Expression of Calcium Channel Subunit Variants in Small Mesenteric Arteries of WKY and SHR

Abstract

Background: Enhanced function of dihydropyridine-sensitive Ca2+ channels (CaV) in hypertensive arterial myocytes (HAM) is well accepted. Increased protein expression of pore forming α1-subunits contributes to this effect, but cannot explain all of the differences in CaV properties in HAM. We hypothesized that differences in expression of CaV subunits and/or their splice variants also contribute.

Methods: RNA, protein, and myocytes were isolated from small mesenteric arteries (SMA) of 20-week-old male WKY and SHR and analyzed by polymerase chain reaction (PCR), sequencing, immunoblotting, and patch clamp methods.

Results: Cav1.2 α1, β2c, and α2δ1d were the dominant subunits expressed in both WKY and SHR with a smaller amount of β3a. Real-time PCR indicated that the mRNA abundance of β3a and α2δ1 but not total Cav1.2 α1 or β2c were significantly larger in SHR. Analysis of alternative splicing of Cav1.2 α1 showed no differences in abundance of mutually exclusive exons1b, 8, 21 and 32 or alternative exons33 and 45. However, inclusion of exon9* was higher and a 73 nucleotide (nt) deletion in exon15 (exon15Δ73) was lower in SHR. Immunoblot analysis showed higher protein levels of Cav1.2 α1 (1.61±0.05), β3 (1.80±0.32), and α2δ1 (1.80±0.24) but not β2 in SHR.

Conclusions: The lower abundance of exon15Δ73 transcripts in SHR results in a larger fraction of total Cav1.2 mRNA coding for full-length CaV protein, and the higher abundance of exon9* transcripts and CaVβ3a protein likely contribute to differences in gating and kinetics of CaV currents in SHR. Functional studies of Ca2+ currents in native SMA myocytes and HEK cells transiently transfected with CaV subunits support these conclusions.

Keywords: Ca2+; alternative splicing; blood pressure; channel subunits; channels.; gene expression; hypertension; hypertensive rats; protein abundance; small mesenteric arteries; voltage gated Ca2+.

Figure 1.

Gene expression of Ca_V subunits in small mesenteric arteries (SMA). (a) Ca_V α₁ subunits. Top: EtBr stained gel of cPCR products from SHR (S), WKY (W), and brain (B). Left: Quantitative PCR results represented as ΔC _t values relative to β-actin. Right: Ratio of abundance of Cav1.2 and Cav1.3 in SHR to WKY represented as $2^{-\Delta \Delta C_t}$. n = 9 for both WKY and SHR. (b) Ca_V β subunits. Top: EtBr stained gel of PCR products for β₁–β₄ subunits in SMA from SHR and WKY, and brain. Left: Quantitative PCR results represented as ΔC _t values relative to β-actin. Right: Ratio of abundance of β₁–β₄ in SHR to WKY represented as $2^{-\Delta \Delta C_t}$. n = 4 for both WKY and SHR. (c) Ca_V α₂δ subunits. Top: EtBr stained gel of PCR products for α₂δ₁–α₂δ₄ subunits in SMA from SHR and WKY, and brain. Left: Quantitative PCR results represented as ΔC _t values relative to β-actin. Right: Ratio of abundance of α₂δ₁ and α₂δ₃ in SHR to WKY represented as $2^{-\Delta \Delta C_t}$. n = 4 for both WKY and SHR. Bars and vertical lines represent the mean and 1 SEM, respectively. The star (*) indicates statistically significant differences between WKY and SHR for ΔC _t analysis (P < 0.05, unpaired t-test) and statistically significant differences from unity for $2^{-\Delta \Delta C_t}$ analysis (P < 0.05, one sample t-test).

Figure 2.

Expression of Cav1.2 splice variants. (a) Exon1 variants. Top: EtBr stained gel of PCR products for Cav1.2 exon1a, 1b, and 1c in SMA from SHR and WKY, and brain. (b) Left: Quantitative PCR results represented as ΔC _t values relative to β-actin. Right: Ratio of abundance of Cav1.2 exon1a, 1b, and 1c in SHR to WKY represented as $2^{-\Delta \Delta C_t}$. n = 7 for both WKY and SHR. (b) Exon21/22. Top: EtBr stained gels of PCR products spanning exons21 and 22 with or without treatment with SpeI for WKY (W) and SHR (S), and brain (B). Bottom: Densitometry analysis of gels for exon21 (SpeI-insensitive) and exon22 abundance (SpeI-sensitive minus null fraction); n = 8 for both WKY and SHR. (c) Exon 9*. Top: EtBr stained gel of PCR products for Cav1.2 spanning exon9* in SMA (M) and TA (T) from SHR and WKY. Bottom: Densitometry analysis of the ratio of +exon 9* to –exon9* transcript abundance in MA and TA from WKY (WM and WT) and SHR (SM and ST); n = 8 for WM, SM, WT, and ST. Bars and vertical lines represent the mean and 1 SEM, respectively. The star (*) indicates statistically different between WKY and SHR (P < 0.05). ΔC _t data in panel a and data in panel c were compared using the unpaired t-test, while values from the $2^{-\Delta \Delta C_t}$ analysis were compared by the one-sample t-Test.

