Voltage and Calcium Use the Same Molecular Determinants to Inactivate Calcium Channels

We have investigated the molecular mechanisms whereby the I-II loop controls voltage-dependent inactivation in P/Q calcium channels. We demonstrate that the I-II loop is localized in a central position to control calcium channel activity through the interaction with several cytoplasmic sequences; including the III-IV loop. Several experiments reveal the crucial role of the interaction between the I-II loop and the III-IV loop in channel inactivation. First, point mutations of two amino acid residues of the I-II loop of Ca v 2.1 (Arg-387 or Glu-388) facilitate voltage-dependent inactivation. Second, overexpression of the III-IV loop, or injection of a peptide derived from this loop, produces a similar inactivation behavior than the mutated channels. Third, the III-IV peptide has no effect on channels mutated in the I-II loop. Thus, both point mutations and overexpression of the III-IV loop appear to act similarly on inactivation, by competing off the native interaction between the I-II and the III-IV loops of Ca v 2.1. As they are known to affect inactivation, we also analyzed the effects of ␤ subunits on these interactions. In experiments in which the ␤ 4 subunit is co-expressed, the III-IV peptide is no longer able to regulate channel inactivation. We conclude that (i) the contribution of the I-II loop to inactivation is partly mediated by an interaction with the III-IV loop and (ii) the ␤ subunits partially control inactivation by modifying this interaction. These data provide novel insights into the mechanisms whereby the ␤ subunit, the I-II loop, and the III-IV loop altogether can contribute to regulate inactivation in high voltage-activated calcium channels.The influx of calcium through voltage-gated calcium channels controls a variety of cellular processes ranging from membrane excitability and synaptic efficacy to gene expression. Both the amplitude and the duration of the calcium influx shape the spatio-temporal efficacy of calcium signaling. A tight control of both processes is needed to avoid long term increases in intracellular calcium levels, which are cytotoxic to neurons. Although the control of calcium entry can be achieved in several ways, inactivation of voltage-gated calcium channels appears to represent a key molecular process. For instance, inactivation is considered as a candidate mechanism for synaptic depression (1, 2). Inasmuch as there are several calcium channel types, there are also several inactivation behaviors. L-type calcium channels inactivate slowly, whereas the neuronal N-, P/Q-, and R-type channels inactivate faster. These fundamental differences are linked to the pore-forming Ca v subunit, which contains the major molecular determinants for inactivation, although auxiliary subunits can play a regulatory function in this process.

Section: Structural Differences Between Aid Sequences and Potentialmentioning

confidence: 64%

mentioning

confidence: 59%

The Interaction between the I-II Loop and the III-IV Loop of Cav2.1 Contributes to Voltage-dependent Inactivation in a β-Dependent Manner

Geib¹,

Sandoz²,

Cornet³

et al. 2002

“…Based on this observation and on the data of Cens et al (41), we have suggested that the domain I-II linker may serve as a physical inactivation gate that functionally interacts with the domain II and IIIS6 regions (27). Our observations with the CecCCC chimera provides further support for a key involvement of the domain I-II linker region in the inactivation process.…”

Section: Discussionmentioning

confidence: 99%

Identification of Inactivation Determinants in the Domain IIS6 Region of High Voltage-activated Calcium Channels

Stotz

Zamponi

2001

We have recently reported that transfer of the domain IIS6 region from rapidly inactivating R-type (␣ 1E ) calcium channels to slowly inactivating L-type (␣ 1C ) calcium channel confers rapid inactivation (Stotz, S. C., Hamid, J., Spaetgens, R. L., Jarvis, S. E., and Zamponi, G. W. (2000) J. Biol. Chem. 275, 24575-24582). Here we have identified individual amino acid residues in the IIS6 regions that are responsible for these effects. In this region, ␣ 1C and ␣ 1E channels differ in seven residues, and exchanging five of those residues individually or in combination did not significantly affect inactivation kinetics. By contrast, replacement of residues Phe-823 or Ile-829 of ␣ 1C with the corresponding ␣ 1E residues significantly accelerated inactivation rates and, when substituted concomitantly, approached the rapid inactivation kinetics of R-type channels. A systematic substitution of these residues with a series of other amino acids revealed that decreasing side chain size at position 823 accelerates inactivation, whereas a dependence of the inactivation kinetics on the degree of hydrophobicity could be observed at position 829. Although these point mutations facilitated rapid entry into the inactivated state of the channel, they had little to no effect on the rate of recovery from inactivation. This suggests that the development of and recovery from inactivation are governed by separate structural determinants. Finally, the effects of mutations that accelerated ␣ 1C inactivation could still be antagonized following coexpression of the rat ␤ 2a subunit or by domain I-II linker substitutions that produce ultra slow inactivation of wild type channels, indicating that the inactivation kinetics seen with the mutants remain subject to regulation by the domain I-II linker. Overall, our results provide novel insights into a complex process underlying calcium channel inactivation.

“…A good candidate for a place in this relay would be the EF-hand-like domain, which is essential for inactivation but does not yet find a place in the switch process (9). Other relaying candidates are suggested by recent observations, demonstrating that the inactivation process involves the I-II intracellular loop and associated ␤ subunit for both Ca v 1.2 and Ca v 2.1 (33)(34)(35). These results suggest that voltage-dependent and Ca 2ϩ -dependent inactivations involve common relays.…”

Section: In the Presence Of Iq The Complex Of Cb With Cam At High Camentioning

confidence: 99%

Interactions of Calmodulin with Two Peptides Derived from the C-terminal Cytoplasmic Domain of the Cav1.2 Ca2+ Channel Provide Evidence for a Molecular Switch Involved in Ca2+-induced Inactivation

Mouton

Feltz

Maulet

2001

When opened by depolarization, L-type calcium channels are rapidly inactivated by the elevation of Ca 2؉ concentration on the cytoplasmic side. Recent studies have shown that the interaction of calmodulin with the proximal part of the cytoplasmic C-terminal tail of the channel plays a prominent role in this modulation. Two motifs interacting with calmodulin in a Ca 2؉ -dependent manner have been described: the IQ sequence and more recently the neighboring CB sequence. Here, using synthetic peptides and fusion proteins derived from the Ca v 1.2 channel combined with biochemical techniques, we show that these two peptides are the only motifs of the cytoplasmic tail susceptible to interact with calmodulin. We determined the K d of the CB interaction with calmodulin to be 12 nM, i.e. below the K d of IQ-calmodulin, thereby precluding a competitive displacement of CB by IQ in the presence of Ca 2؉ . In place, we demonstrated that a ternary complex is formed at high Ca 2؉ concentration, provided that calmodulin and the peptides are initially allowed to interact at a low Ca 2؉ concentration. These results provide evidence that CB and IQ motifs interacting together with calmodulin constitute a minimal molecular switch leading to Ca 2؉ -induced inactivation. In addition, we suggest that they could also be the tethering site of calmodulin.