The peptide fragments used in this study were derived from voltage-gated calcium channel auxiliary subunits β1, β2, β3, and β4; their protein accession numbers are P54283.1, Q8VGC3.2, P54287.1, and Q8R0S4.2, respectively (Table 1). The peptide sequences are listed in Table 1, and most of them are patented in Korea (Patent no. 10-2425548, year 2022). All the peptides were synthesized by a peptide synthesis company (Peptron, Inc.), and the purity of each peptide (> 95%) was determined by high-performance liquid chromatography (according to the information provided by the company).

All procedures with animals were approved by the Institutional Animal and Use Committee of Kyungpook National University (Approval no. 2019-008-2). TG neurons were collected from the TG of Sprague-Dawley rats (7–16 day-old; male or female). Initially, the ganglia were incubated at 37°C for 15 min in prewarmed L-15 media containing collagenase D (1 mg/ml; Roche Diagnostics GmBH). After trituration, ganglion neurons were harvested by washing twice with 1× phosphate-buffered saline and plated on poly-D-lysine/laminin-coated coverslips (diameter, 25 mm) in DMEM (Sigma) containing 10% fetal bovine serum and 1% penicillin/streptomycin. Neurons were used for whole-cell recordings within 1–6 h after maintaining at 37°C with 5% CO₂.

To record Ca²⁺ currents in the isolated TG neuron, whole-cell voltage-clamp recordings were performed with 3–5 MΩ heat-polished borosilicate patch pipettes (TW150F; WPI) on the inverted microscope (IX51; Olympus) at room temperature (24°C–26°C). The internal solution was composed of CsCl (135 mM), HEPES (10 mM), EGTA (1 mM), EDTA (1 mM), and MgCl₂ (4 mM), and was adjusted to pH 7.2 with 20% tetraethylammonium (TEA) hydroxide. The bath solution contained TEA-Cl (135 mM), CaCl₂ (2 mM), HEPES (10 mM), and tetrodotoxin (TTX) (0.0001 mM) (pH 7.2; adjusted by with 20% TEA hydroxide). TEA and TTX was added to the internal and/or external solution to suppress the induction of K⁺ or Na⁺ currents, respectively. The TG neurons were held at holding potential (V_h) of –60 mV, and voltage-activated currents were evoked every 15 sec by depolarizing voltage steps (from –50 mV to +40 mV in 10 mV increment for 250 or 500 ms). The recorded currents were amplified with Axopatch 1D (Molecular Devices) and filtered at 2 kHz (sampling rate, 5 kHz) using pClamp software (Molecular Devices). Transients due to whole-cell capacitance and series resistance were cancelled, and leak currents were subtracted using scaled hyperpolarizing currents (-P/4 protocol). The peak amplitudes of currents were adjusted by the whole-cell capacitance (pA/pF), and neurons with > 10 pA/pF were included for analysis. The peptides were applied through the internal solution.

Arterial blood pressure and peptide solution infusion were measured using a catheter system in the femoral artery and femoral vein, respectively, in Sprague-Dawley rats (330–430 g) of both sexes. Initially, the rats were anesthetized with isoflurane (2%) vaporized in oxygen (1%–1.5%), and the anesthesia was maintained by urethane (1 g/kg, intraperitoneal injection) and isoflurane (0.8%–1%) via snout mask. To expose the femoral neurovascular bundle, the skin in the femoral region was incised, and the soft tissue was dissected. The femoral sheath was opened using blunt dissection directed along its long axis between the femoral artery and vein (dark red). The femoral artery was retracted laterally, and the femoral vein was retracted medially. Then, two pieces of silk (4-0) were inserted under the artery just below the arteria profunda femoris, and one piece after a loose ligature (for upcoming proximal ligation) was pulled toward the body and the other was tightly ligated by a triple knot as far as possible toward the leg (distal ligation). Similarly, two pieces of silk (4-0) were inserted under the vein below the vena profunda femoris, and a loose ligature for the upcoming proximal ligation and a distal tight ligature were made. To insert catheters (PE50 tube) into the femoral artery and femoral vein, a small incision using micro-dissecting scissors was made approximately 2 mm proximal to the distal ligation. The catheter, pre-filled with a saline solution containing 300 U/L of heparin with a syringe (1 ml), was then partly inserted through the incision. The pulled proximal ligature was then loosened, and the catheter was fully inserted (> 1 cm). Then, proximal ligation was accomplished using a triple knot around the artery and catheter or the vein and catheter to secure the catheter to the vessel. Catheter patency was further confirmed by identifying the blood inside the inserted catheter and flushing the heparinized saline solution.

