Korean J Physiol Pharmacol.  2023 Sep;27(5):481-491. 10.4196/kjpp.2023.27.5.481.

Peptides derived from high voltage-gated calcium channel ββ subunit reduce blood pressure in rats

• Kim HK
• Jun J
• Kim TW
• Youn Dh

• KMID: 2545538
• DOI: http://doi.org/10.4196/kjpp.2023.27.5.481

Abstract

The β subunits of high voltage-gated calcium channels (HGCCs) are essential for optimal channel functions such as channel gating, activation-inactivation kinetics, and trafficking to the membrane. In this study, we report for the first time the potent blood pressure-reducing effects of peptide fragments derived from the β subunits in anesthetized and non-anesthetized rats. Intravenous administration of 16-mer peptide fragments derived from the interacting regions of the β1 [cacb1(344–359)], β2 [cacb2(392–407)], β3 [cacb3(292–307)], and β4 [cacb4(333– 348)] subunits with the main α-subunit of HGCC decreased arterial blood pressure in a dose-dependent manner for 5–8 min in anesthetized rats. In contrast, the peptides had no effect on the peak amplitudes of voltage-activated Ca²⁺ current upon their intracellular application into the acutely isolated trigeminal ganglion neurons. Further, a single mutated peptide of cacb1(344–359)—cacb1(344–359)_K357R —showed consistent and potent effects and was crippled by a two-amino acid-truncation at the Nterminal or C-terminal end. By conjugating palmitic acid with the second amino acid (lysine) of cacb1(344–359)_K357R (named K2-palm), we extended the blood pressure reduction to several hours without losing potency. This prolonged effect on the arterial blood pressure was also observed in non-anesthetized rats. On the other hand, the intrathecal administration of acetylated and amidated cacb1(344–359)_K357R peptide did not change acute nociceptive responses induced by the intradermal formalin injection in the plantar surface of rat hindpaw. Overall, these findings will be useful for developing antihypertensives.

Keyword

β subunit; Blood pressure; High voltage-gated calcium channel; Pain; Trigeminal ganglion neurons

Figure

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  Fig. 1 Peptides derived from high voltage-gated calcium channel β subunits do not change the peak amplitude of high voltage-activated Ca2+ currents recorded in the acutely isolated trigeminal ganglion neurons. (A) Voltage step (from –60 mV to –50–40 mV, 500 ms)-activated Ca2+ currents were recorded in the isolated trigeminal ganglion (TG) neurons with the internal whole-cell patch solution containing β1 [cacb1(344-359)], β2 [Cacb2(392-407)], β3 [cacb3(292-307)], and β4 [cacb4(333-348)] subunits-derived peptides. (B) Summarized current-voltage relationships depicted by whole-cell recordings with internally applied individual or all peptides. Numbers indicate the number of recorded TG neurons. Values are presented as mean ± SEM.

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  Fig. 2 Decrease of the arterial blood pressure (BP) by a peptide fragment derived from high voltage-gated calcium channel (HGCC) β1 subunit in anesthetized rats. (A) Intravenous injection (0.15 mg/kg, 0.2 ml, IV) of a 16-mer peptide IKITSPKVLQRLIKSR, which is a part of HGCC β1 subunit [cacb1(344–359)], decreased the arterial BP from the femoral artery. The intravenous injection was performed through a catheter inserted into the femoral vein. The decreasing effect of cacb1(344–359) on arterial BP was more intense and longer when the amount of peptide was increased to 1 mg/kg. (B) The decreased systolic, diastolic, mean arterial and pulse pressure are shown in the extended version of the recording. Numbers indicate the time sampled on the pressure recording shown in A. (C) Intravenous injection of saline or the 16-mer peptide LDENQLEDACEHLAEY, which is a different part of the β1 subunit [cacb1(389–404)] and was used as a control, did not change arterial BP.

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  Fig. 3 Decrease of the arterial blood pressure (BP) by peptide fragments of high voltage-gated calcium channel (HGCC) β2, β3, and β4 subunits, which are similar to β1. (A) Summary of the decreases of systolic BP by various 16-mer peptides originated from HGCC β1 [cacb1(344–359)], β2 [cacb2(392–407)], β3 [cacb3(292–307)], and β4 [cacb4(333–348)] subunits. The effects of the 16-mer peptides were dose-dependent, except for that of the control peptide cacb1(389–404). (B) Average decreases in systolic, diastolic, and mean arterial pressure (MAP), and heart rate (HR). Numbers in parentheses indicate the number of rats used. *p < 0.05 and **p < 0.01, vs. cacb1(389–404).

