Novel functional properties of Ca(2+) channel beta subunits revealed by their expression in adult rat heart cells

Abstract

Recombinant adenoviruses were used to overexpress green fluorescent protein (GFP)-fused auxiliary Ca(2+) channel beta subunits (beta(1)-beta(4)) in cultured adult rat heart cells, to explore new dimensions of beta subunit functions in vivo. Distinct beta-GFP subunits distributed differentially between the surface sarcolemma, transverse elements, and nucleus in single heart cells. All beta-GFP subunits increased the native cardiac whole-cell L-type Ca(2+) channel current density, but produced distinctive effects on channel inactivation kinetics. The degree of enhancement of whole-cell current density was non-uniform between beta subunits, with a rank order of potency beta(2a) approximately equal to beta(4) > beta(1b) > beta(3). For each beta subunit, the increase in L-type current density was accompanied by a correlative increase in the maximal gating charge (Q(max)) moved with depolarization. However, beta subunits produced characteristic effects on single L-type channel gating, resulting in divergent effects on channel open probability (P(o)). Quantitative analysis and modelling of single-channel data provided a kinetic signature for each channel type. Spurred on by ambiguities regarding the molecular identity of the actual endogenous cardiac L-type channel beta subunit, we cloned a new rat beta(2) splice variant, beta(2b), from heart using 5' rapid amplification of cDNA ends (RACE) PCR. By contrast with beta(2a), expression of beta(2b) in heart cells yielded channels with a microscopic gating signature virtually identical to that of native unmodified channels. Our results provide novel insights into beta subunit functions that are unattainable in traditional heterologous expression studies, and also provide new perspectives on the molecular identity of the beta subunit component of cardiac L-type Ca(2+) channels. Overall, the work establishes a powerful experimental paradigm to explore novel functions of ion channel subunits in their native environments.

Figure 4. Distinctive effects of β-GFP subunits on single L-type Ca²⁺ channel gating

Aa-Da, top, voltage protocols, single-channel currents were elicited by test depolarizations to +40 mV, from a holding potential of −60 mV. Lower traces, exemplar single-channel records from cells over-expressing GFP (Aa) or β-GFP proteins as indicated (Ba-Da). Records shown are consecutive sweeps from patches containing a single active channel. Mean unitary current amplitudes were: GFP, 0.71 ± 0.03 pA, n = 4; β_1b-GFP, 0.70 ± 0.02 pA, n = 4; β_2a-GFP, 0.72 ± 0.01 pA, n = 4; β₃-GFP, 0.71 ± 0.01 pA, n = 3. Ab-Db, ensemble currents (grey traces) averaged from the indicated number of patches were obtained from all sweeps, including nulls. Ac-Dc, first latency (FL) distributions (thick grey traces) obtained from all sweeps, and averaged from the same number of patches as indicated in Ab-Db, above. The FL histogram for GFP (Ac) is reproduced in Bc-Dc (dashed line) to permit direct comparison between the different infection conditions. Ad-Dd, conditional open probability (P_OO) distributions (thick grey traces) averaged from the indicated number of patches. The steady-state P_OO value for channels from GFP-expressing cells was 0.18, and this value is reproduced in Bd-Dd (dashed line) to facilitate direct comparison with values obtained in cells expressing β-GFP subunits. Ab-Dd, continuous black lines through the data are fits generated from the kinetic model shown in Fig. 5C, with parameters given in Table 1.

Figure 5. Open and closed time distributions, and quantitative modelling provide insights into distinctive β subunit effects on L-type channel gating

A, open time distributions (thick grey traces) of single L-type channels generated from cells expressing GFP (27, 321), β_1b-GFP (8, 723), β_2a-GFP (71, 773), or β₃-GFP (12, 082) as indicated. Numbers in parentheses represent the total number of events used to generate the distributions. When reproduced onto the other plots the GFP distribution (dotted traces) was coincident with β-GFP distributions, demonstrating that β subunits did not affect channel open times. Continuous black curves through the data are single exponential fits utilizing the weighted rate constant from the open to closed transition (δ = 2.18 ms⁻¹) in the kinetic model shown in C. B, closed time distributions (thick grey traces) were generated from the following number of events: GFP (27, 031), β_1b-GFP (8, 520), β_2a-GFP (71, 556), and β₃-GFP (11, 845). When reproduced onto the other plots, the GFP closed time distribution (dotted traces) was discordant with β-GFP distributions, demonstrating that each β subunit uniquely impacted L-type channel closed times. Continuous curves through the data are fits generated from the quantitative model in Fig. 5C, with the parameters shown in Table 1. C, top, kinetic scheme used to model β subunit effects on L-type channel gating. Details of the model are presented in Methods. Bottom, fold changes in forward equilibria for activation and inactivation transitions for β-GFP channels, when compared to control (GFP) channels. The forward equilibrium for each transition was normalized to the corresponding GFP value. Parameter values used to generate fits in Figs 4 and 5 are shown in Table 1.

