Tetraspanin-13 modulates voltage-gated CaV2.2 Ca2+ channels

Abstract

Integration of voltage-gated Ca(2+) channels in a network of protein-interactions is a crucial requirement for proper regulation of channel activity. In this study, we took advantage of the specific properties of the yeast split-ubiquitin system to search for and characterize so far unknown interaction partners of CaV2 Ca(2+) channels. We identified tetraspanin-13 (TSPAN-13) as an interaction partner of the α1 subunit of N-type CaV2.2, but not of P/Q-type CaV2.1 or L- and T-type Ca(2+) channels. Interaction could be located between domain IV of CaV2.2 and transmembrane segments S1 and S2 of TSPAN-13. Electrophysiological analysis revealed that TSPAN-13 specifically modulates the efficiency of coupling between voltage sensor activation and pore opening of the channel and accelerates the voltage-dependent activation and inactivation of the Ba(2+) current through CaV2.2. These data indicate that TSPAN-13 might regulate CaV2.2 Ca(2+) channel activity in defined synaptic membrane compartments and thereby influences transmitter release.

Figure 1. Co-immunoprecipitation analysis using solubilisates of membrane-fractions obtained from CHO cells expressing Ca_V2.2-EGFP and transiently transfected with TSPAN-13-Myc.

Eluates from co-immunoprecipitations with either anti-Myc (left) or IgG-control antibodies (middle) and the input controls (right) were separated on 7% (Ca_V2.2-EGFP) or 12% (TSPAN-13-Myc) SDS-polyacrylamide gels, respectively. Shown is a representative example of three independent co-immunoprecipitation experiments. The figure shows the relevant area of the antibody-staining.

Figure 2. Functional characterization of the interaction of TSPAN-13 with different classes of voltage gated Ca²⁺ channels.

(a) Representative examples of current traces recorded from differentiated NG108-15 cells transfected either with an EGFP control plasmid or with a plasmid encoding TSPAN-13-EGFP. Current traces activated by depolarizing pulses with amplitudes between −70 mV and −10 mV and between 0 mV and +40 mV are presented separately. (b) Averaged current-voltage (I-V) relationships constructed from peak current amplitudes measured as illustrated in panel A. - cells transfected with an EGFP control plasmid (n = 26); - cells transfected with a plasmid encoding TSPAN-13-EGFP (n = 26). (c) I-V relationship for the recombinant Ca_V3.1 channel stably expressed in a HEK 293 cell line. - cells co-transfected with an EGFP control plasmid (n = 22); - cells co-transfected with a plasmid encoding TSPAN-13-EGFP (n = 19). (d) I-V relationship for the recombinant Ca_V1.2 channel stably expressed in a HEK 293 cell. - cells co-transfected with an EGFP control plasmid (n = 18); - cells co-transfected with a plasmid encoding TSPAN-13-EGFP (n = 20). (e) I-V relationship for the recombinant Ca_V2.2 channel stably expressed in a CHO cell line. - cells co-transfected with an EGFP control plasmid (n = 16); - cells co-transfected with a plasmid encoding TSPAN-13-EGFP (n = 15).

Figure 3. Activation and inactivation kinetics of the current through Ca_V2.2 channels stably expressed in CHO cells.

(a) Examples of current traces recorded by 10 ms long depolarisations from a holding potential of −80 mV to potentials increasing from −10 mV up to +50 mV with a step of 10 mV. Ba²⁺ was used as charge carrier. Cells were either transfected with an EGFP control plasmid (n = 18) or with a plasmid encoding TSPAN-13-EGFP (n = 19). Current traces activated by depolarisations to +10 mV and higher were fitted by a single exponential and corresponding time constants were averaged and plotted in the graph. The significance of the difference between time constants determined for individual depolarisations was tested by the unpaired two-tail t-test (+10 mV, p = 0.018; +20 mV, p = 0.0017; +30 mV, p = 0.0016; +40 mV, p = 0.0027; +50 mV, p = 0.0100). (b) Averaged current traces recorded by 2 s long depolarizations from a holding potential of −80 mV to +20 mV (peak of an IV). Ba²⁺ was used as charge carrier. Cells were either transfected with an EGFP control plasmid (solid line; n = 22) or with a plasmid encoding TSPAN-13-EGFP (dashed line; n = 20). Each trace was fitted by a sum of two exponentials. The right part of the panel shows both, the fast and slow components which were simulated using the averaged time constants and relative amplitudes listed in table 1. (c) Examples of current traces recorded using the same protocol as in (a). Ca²⁺ was used as charge carrier. Cells were either transfected with an EGFP control plasmid (n = 11) or with a plasmid encoding TSPAN-13-EGFP (n = 14). Current traces activated by depolarisations to +10 mV and higher were fitted by a single exponential and corresponding time constants were averaged and plotted in the graph. (d) Averaged current traces recorded by 2 s long depolarisations from a holding potential of −80 mV to +20 mV (peak of an IV). Ca²⁺ was used as charge carrier. Cells were either transfected with an EGFP control plasmid (solid line; n = 8) or with a plasmid encoding TSPAN-13-EGFP (dashed line; n = 12).

Figure 4. Effect of TSPAN-13 on gating currents of Ca_V2.2 channels expressed in CHO cells and determination of the G_max/Q_max ratio.

(a) Examples of current traces recorded by depolarization from a holding potential of −80 mV to the potential corresponding to the reversal potential of the investigated cell. Data represent the average of five runs. Cells were either transfected with an EGFP control plasmid or with a plasmid encoding TSPAN-13-EGFP. (b) G_max versus Q_max for control cells () and cells transfected with TSPAN-13 (). Straight lines represent linear fits of experimental data. (c) G_max/Q_max ratio for TSPAN-13 and various TSPAN-13 constructs. Number of cells tested is indicated at each column. ** - significantly different from control, p < 0.01. Abbreviations: FL-TSPAN-13, full-length-TSPAN-13; δ, regions that are deleted in TSPAN-13 constructs.

Figure 5. Intracellular localisation of full-length TSPAN-13-EGFP and mutant forms of TSPAN-13-EGFP expressed in HEK293 cells using pAkt-PH-mCherry as membrane marker.

In contrast to full-length TSPAN-13, the Δ(S3-C-term) mutant and TSPAN-13(F55A,F57A) mutant, the N-terminal TSPAN-13 deletion mutant does not localise to the plasma membrane. First column: TSPAN-13-EGFP constructs used in this study; second column: EGFP fluorescence of TSPAN-13 constructs; third column: red fluorescence of pAkt-PH-mCherry membrane marker; forth column: merged images. Confocal images were recorded 48 h after transfection.