Modulation of large-conductance Ca^(2+) activated K^(+) channels by intracellular Na^(+), K^(+) and Na^(+)/Ca^(2+) exchanger inhibitors

Abstract

In this thesis, modulation effects of Na+ and K+ on large-conductance Ca2+ activated K+ (BKCa) current and pharmacological modulation of BKCa channels by Na+/Ca2+ exchanger (NCX) inhibitors are investigated by using an in vitro electrophysiological method. BKCa currents were markedly increased by extracellular Na+ depletion in Ca2+- and voltage-clamped cells even in extracellular Ca2+-free condition. Background Na+ influx, as a dominant determination factor of resting membrane potential (VM), made more positive resting VM of HUVECs than other types of cells and it was also regulated by VM or decreased by extracellular Na+ depletion. Specifically, in current-clamped patches, extracellular Na+ replacement by NMDG+ or mannitol hyperpolarized cells, and in voltage-clamped patches, changing VM from 0 mV to negative potentials increased intracellular Na+ concentration ([Na+]i) and vice versa. Further single-channel study showed that in inside-out patches, increasing [Na+]i from 0 to 20 or 40 mM affected BKCa channel activity: single channel conductance was significantly reduced and open probability (NPo) was not changed. BKCa channels also exhibit a intracellular K+ concentration ([K+]i) dependent inactivation, e.g. decrease of [K+]i gradually from 140 to 70 mM markedly reduced both single channel conductance and NPo. Exposure of inside-out patches to several combination of bath solutions, e.g. increasing [Na+]i gradually from 0 to 70 mM by replacing [K+]i, reduced both of single BKCa channel conductance and NPo in [Na+]i dependent manner. In whole-cell patches, extracellular application of Ni2+ or KB-R7943, NCX blockers, decreased [Na+]i and increased BKCa currents simultaneously, and the Na+ ionophore monensin completely inhibited BKCa currents. BKCa currents were significantly augmented by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM and significantly reduced by decreasing [K+]o from 12 or 6 to 0 mM or applying the Na+-K+ pump inhibitor ouabain. It was suggested that [Na+]i which determined by Na+ influx modulates BKCa currents due to change of single BKCa channel conductance not channel activity. Meanwhile increase of [K+]i, one more factor which also affected by Na+ influx, increases both of single channel conductance and NPo of BKCa channel. In the next following study, it was found that some selective NCX blockers can also modulate BKCa channels of HUVECs and MASMCs. Therefore, pharmacological modulation effects of 2-[2-[4-(4-nitrobenzyloxy)phenyl]] isothiourea methanes (KB-R7943), dichlorobenzamil (DCB) and SN-6 on BKCa channels are studied by using the patch clamp technique. It was investigated that both KB-R7943 and DCB drugs reversibly activated BKCa currents in whole-cell clamped HUVECs or MASMCs. The EC50 of KB-R7943 and 2',4'-DCB for BKCa current activation in HUVECs were 6.78 ± 0.7 μM and 2.64 ± 0.10 μM respectively. In inside-out and