The catheter inserted through the femoral artery was connected to a pressure transducer placed at the same height as the heart of the rat. The transducer was further connected to a PowerLab data acquisition system (ADInstruments). Arterial blood pressure data were recorded and analyzed using LabChart (version 7; ADInstruments). From the cyclic records of arterial blood pressure, systolic pressure was identified by the peak and diastolic pressure by the valley between peaks. Mean arterial pressure was calculated by adding one-third of the difference between the systolic and diastolic pressures to the diastolic pressure. Heart rate was determined as the number of cycles per minute. The ∆ blood pressure was calculated by subtracting the blood pressure after the injection of a peptide solution from the blood pressure before the injection.

Rats were habituated to the apparatus and environment to measure their systolic pressure. A rat was warmed at 30°C for 30 min in an isothermal container and constrained by an acryl holder with a tail-cuff sensor. Systolic pressure was measured in the non-anesthetized state using a non-invasive tail-cuff blood pressure system (Model 47; IITC Life Science). The average of three consecutive measurements was calculated. Peptide solutions or saline was administered through the tail vein of rats after light isoflurane anesthesia in a closed container.

A PE-10 catheter was inserted into the L3-4 lumbar subarachnoid space of a 7–8 week-old rat (male or female) under deep isoflurane anesthesia (2.5%–3%). The rat was recovered for a week, and was subjected to formalin test after adapting to the test environment for 0.5–1 h. The peptide solution (10 µl) was intrathecally administered through the catheter 30 min before the formalin test. The formalin test was conducted using the formalin solution (5%, 50 µl) that was intradermally injected into the plantar surface in one of rat hindpaws (typically left hindpaw). Immediately after the formalin injection, pain behaviors, such as paw withdrawal or licking, were counted and presented in 5-min blocks up to 65 min.

Statistical comparisons were performed using an unpaired Student’s t-test (*p < 0.05, **p < 0.01). Data are reported as mean ± standard error of the mean (SEM).

In this study, we tested the 16-mer peptide fragments derived from HGCC β1 (cacb1), β2 (cacb2), β3 (cacb3), and β4 (cacb4) subunits to determine whether they affected the function of HGCCs and, therefore, arterial blood pressure in rats. In an electrophysiological experiment using whole-cell voltage-clamp recordings in acutely isolated TG neurons [13], the synthesized peptides (water solubility, > 10 mg/ml) did not change the amplitudes of voltage-activated Ca²⁺ current upon their intracellular application through recording patch pipettes (Fig. 1). However, when the peptides were intravenously administered at 0.15 mg/kg (volume, 0.2 ml), the arterial blood pressure from the femoral artery started to decrease and peaked within a minute (the case of β1 subunit, cacb1(344–359); Fig. 2A). As the dose was increased to 1 mg/kg, the magnitude of the reduction in arterial pressure became more intense and longer in duration (Fig. 2A), and pulse pressure slightly decreased (Fig. 2B). Thus, the effect of cacb1(344–359) on systolic pressure was dose-dependent (Fig. 3A), and the systolic, diastolic, and mean arterial pressure were decreased by 23.7 ± 10.4 mmHg, 22.0 ± 8.6 mmHg, and 22.6 ± 9.2 mmHg, respectively, at a dose of 0.5 mg/kg in three rats, and the heart rate was slowed by 36.0 ± 22.4 beats per minute (Fig. 3B). However, the arterial blood pressure remained unchanged by intravenous injection of either saline or another 16-mer control peptide [cacb1(389–404)], derived from a different part of the β1 subunit (Fig. 2C) (average changes from five rats at 0.5 mg/kg: systolic pressure, −0.4 ± 1.2 mmHg; diastolic pressure, −0.2 ± 1.2 mmHg; mean arterial pressure, −0.3 ± 1.1 mmHg; Fig. 3B). Peptides from the β2 [cacb2(392–407)], β3 [cacb3(292–307)], and β4 [cacb4(333–348)] subtypes, which are the same part but have slightly different amino acid sequences compared to β1, also decreased arterial blood pressure in rats (Fig. 3A). The effects of these 16-mer peptides on systolic blood pressure were dose-dependent; the effects on systolic, diastolic, and mean arterial pressure were significantly different at 0.5 mg/kg compared to that of the control cacb1(389–404) peptide (Fig. 3B).

