Abstract P252: Angiotensin II Type 1 Receptor Autoantibody Inhibited Bk Channels In Vascular Smooth Muscle Cells

Abstract

Aims: The angiotensin II type 1 receptor autoantibody (AT1-AA) leads pathological sustainable vasoconstriction. Large-conductance Ca 2+ -and voltage-activated potassium (BK) channel, mainly consist by functional α subunits and modulatory β1 subunits in vascular smooth muscle cells (SMCs), plays a vital role on vascular relaxation. Recently, it has been found that AT1-AA inhibited BK channel’s function. However, the specific mechanisms remain unclear. In this study, we further investigated the open probability, Ca 2+ sensitivity and β1 subunits of BK channel after AT1-AA stimulation. Methods and Results: BKα subunit has several splicing variants. Immunofluorescence and PCR results showed that BK channels widely distributed among mesenteric artery (MA), most of them belong to zero-type. SMCs from MA were isolated freshly and used for patch clamp within 6 hours. AT1-AA downregulated the open probability of BK channel to 57.41±21.36% (the corresponding data of baseline was considered as 100%), specifically reduced the number of simultaneous opened channels (level 4 to level 2) instead of dwell time (T 1 =0.45 ms, T 2 = 3.39 ms to T 1 =0.58 ms, T 2 =4.99 ms) and amplitude (187.4 ±16.23 to 187.3 ±13.80 pS). This phenomenon could be reversed by BK channel agonist (NS1619) (enhanced to 124%). Noteworthy, different from Ang II, AT1-AA suppressed BK channel’s open probability consistently even after 15min (Ang II: recovered to 164.8%; AT1-AA, kept at 66.0%). The activated effect of Ca 2+ on BK channel nearly disappeared after bathing with AT1-AA in inside-out recording mode (vehicle: 100% to 471.6%; AT1-AA: 100% to 163.9%). Furthermore, to explore whether AT1-AA’s inhibitory effect on BK channel was independent from β1 subunit, only ZERO-BKα-GFP subunits were transfected into HEK293T cells. AT1-AA still diminished BK channels’ function to 57.35±5.4%. However, the inhibition of AT1-AA on BK channels was almost non-existent (to 92.1±12.81%) in AT1 receptor knock out rats. Conclusion: These results demonstrated that AT1-AA sustainably decreased open probability and Ca 2+ sensitivity of BK channels even if β1 subunit were absence. This effect was AT1 receptor dependent. It remains us that BK channel may become a novel target for improving AT1-AA related hypertension.