2a subunit increased the maximum open probability. The higher z using variance analysis in alpha 1E and alpha 1E beta 1a can be explained by a set of charges not coupled to pore opening. This set of charges moves in transitions leading to nulls thus not contributing to the ionic current fluctuations but eliciting gating currents. Coexpression of the beta 2a subunit would minimize the fraction of nulls leading to the correct estimation of the number of channels and z.

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Partners in Crime: Towards New Ways of Targeting Calcium Channels

International Journal of Molecular Sciences ◽

10.3390/ijms20246344 ◽

2019 ◽

Vol 20 (24) ◽

pp. 6344 ◽

Cited By ~ 3

Author(s):

Lucile Noyer ◽

Loic Lemonnier ◽

Pascal Mariot ◽

Dimitra Gkika

Keyword(s):

Calcium Channels ◽

Calcium Channel ◽

Transient Receptor Potential ◽

Receptor Potential ◽

Interaction Domain ◽

Domain Structures ◽

Voltage Dependent ◽

Voltage Dependent Calcium Channels ◽

Transient Receptor ◽

Partner Proteins

The characterization of calcium channel interactome in the last decades opened a new way of perceiving ion channel function and regulation. Partner proteins of ion channels can now be considered as major components of the calcium homeostatic mechanisms, while the reinforcement or disruption of their interaction with the channel units now represents an attractive target in research and therapeutics. In this review we will focus on the targeting of calcium channel partner proteins in order to act on the channel activity, and on its consequences for cell and organism physiology. Given the recent advances in the partner proteins’ identification, characterization, as well as in the resolution of their interaction domain structures, we will develop the latest findings on the interacting proteins of the following channels: voltage-dependent calcium channels, transient receptor potential and ORAI channels, and inositol 1,4,5-trisphosphate receptor.

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Antibodies against the β subunit of voltage-dependent calcium channels in Lambert–Eaton Myasthenic Syndrome

Neuroscience ◽

10.1016/s0306-4522(98)00378-9 ◽

1999 ◽

Vol 90 (1) ◽

pp. 269-277 ◽

Cited By ~ 17

Author(s):

C Raymond ◽

D Walker ◽

D Bichet ◽

C Iborra ◽

N Martin-Moutot ◽

...

Keyword(s):

Calcium Channels ◽

Β Subunit ◽

Voltage Dependent ◽

Voltage Dependent Calcium Channels ◽

Myasthenic Syndrome

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Mechanism of Auxiliary Subunit Modulation of Neuronal α1E Calcium Channels

The Journal of General Physiology ◽

10.1085/jgp.112.2.125 ◽

1998 ◽

Vol 112 (2) ◽

pp. 125-143 ◽

Cited By ~ 85

Author(s):

Lisa P. Jones ◽

Shao-kui Wei ◽

David T. Yue

Keyword(s):

Calcium Channels ◽

Ionic Current ◽

Charge Movement ◽

Open Probability ◽

Hek 293 ◽

Gating Model ◽

Auxiliary Subunit ◽

Activation Gating ◽

Α2δ Subunit ◽

Current Activation

Voltage-gated calcium channels are composed of a main pore-forming α1 moiety, and one or more auxiliary subunits (β, α2δ) that modulate channel properties. Because modulatory properties may vary greatly with different channels, expression systems, and protocols, it is advantageous to study subunit regulation with a uniform experimental strategy. Here, in HEK 293 cells, we examine the expression and activation gating of α1E calcium channels in combination with a β (β1–β4) and/or the α2δ subunit, exploiting both ionic- and gating-current measurements. Furthermore, to explore whether more than one auxiliary subunit can concomitantly specify gating properties, we investigate the effects of cotransfecting α2δ with β subunits, of transfecting two different β subunits simultaneously, and of COOH-terminal truncation of α1E to remove a second β binding site. The main results are as follows. (a) The α2δ and β subunits modulate α1E in fundamentally different ways. The sole effect of α2δ is to increase current density by elevating channel density. By contrast, though β subunits also increase functional channel number, they also enhance maximum open probability (Gmax/Qmax) and hyperpolarize the voltage dependence of ionic-current activation and gating-charge movement, all without discernible effect on activation kinetics. Different β isoforms produce nearly indistinguishable effects on activation. However, β subunits produced clear, isoform-specific effects on inactivation properties. (b) All the β subunit effects can be explained by a gating model in which subunits act only on weakly voltage-dependent steps near the open state. (c) We find no clear evidence for simultaneous modulation by two different β subunits. (d) The modulatory features found here for α1E do not generalize uniformly to other α1 channel types, as α1C activation gating shows marked β isoform dependence that is absent for α1E. Together, these results help to establish a more comprehensive picture of auxiliary-subunit regulation of α1E calcium channels.

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