Abstract 37: Loss Of BK Ca - IP3R Functional Coupling Underlies Vascular Hypercontractility In Spontaneously Hypertensive Rats

The cellular mechanisms underlying enhanced vascular contractility in hypertension are still not fully clear. Recent studies have demonstrated that functional coupling between the large-conductance Ca 2+ -activated K + (BK Ca ) channel in the cell membrane and inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum is involved in the regulation of intracellular Ca 2+ in vascular smooth muscle cells (VSMC) and vascular contractility. Here, we examined the role of BK Ca -IP3R coupling in vascular hypercontractility in Spontaneously Hypertensive Rats (SHR), as compared with Sprague Dawley (SD) rats. In pressurized mesenteric arteries, paxilline (1μM), a selective BK Ca channel blocker, significantly increased norepinephrine (1μM)-induced vasoconstriction in both SHR (% initial diameter: 71.6±6.8% [95.9±13 of 132.8±6.2 μm] and 65.0±4.8% [80.6±7.7 of 123.6±3.9 μm] in arteries treated with vehicle and paxilline respectively, n=4, P<0.05) and in SD rats (% initial diameter: 84.2±7.9% [108.4±12.3 of 128.5±2.5 μm] and 60.5±0.2% [77.4±0.7 of 127.9±0.6 μm] in arteries treated with vehicle and paxilline respectively, n=3, P<0.05). Paxilline-induced increases in vasoconstriction were significantly greater in arteries of SD as compared with SHR rats, suggesting that BK Ca channels are involved in hypercontractility in SHR. In inside-out patch-clamp, BK Ca channels in VSMCs isolated from mesenteric arteries of SHR and SD rats displayed identical calcium and voltage sensitivity. We next examined the effect of the selective IP3R-agonist, Adenophostin A, on BK Ca current density in VSMCs, using patch-clamp whole-cell mode. The results demonstrated that Adenophostin A (5μM) significantly increased BK Ca current density in VSMCs from both SHR (from 41.9± 5.7 to 61.5±9.1 pA/pF; n=6 cells, p<0.05) and SD rats (from 29.9 ± 8.0 to 92.6 ± 9.1 pA/pF, n=5 cells, P<0.01). There was no significant difference in basal BKca current density between SHR and SD rats; however, the Adenophostin-induced elevation in BK Ca current density was significantly diminished in VSMCs of SHR as compared with SD rats. In conclusion, the results indicate that loss of functional coupling between BKca and IP3R is involved in vascular hypercontractility in SHR arteries.

Cryo-EM Reveals Ligand Induced Allostery Underlying InsP3R Channel Gating

Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are cation channels that mobilize Ca2+ from intracellular stores in response to a wide range of cellular stimuli. The paradigm of InsP3R activation is the coupled interplay between binding of InsP3 and Ca2+ that switches the ion conduction pathway between closed and open states to enable the passage of Ca2+ through the channel. However, the molecular mechanism of how the receptor senses and decodes ligand-binding signals into gating motion remains unknown. Here we present the electron cryo-microscopy structure of InsP3R1 from rat cerebellum determined to 4.1 Å resolution in the presence of activating concentrations of Ca2+ and adenophostin A (AdA), a structural mimetic of InsP3 and the most potent known agonist of the channel. Comparison with the 3.9 Å-resolution structure of InsP3R1 in the Apo-state, also reported herein, reveals the binding arrangement of AdA in the tetrameric channel assembly and striking ligand-induced conformational rearrangements within cytoplasmic domains coupled to the dilation of a hydrophobic constriction at the gate. Together, our results provide critical insights into the mechanistic principles by which ligand-binding allosterically gates InsP3R channel.

Synthesis of dimeric analogs of adenophostin A that potently evoke Ca2+release through IP3receptors

Syntheses and Ca2+release potentials of four dimeric analogs of adenophostin A (AdA) through activation of type 1 IP3R are reported. These analogs are full agonists of IP3R and are equipotent to AdA, the most potent agonist of IP3R.