Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Nov;468(11-12):1895-1907.
doi: 10.1007/s00424-016-1895-5. Epub 2016 Oct 29.

Upward movement of IS4 and IIIS4 is a rate-limiting stage in Cav1.2 activation

Stanislav Beyl  1 , Annette Hohaus  1 , Stanislav Andranovits  1 , Eugen Timin  1 , Steffen Hering  2
Affiliations

Upward movement of IS4 and IIIS4 is a rate-limiting stage in Cav1.2 activation

Stanislav Beyl et al. Pflugers Arch. 2016 Nov.

Abstract

In order to specify the role of individual S4 segments in CaV1.2 gating, charged residues of segments IS4-IVS4 were replaced by glutamine and the corresponding effects on activation/deactivation of calcium channel currents were analysed. Almost all replacements of charges in IS4 and IIIS4 decreased the slope of the Boltzmann curve of channel activation (activation curve) while charge neutralisations in IIS4 and IVS4 did not significantly affect the slope. S4 mutations caused either left or rightward shifts of the activation curve, and in wild-type channels, these S4 mutations hardly affected current kinetics.In slowly gating pore (S6) mutants (G432W, A780T, G1193T or A1503G), neutralisations in S4 segments significantly accelerated current kinetics. Likewise in wild type, charge replacements in IS4 and IIIS4 of pore mutants reduced the slope of the activation curves while substitutions of charges in IIS4 and IVS4 had less or no impact. We propose a gating model where the structurally different S4 segments leave their resting positions not simultaneously. Upward movement of segments IS4 and (to a lesser extend) IIIS4 appear to be a rate-limiting stage for releasing the pore gates. These segments carry most of the effective charge for channel activation. Our study suggests that S4 segments of CaV1.2 control the closed state in domain specific manner while stabilizing the open state in a non-specific manner.