outside-out patches, KB-R7943 and 2',4'-DCB remarkably increased BKCa channels activity by increasing channel open frequency, but not by modulation of mean open time of channels. In inside-out patches, both of KB-R7943 and 2',4'-DCB shifted the relationship curve between [Ca2+]i and NPo to the left; the [Ca2+]i required to evoke half-maximal activation changed from 1087 ± 142 nM to 500 ± 66 nM by 10 μM 2',4'-DCB and from 1220 ± 68 nM to 620 ± 199 nM by 10 μM KB-R7943, respectively. In addition, KB-R7943 and 2',4'-DCB shifted the relationship curve between VM and NPo to more negative potentials; the VM to evoke half-maximal activation changed from 81 ± 2 mV to 65 ± 3 mV by 10 μM 2',4'-DCB and changed from 77 ± 1 mV to 50 ± 3 mV by 10 μM KB-R7943. Moreover, no significant difference was found between DCB effects on BKCa channels inside-out and outside-out patches. These results suggested that KB-R7943 and DCB activate BKCa channels activity in HUVECs and mouse aortic smooth muscle cells (MASMCs) by increasing the sensitivity of BKCa channels to cytosolic free Ca2+ and VM. It was also found that SN-6 produced the opposite effects of KB-R7943 or DCB, a decrease in BKCa channel activity. These reports would provide a consideration if KB-R7943, DCB and SN-6 are used as NCX blockers in vascular endothelial cells or smooth muscle cells. This thesis provides supporting evidences and helps understanding the mechanism of physiological BKCa channel modulation by monovalent cations and pharmacological modulation by NCX inhibitors.;본 연구에서는 인간제대정맥내피세포(human umbilical vein endothelial cells (HUVECs))와 흰쥐 대동맥 평활근세포(mouse aortic smooth muscle cells (MASMCs))에서 large-conductance Ca2+-activated K+ (BKCa)채널에 미치는 K+, Na+ 등 1가 양이온과 Na+-Ca2+ exchanger (NCX)억제제에 의한 조절 작용 및 그 기전에 대하여 전기생리학적 연구방법을 사용하여 알아보았다. HUVECs의 안정막전위(resting membrane potential)는 -12.7 ± 1.7 mV이었으며 세포막을 통과할 수 없는 mannitol, NMDG로 Na+을 대체하면 막전압이 과분극 되었으며 세포막을 통과할 수 있는 Li+으로 대체하면 변화가 없었다. 막전압을 고정(voltage-clamped patch)을 한 세포의 막전압을 0 mV에서 +40 mV로 저분극 시키면 세포 내 Na+ 농도 ([Na+]i)는 증가하였고 막전압을 -100mV로 과분극 시키면 [Na+]i는 감소하였다. 세포 내 Ca2+ 농도를 여러 가지 농도에 고정시켜 활성화 시킨 BKCa 전류는 세포 밖 Na+을 고갈시키면 현저히 증가되었다. 이러한 세포외 Na+고갈에 의한 BKCa 전류 활성화는 세포 밖 Ca2+을 제거하여도 발생하였다. 단일 채널수준 연구에 의하면, inside-out patch에서는 [Na+]i를 0 mM 에서 20, 40 mM로 증가시키면 BKCa채널의 conductance와 열릴 확률(open probability)이 감소하였다. 그리고 세포 내 K+농도를 140 mM에서 70 mM로 감소시키면 BKCa 채널의 conductance와 열릴 확률이 모두 감소하였다. [Na+]i와 BKCa 전류를 동시에 측정하면, 세포 내 Ca2+을 0.5 μM에 고정하여 활성화시킨 BKCa 전류는 Ni2+ 또는 KB-R7943 등 NCX억제제로 [Na+]i을 감소시키면 증가하였고 NCX억제제를 제거하여 [Na+]i을 증가시키면 감소하였다. 그리고 Na+ 이온 투과 담체인 monensin은 BKCa 전류를 완전히 억제하였다. 한편 일부 NCX 억제제들 (KB-R7943, DCB, SN-6)은 HUVECs과 MASMCs에서 BKCa 채널의 활성을 조절하였다. KB-R7943과 DCB은 BKCa전류를 증가시켰는데 EC50는 각각 6.78 ± 0.7 μM과 2.64 ± 0.1 μM 이었다. KB-R7943과 DCB 모두 BKCa채널의 mean open time에는 영향을 주지 않았으나 channel open frequency를 유의하게 증가시켰으며, Ca2+에 대한 민감성과 막전압에 대한 민감성을 모두 증가 시켰다. 그리고 이러한 효과는 inside-out, outside-out patch에서 차이가 없었다. 그러나 SN-6는 BKCa전류를 억제하였다. 이상의 결과에서 BKCa 채널의 활성 및 BKCa전류는 세포 내 Na+, K+이온의 농도와 일부 NCX 억제제에 의하여 조절 된다는 것을 알 수 있다.