The peptide sequence comparison showed that cacb2(392–407) had mutations at I344 (to V) and T347 (to S) of cacb1(344–359), cacb4(333–348) at I344 (to V), I346 (to V), and T347 (to S) of cacb1(344–359), and cacb3(292–307) at I344 (to V), I346 (to V), T347 (to S), and K357 (to R) of cacb1(344–359) (Table 1). Thus, we tested cacb1(344–359)_I344V with a single mutation (to V) only at the I344 position of cacb1(344–359) and cacb1(344–359)_I344V/K357R with double mutations at both the I344 and K357 positions, which also decreased arterial blood pressure (Fig. 4). In addition, a peptide with only the K/R mutation at K357 of cacb1(344–359) [IKITSPKVLQRLIRSR, cacb1(344–359)_K357R] also decreased arterial blood pressure (Fig. 4). On the other hand, a single K405R mutant of cacb2(392–407) [VKISSPKVLQRLIRSR, cacb2(392–407)_K405R; Fig. 3A] decreased arterial blood pressure, similar to the cacb3(292–307) peptide, which is also a K/R mutation from cacb4(333–348). The dose-dependent effects of peptides mutated at the first, fourteenth, or both positions of cacb1(344–359) on systolic pressure are summarized (Fig. 4A). The magnitudes of these peptides mutated from cacb1(344–359) at 0.5 mg/kg were individually plotted in systolic pressure (Fig. 4B), indicating that the reduction in blood pressure was independent of the blood pressure before peptide administration. Additionally, the effects of cacb1(344–359) and its mutant peptides on systolic, diastolic, and mean arterial blood pressure were significantly different from those of other 16-mer control cacb1(389–404) peptide (Fig. 4C). The cacb1(344–359)_K357R peptide was selected for further study because it was slightly more potent and consistent at the dose of 0.5 mg/kg than the other peptides and produced relatively constant blood-reducing effect (Figs. 3 and 4).

To further explore the essential amino acids involved in the blood pressure-reducing effects of the peptides, we performed a truncation study in which two amino acids were continuously cut starting from the N-terminal or C-terminal ends of the cacb1(344–359)_K357R peptide. Interestingly, the first truncation of two amino acids at either the N-terminal or C-terminal end almost abolished the blood pressure-reducing effect of cacb1(344–359)_K357R (Fig. 5); likewise, the second and third truncations by each of the two amino acids negated the effect of peptides. This indicates that all 16 amino acids are necessary for the effect of cacb1(344–359)_K357R on blood pressure.

Despite the effectiveness of cacb1(344–359) and its derivatives in reducing arterial blood pressure, the duration of the effect was relatively short, reaching only 7–8 min (Fig. 6A). Thus, we conjugated palmitic acid, an albumin ligand, to lysine (K) at the 2nd or 7th amino acid position (from the N-terminal) of cacb1(344–359)_K357R, respectively producing IK(palmitoyl)ITSPKVLQRLIRSR (named K2-palm) and IKITSPK(palmitoyl)VLQRLIRSR (named K7-palm). Intravenous injection of K2-palm at a dose of 0.5–2 mg/kg substantially prolonged the effect on arterial blood pressure, reaching a maximum duration of over 4 h (Fig. 6B and Fig. 7). K7-palm prolonged the effect at a dose of 2 mg/kg, demonstrating a lower potency than the K2-palm peptide (Fig. 7). In addition, we constructed a fusion peptide of cacb1(344–359)_K357R by adding EYEKEYE peptide at its C-terminal end. Then, palmitic acid was conjugated to the lysine side chain of the EYEKEYE sequence (named K20-palm, because of the 20th residue). This peptide also showed a long blood pressure-reducing effect (Fig. 8), but only at a dose of 2 mg/kg, which is four times higher than that of cacb1(344–359)_K357R. Therefore, we conclude that the K2-palm peptide is effective in terms of potency and prolongation of the blood-reducing effect.

Because the blood pressure-reducing effect of cacb1(344–359)_K357R was significantly prolonged by palmitic acid conjugation at its lysine amino acid (K2-palm), we further tested whether the peptide reduced blood pressure for more than a day. For this, we measured the blood pressure in non-anesthetized rats using the indirect tail-cuff method. In this experiment, K2-palm was further protected from peptidases by N-terminal acetylation, and the acetylated K2-palm peptide maintained its efficacy against systolic, diastolic, and mean arterial pressure (Fig. 9A). When the acetylated K2-palm peptide was administered through the tail vein in non-anesthetized rats, the blood pressure, indirectly measured by tail-cuff, was significantly reduced at 3 h after administration, but after 24 h, it returned to the baseline (Fig. 9B). This indicates that the several-hour blood pressure reducing-effect of K2-palm can be reproduced in non-anesthetized rats.

Despite of the potent antihypertensive effects of the peptide fragments derived from HGCC auxiliary β subunits, we further tested whether they have any effect on acutely-evoked inflammatory pain. As a result, the intrathecal administration of the Ac-cacb1(344-359)_K357R-NH₂ peptide (10 µl at 10 mM), which was acetylated at N-terminal and amidated at C-terminal for enzymatic protection, did not change the spontaneous pain behaviors (paw withdrawal and licking) induced by intradermal injection of formalin solution (5%, 50 µl), compared to those induced in the rats administered with the control scrambled peptide (Fig. 10).