Keywords: Calcium channel; Electrophysiology; Gating; Heart; Mutational analysis; Patch clamp; Pore; Voltage sensor.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Voltage-sensing S4 and pore-forming S6 segments of CaV1.2. a Schematic representation of pore-forming α1 subunit of CaV1.2 with S4 (blue) and S6 (red) segments highlighted. b, c Alignments of S4 (b) and S6 (c) segments of CaV1.2 (accession number, P15381) compared with other channel types. Charged residues are shown in blue. A ring of glycine or alanine residues on pore forming IS6–IVS6 (‘GAGA’ G432, A780, G1134, A1503) is highlighted in red. d Voltage-sensing S4 (blue) and pore-forming S6 (red) segments of CaV1.1 (pdb: 3JBR) channel shown as structural representation of corresponding segments of CaV1.2 α1 subunit. GAGA residues are assumed to participate in stabilisation of the closed gates [1]. Charged S4 residues and the 'GAGA ring' in the pore are highlighted. (Colour figure online)
Fig. 2
Fig. 2
Modulation of CaV1.2 gating by S4 charge neutralisations. Activation (a) and deactivation (b, tail current) of I Ba through wild-type and mutant channel constructs with partially neutralised S4 segments (IS4N+R276, IIS4N+R662, IIIS4N+R1041+R1037). The deactivation was measured by applying the voltage steps to different potentials after a short (20 ms) activating pulse (b). Note that neutralisation of S4 charges has minor effects on activation and deactivation kinetics of CaV1.2. c–f Averaged activation curves of wild-type, IS4N+R276, IS4N+R276+R273, IIS4N+R662, IIS4N+R662+R652, IIIS4N+R1041+R1037 and R1359Q channel constructs. The slope of IS4N+R267 (c) and IIIS4N+R1041+R1037 (e) activation curves was significantly reduced (k act = 5.9 ± 0.9 mV in WT vs 8.5 ± 0.6 mV and 9.9 ± 0.7 mV in IS4N+R267 and IIIS4N+R1041+R1037 respectively)
Fig. 3
Fig. 3
Neutralisations in IS4–IVS4 differently affect the slope of the CaV1.2 activation curve. Compared to wild-type CaV1.2, slope factors of the activation curves were significantly (*p < 0.05) increased by charge neutralisations in IS4 and IIIS4 but not by neutralisations of charged residues in segments IIS4 and IVS4
Fig. 4
Fig. 4
Modulation of pore mutant A780T by IS4 and IIS4 carrying a single charge (IS4N+R276 and IIS4N+R662). Activation (a) and deactivation (b, tail current) of I Ba through pore mutant A780T and corresponding constructs with partially neutralised segments IS4 (IS4N+R276, four out of five IS4 charges are substituted by glutamines) and IIS4 (IIS4N+R267, four out of five IIS4 charges are substituted by glutamines). The deactivation was measured by applying the voltage steps to different potentials after a short (20 ms) activating pulse (b, voltage protocol above current traces). Partial neutralisation of IS4 or IIS4 charges had minor effects on activation and deactivating kinetics (compare with corresponding traces on Fig. 2) but caused a significant acceleration of current kinetics of the slowly gating A780T (constructs A780T/IS4N+R267 and A780T/IIS4N+R662, arrows on d and f. c, e Averaged activation curves of WT, A780T, A780T/IS4N+R267 and A780T/IIS4N+R662 channels. The slope of A780T/IS4N+R267 was significantly reduced (from k act = 5.1 ± 0.6 mV in A780T to k act = 11.9 ± 1.0 mV in A780T/IS4N+R267). The slope of A780T/IIS4N+R662 activation curve was not changed while the activation curve was shifted by +10.3 ± 0.8 mV (see Table 1). Analogous gating disturbances induced by mutations in the conserved ring of small residues (either glycines or alanines, Fig. 1d) and the effect of S4 charge neutralisation are presented on Figs. S1, S2, S3, and S4 (Supplemental Materials) and also Table 1. d, f Voltage-dependent time constants of channel activation/deactivation. Red arrows highlight the acceleration of current kinetics when A780T was combined with a ‘neutralised’ S4 segment. (Colour figure online)
Fig. 5
Fig. 5
Neutralisation of single IS4 and IIS4 charges differently affects gating of pore mutants A780T and G1193T. a Averaged activation curves of WT; A780T and G1193T and constructs A780T/R267Q, G1193T/R267Q (IS4), A780T/R650Q, G1193T/R650Q (IIS4). Red arrows illustrate the rightward shift of the activation curves upon neutralisation of IS4 R267 (R267Q) and IIIS4 R267 (R267Q) charges. Note that neutralisation of single IS4 and IIIS4 charges caused significant reductions in slope of the activation curves (red curves, see also Table 1). b Slope factors of the activation curves of pore mutants (grey bars) are significantly increased by single charge neutralisation in IS4 (red bars) but not in IIS4 (blue bars). See also Table 1 and Figs. S2, S3, and S4 (Supplemental Materials) for other constructs. c, d Voltage-dependent time constants of channel activation/deactivation (c) and the slowest activation time constant at the peak of the bell-shaped curve (τ max) (d). Note that voltage-sensor neutralisations in IS4 (red) or IIS4 (blue) significantly accelerate the kinetics of ‘slowly’ gating pore mutants. (Colour figure online)
Fig. 6
Fig. 6
Modulation of gating by neutralisation of S4 segments. a–d Averaged activation curves of wild-type and indicated mutant channels. Activation curves of constructs with reduced IS4 or IIIS4 charges are shifted to more negative potentials, and the slope factors of the activation curve of channels with partially charged IS4 or IIIS4 are reduced (see also Table 1). e–h Mean effective charges of all constructs (wild-type CaV1.2 and pore mutants) were estimated from the slopes of the activation curves (Table 1) and plotted versus the number of charged residues in IS4 (e), IIS4 (f), IIIS4 (g) and IVS4 (h). To enable ‘back extrapolation’, data (e–h) were fitted to a non-linear regression. Back extrapolation of the exponential function yields an apparent mean effective charge of about two elementary units for IS4 (e), about 0.5 elementary units for IIS4 (f) and ≈1.5 elementary units for IIIS4 (g). Choosing an exponential function for analysis does not imply that S4 charges make an exponential contribution to the activation slope
Fig. 7
Fig. 7
Different impacts of S4 segments on CaV1.2 activation. Cartoon illustrating the hypothetical role of IS4 and IIIS4 in locking the channel pore in the closed conformation. Their upward movement correspondingly ‘releases’ the activation gates (enables a concerted pore opening) and stabilises the pore in the open conformation. Both S4 segments together contribute ≈90 % to the effective charge (Fig. 6e, g). Segment IIS4 contributes only 10 % of the effective charge for activating the channel (Fig. 6f). All four S4 stabilise the open state. IS4 and IIIS4 determine the voltage dependence to both the opening (‘unlocking the closed state’) and the closing of gate structures.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Beyl S, Depil K, Hohaus A, Stary-Weinzinger A, Linder T, Timin E, Hering S. Neutralisation of a single voltage sensor affects gating determinants in all four pore-forming S6 segments of CaV1.2: a cooperative gating model. Pflugers Arch - Eur J Physiol. 2012;464:391–401. doi: 10.1007/s00424-012-1144-5. - DOI - PMC - PubMed
    1. Beyl S, Kügler P, Hohaus A, Depil K, Hering S, Timin E. Methods for quantification of pore–voltage sensor interaction in CaV1.2. Pflugers Arch - Eur J Physiol. 2014;466:265–274. doi: 10.1007/s00424-013-1319-8. - DOI - PMC - PubMed
    1. Beyl S, Kügler P, Kudrnac M, Hohaus A, Hering S, Timin E. Different pathways for activation and deactivation in CaV1.2: a minimal gating model. J Gen Physiol. 2009;134:231–241. doi: 10.1085/jgp.200910272. - DOI - PMC - PubMed
    1. Bezanilla F, Perozo E, Stefani E. Gating of Shaker K+ channels: II. The components of gating currents and a model of channel activation. Biophys J. 1994;66:1011–1021. doi: 10.1016/S0006-3495(94)80882-3. - DOI - PMC - PubMed
    1. Capes DL, Goldschen-Ohm MP, Arcisio-Miranda M, Bezanilla F, Chanda B. Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels. J Gen Physiol. 2013;142:101–112. doi: 10.1085/jgp.201310998. - DOI - PMC - PubMed

Publication types

Substances

LinkOut - more resources

-