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  Fig. 4 Decrease of the arterial blood pressure (BP) by mutant peptides of cacb1(344–359). (A) Decrease of systolic BP by mutant peptides of cacb1(344–359). The effects of the mutant peptides were dose-dependent, except for that of the control peptide cacb1(389–404). (B) Scatterplot of the systolic pressure before and after intravenous injection of three mutant peptides (I334V, I344V/K357R, and K357R) at the dose of 0.5 mg/kg. All plots are below the dotted line, indicating that the systolic BP decreased after the injection of peptides. The chart legends of (A) and (B) are the same. (C) Histogram of the average pressure decreases by cacb1(344–359) mutant peptides in systolic, diastolic, and mean arterial pressure (MAP), and of the variation in heart rate (HR). Numbers in parentheses indicate the number of rats used. *p < 0.05 and **p < 0.01, vs. cacb1(389–404).

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  Fig. 5 Loss of the blood pressure (BP)-reducing effects of cacb1 (344–359)K357R by N-terminal or C-terminal truncation. The decrease of BP by cacb1(344–359)K357R was significantly reduced when two amino acids were removed from its N-terminal or C-terminal end. The arrows on the inset above the histogram indicate the cut sites. *p < 0.05 and **p < 0.01, vs. cacb1(344–359)K357R.

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  Fig. 6 Prolongation of the blood pressure (BP)-reducing effect by conjugation of palmitic acid with the 2nd lysine of cacb1(344–359)K357R. (A) The mutant peptide cacb1(344–359)K357R caused a dose-dependent decrease of arterial BP with a short duration (~5–8 min). (B) Intravenous injection of cacb1(344–359)K357R conjugated with palmitic acid at its 2nd lysine (K2-palm, 1 mg/kg) prolonged the decrease of arterial BP for more than 2 h.

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  Fig. 7 Prolongation of the blood pressure (BP)-reducing effect by conjugation of palmitic acid with the lysine of cacb1(344–359)K357R. (A) Histogram of the effects of the palmitic acid-conjugated cacb1(344–359)K357R peptide (“K2-palm”, because of the conjugation at the 2nd amino acid, lysine) on systolic, diastolic, and mean arterial pressure (MAP) and heart rate (HR). The BP-reducing effects of K2-palm were dose-dependent, with a significant increase at 2 mg/kg of the systolic pressure (**p < 0.01 vs. K357R). The cacb1(344–359)K357R peptide conjugated with palmitic acid at its 7th residue (K7-palm) at the dose of 2 mg/kg produced an effect similar to that of the K2 palm peptide at the dose of ~0.5–1 mg/kg. Numbers in parentheses indicate the number of rats used. (B) Scatterplot of the duration of the BP-reducing effect by palmitic acid-conjugated peptides with changes in systolic pressure in each rat (K2-palm, 1 mg/kg, open circle; K2-palm, 2 mg/kg, closed circle; K7-palm, 2 mg/kg, closed triangle).

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  Fig. 8 Effect of the EYEKEYE-added peptide with the conjugation of palmitic acid at the 20th lysine. (A) Intravenous injection of the fusion peptide, cacb1(344–359)K357R-EYEKEYE, with conjugation of palmitic acid at its 20th residue (K20-palm) prolonged the decrease of arterial blood pressure (BP) for at least 90 min. (B) Effect of the fusion peptide cacb1(344–359)K357R-EYEKEYE and the palmitic acid-conjugated fusion peptide (K20-palm) on systolic, diastolic, and mean arterial pressure (MAP) and heart rate (HR). The fusion peptide generated a less potent decrease in BP than the cacb1(344–359)K357R peptide (K357R). Numbers in parentheses indicate the number of rats used.

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  Fig. 9 Effect of the palmitic acid-conjugated cacb1(344–359)K357R peptide on the arterial systolic blood pressure (BP) of non-anesthetized rats. (A) N-terminal acetylation of the palmitic acid-conjugated cacb1(344–359)K357R peptide (K2-palm) did not alter the BP-reducing effect in the anesthetized rats. (B) Intravenous (tail vein) administration (first and second) of the acetylated K2-palm peptide (Ac-K2-palm) decreased systolic BP in non-anesthetized rats recorded by the tail-cuff method. *p < 0.05 and **p < 0.01 vs. before the administration; #p < 0.05 and ##p < 0.01 vs. saline control. Values are presented as mean ± SEM.MAP, mean arterial pressure; HR, heart rate.

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  Fig. 10 Effect of Ac-cacb1(344–359)K357R-NH2 peptide on the formalin-induced acute pain. Intrathecal administration of acetylated (Ac) and amidated (NH2) cacb1(344–359)K357R peptide for protection in vivo did not change the pain behaviors induced by intradermal formalin injection (5%, 50 µl) on the plantar surface of rat hindpaw. Values are presented as mean ± SEM.

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