Figure 1. Expression and subcellular targeting of β-GFP subunits in adult rat heart cells

A, Western blots of whole heart cell lysates probed with the GFP antibody. Cells were harvested 48 h post-infection with recombinant adenovirus, except for β_2a-GFP-expressing cells, which were harvested after 18 h, because of toxicity. The higher molecular weight band in the GFP lane corresponds to GFP dimer and disappears with more intense boiling of the protein sample before electrophoresis (not shown). B, Western blots of whole-cell lysates probed with the β_GEN antibody, showing marked over-expression of β₃- and β₄-GFP compared with the endogenous β subunit. From this perspective, some proteolysis of β₃-GFP was evident, however, this did not result in a proteolytic fragment that was recognized by the GFP antibody (see A). C, confocal images showing differential targeting of Ca²⁺ channel β-GFP subunits in heart cells. Images were obtained in live cells < 24 h post infection, and represent a section through the centre of the cardiomyocytes. Images are representative of observations made in > 100 cells for each β-GFP subunit.

Figure 2. Tuning of L-type Ca²⁺ channel whole-cell current properties in ventricular myocytes over-expressing recombinant Ca²⁺ channel β subunits

Aa-Ea, current density waveforms averaged from multiple ventricular cells expressing GFP alone, or various β-GFP constructs. Black traces plot the mean, and grey traces above and below the s.e.m. confidence range. Averaged traces were elicited by 0 mV step depolarizations from a holding potential of −90 mV. Compared with GFP cells, peak current density amplitudes were markedly elevated for channels expressing β-GFP subunits, albeit to different extents. Ab-Eb, current density (J) vs. step voltage (V) relationships for GFP- and β-GFP-expressing cells. Dashed traces in Bb-Eb reproduce the trace in Ab to facilitate direct visual comparison between the data from β-GFP-expressing cells and control (GFP-expressing) cells. Traces show that the enhancement of native cardiac L-type channel current density extends across a wide range of voltages. Ac-Ec, plots of the fraction of current remaining after a 300 ms depolarization (r₃₀₀) vs. step voltage (V). Dashed lines in Bc-Ec reproduce the trace in Ac to facilitate visual comparison between the data from GFP- and β-GFP-expressing cells. Faster inactivation rates result in lower r₃₀₀ values. Therefore, β_2a- and β₄-GFP expression in heart cells markedly slow L-type current inactivation rates over a broad range of voltages. *P < 0.05, n = 4-10 for each point.

Figure 3. Exogenous β subunits increase L-type channel gating currents in heart cells

Aa-Ea, top, voltage protocols, 20 ms step depolarizations from a holding potential of −50 mV were used to elicit gating currents. Ionic currents were blocked by including 2 mm Cd²⁺/0.1 mm La³⁺ in the bath solution. Lower traces, exemplar Ca²⁺ channel gating currents elicited by voltage steps to −30, −10 and +10 mV. Ab-Eb, plot of the integral of the normalized on-gating charge (Q_on) vs. V. The Q_on-V relationship for GFP (Ab) has been reproduced in Bb-Eb (dashed lines) to facilitate direct comparison with the Q_on-V relationship for β-GFP-expressing cells. Continuous curves through the data were derived from least-squares fits to a single Boltzmann function, with the following parameters (Q_max, V_1/2 and k, respectively). GFP (Ab): 4.7 fC pF⁻¹, −17.1 mV and 8.5; β_1b-GFP (Bb): 6.9 fC pF⁻¹, −19.6 mV and 7.2; β_2a-GFP (Cb): 8.7 fC pF⁻¹, −18.6 mV and 7.6; β₃-GFP (Db): 7.1 fC pF⁻¹, −17.3 mV and 7.5; β₄-GFP (Eb) 11.4 fC pF⁻¹, −15.3 mV and 6.5.

Figure 6. Identification and functional characterization of a novel rat heart β₂ splice variant

A, sequence comparison of the amino terminus (D1 domain) of newly cloned rat β₂ variant β_2b (GenBank Accession number AF423193) to the corresponding region in orthologues from other species. B, exemplar Ba²⁺ currents from recombinant L-type channels (α_1C α₂δ) reconstituted in HEK 293 cells with either β_2a (black trace) or β_2b (grey trace) indicates greater inactivation with β_2b. Currents were elicited with a 300 ms depolarization to +10 mV. C, averaged Ba²⁺ currents from heart cells infected with GFP virus (black trace, n = 5), or a recombinant virus encoding β_2b and GFP in a bicistronic cassette (grey trace, n = 5). The GFP trace has been scaled up so as to match current amplitudes at the end of the test pulse, to permit explicit comparison of current waveforms. Currents were evoked with a 300 ms depolarization to +10 mV. Da-Df, single L-type channel gating properties in heart cells over-expressing β_2b. Exemplar currents (Da) are consecutive traces from a patch containing one channel. Ensemble currents (Db, grey trace) were averaged from four single-channel patches, as were FL (Dc), P_oo (Dd), open time histograms (De), and closed time histograms (Df). Continuous traces through the data are model fits using the same parameters as for GFP (Table 1), except that the percentage of cells initially in state C₁ was 57 %.

Figure 7. Molecular basis of Ca²⁺ channel β₂ subunit diversity

Top, generic modular domain structure proposed for Ca²⁺ channel β subunits in which three variable domains (D1, D3 and D5) are interspersed by two conserved regions (D2 and D4). Bottom, analysis of human chromosome 10 genomic DNA sequence (contig NT-008682) clarifies the molecular basis of β₂ subunit diversity. Five D1 variants arise from alternative splicing of six exons and have been designated β_2a-β_2e. Three D3 variants arise from mutually exclusive splicing of exons 11, 12 and 13. β_2a-e transcripts all contain exon 11.