Figure 3.

Abundance of Cav1.2Δ73 transcripts. (a) Fraction of clones. Top: EtBr stained gel of PCR products for Cav1.2 spanning exon15 from left ventricle, SMA, and brain. (b) Bottom: Fraction of full length (exon15+) or truncated (exon15−) clones relative to the total number in WKY (n = 49) and SHR (n = 41) SMA analyzed by sequencing. The star (*) indicates a statistically significant difference between WKY and SHR (Fisher exact test; P = 0.05). (b) Quantitative PCR. Top: Schematic representation of the location of the forward (FP) and reverse (RP) qPCR primers, and the probe for the 73 nucleotide (nt) deletion (Δ73) in exon15 plus full length transcripts with the location of the EcoRI site in exon15. Bottom: Analysis of abundance of the 73 nt deletion shown as the ratio of deleted to total Cav1.2 transcripts (Δ73/(Δ73 + FL)). Bars and vertical lines represent the mean and 1 SEM (n = 7), respectively, and star (*) indicates values statistically different from unity (unpaired t-test; P < 0.05). (c) Combinatorial analysis of expression of exons9* and exon15Δ73. The fraction of clones with and/or without exon9* and/or exon15Δ73 are shown for WKY and SHR. The “FL or +” indicate full length clones and the “−” clones with the 73 nt deletion. The star (*) indicates statistically significant difference between WKY and SHR (χ²-test; P = 0.03 for 1592 and P = 0.01 for 1519).

Figure 4.

Ca_V subunit protein expression. Left: Western blot of protein lysates from WKY (W) and SHR (S) probed with antibodies to (a) the II-III intracellular loop of Cav1.2 α₁ (top) and β-actin (bottom); (b) Ca_Vβ₂ and Ca_Vβ₃ (top), and β-actin (bottom); and (c) Ca_Vα₂δ₁ (top) and β-actin (bottom). Right: Graphs summarizing Ca_V subunit protein expression determined by densitometry normalized to β-actin in SHR vs. WKY for (A) Cav1.2 α₁ with exon1b (n = 5) and II-III loop (n = 10) antibodies; (B) Ca_Vβ₂ (n = 8) and Ca_Vβ₃ (n = 8), and (c) Ca_Vα₂δ₁ (n = 8). Bars and vertical lines represent mean and 1 SEM, respectively, and star (*) indicates values statistically different from unity (P < 0.05, one sample t-test).

Figure 5.

Functional effects of Ca_V subunit differences. (a) I _Ca (pA/pF) was larger in isolated SHR myocytes that have lower expression levels of Cav1.2Δ73 compared to WKY (data were replotted from Figure 3 in ref. with permission). (b) Increasing expression of Cav1.2Δ73 decreased I _Ca (pA/pF) in ASMC (data reproduced from Figure 5 in ref.). (c) Voltage dependence of I _Ca normalized to the maximum value is shifted in the negative voltage direction in SHR myocytes which have a higher level of expression of exon9* containing transcripts (data were replotted from ref. with permission). (d) Voltage dependence of I _Ca in HEK cells transfected with Cav1.2 constructs with (77–9*) or without (77-WT) exon9* (data were replotted from Figure 4 in ref.). (e) I _Ca inactivation represented as r₄₀₀ (current measured 400 milliseconds after the voltage clamp step divided by the peak value) was faster in SHR myocytes that have a higher level of Ca_Vβ₃ expression. (f) In transiently transfected HEK cells inactivation was faster (smaller r₄₀₀) in cells expressing Ca_Vβ₃ compared to Ca_Vβ_2c along with the same Cav1.2 α₁ and α₂δ₁ constructs. For (e) and (f), families of Ca²⁺ currents recorded at voltages from −60 to +40 mV in 10 mV, 500-ms-long voltage steps from a holding potential of −80 mV are shown for each condition. For all panels, symbols and vertical lines represent the mean and ±1 SEM, respectively.