The Effects Of HCN1 Channels Knockout On The Expression And Function Of BK Channels On ICCs Of Mouse Bladder

Background and ObjectiveThe abnormal of detrusor excitability is the common nosogenesis of some urological diseases such as Overactive Bladder(OAB), neurogenic bladder, etc. Illuminating the mechanism underlying the regulation of bladder excitability is important to make clear the pathogenesis of these diseases. “Neurogenic theory” and “myogenic theory” are both involved in the regulation of bladder excitability. Since the discovery of interstitial cells of Cajal(ICCs) in the bladder which is similar to the ICCs in the gastrointestinal tract, the “myogenic theory” is accepted gradually and is attached greater importance to.There are a variety of ion channels and receptors playing important roles in operating cellular functions on ICCs. Among them, the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels(HCN Channels) which mediate pacemaker current make the ICCs possible to be the pacemaker of bladder thus account for the core status. Many neuroreceptors such as Muscarinic Receptors and Purinergic P2X3 Receptors(P2X3 Receptors) can regulate the functions of ICCs following the neural signal. Mechanical stretch takes part in the regulation of the functions of ICCs as well. Besides, gap junctions between detrusor smooth muscle cells(DSMCs) or between ICCs and DSMCs were confirmed in the bladder. We come to a conclusion that the ICCs may integrate the signals from nerves and mechanical stimulus and initiate a HCN-mediated pacemaker current that spreads to detrusor via ion channels, receptors, and intercellular communication. In this process, many ion channels related to calcium ion such as Large-Conductance Calcium-Activated Potassium Channels(BK channels) play important roles.It’s acknowledged that HCN channels can mediate pacemaker current, and different subtypes of HCN channels play outstanding roles in the control of cardiac and nervous rhythm. It’s reported that HCN channels expressed on ICCs in both human and rat bladder, and the pacemaker current named If was recorded, the expression of HCN channels increased in detrusor overactivity(DO). BK channels have no permeability for calcium ion, the current mediated by it is the outflow of potassium ions. The potassium outflow current can reduce the calcium afflux via a negative feedback pathway. The activation of BK channels could limit depolarization and thus suppress the contraction of smooth muscle. Interventions of BK channels could impact the bladder excitability and lead to various bladder dysfunction. BK channels also expressed on ICCs in the bladder, its expression increased in DO.There are possible interactions between HCN channels and BK channels, both of them are essential to the function regulation of ICCs. The alterations of BK channels after intervention of HCN channels attract our attention. HCN1 is the main subtype of HCN channels in murine bladder, this research is aimed to investigate the effects of HCN1 channels knockout on the expression and function of BK channels on ICCs of the mouse bladder, and discuss the significance of the effects to the regulation of bladder excitability.Methods1. HCN1 wild type(WT) and HCN1 knockout(KO) mice were used in this research and the mice were divided into normal and KO groups.2. The alteration of expression of BK channels in the bladder was detected in m RNA and protein levels by Reverse Transcription Polymerase Chain Reaction(RT-PCR), Realtime Fluores-cence Quantitative Transcription Polymerase Chain Reaction(Q-PCR) and Western blot(WB). Isolated ICCs were obtained using “improved acute enzyme digestion” method. The alteration of expression of α subunit of BK channels on ICCs was detected by immunofluorescence staining.3. The inhibitor and agonist of BK channels, Iberiotoxin(IBTX) and NS1619, were used to detect the function alteration of BK channels. At first, the contraction function of bladder was compared between normal and KO groups by detrusor muscle strip test, the sensitivity of muscle strips on IBTX was examined to evaluate the function alteration of BK channels. The laser confocal fluorescence microscopy was used for intracellular calcium concentration measurement, The actions of NS1619 and IBTX on the intracellular calcium concentration of ICCs were detected to evaluate the function alteration of BK channels.Results1. RT-PCR, Q-PCR revealed that the expressions of five BK channels’ subunits, α, β1, β2, β3 and β4, in the bladder of KO group were lower than that of normal group(P < 0.01).2. WB revealed that the expressions of five BK channels’ subunits, α, β1, β2, β3 and β4, in the bladder of KO group were lower than that of normal group(α, β3, β4: P < 0.01; β1, β2: P < 0.05).3. Isolated ICCs were identified by morphological characteristics and the primary cultured ICCs were in good condition.4. The immunofluorescence staining revealed that the expression of α subunit of BK channels on ICCs of KO group was lower than that of normal group(P < 0.01).5. The detrusor strip test revealed that the amplitude and frequency of detrusor contraction in KO group were both lower than that in normal group(P < 0.05, P < 0.01). IBTX generated an increase in amplitude of detrusor contraction in both KO and normal groups(P < 0.01, P < 0.05) along with no significant change in frequency(P > 0.05), but the KO group’s detrusor had a lower sensitivity on IBTX(P < 0.05).6. NS1619 and IBTX respectively decrease and increase the intracellular calcium concentration of ICCs in both normal group(P < 0.05, P < 0.01) and KO group(P < 0.01), but the actions of both NS1619 and IBTX on KO group were lower than that on normal group(P <0.01).Conclusions1. The contraction function of bladder in KO group was lower than that in normal group.2. The knockout of HCN1 channels resulted in a down-regulation of the expression of BK channels on ICCs of mouse bladder.3. The knockout of HCN1 channels resulted in a down-regulation of the function of BK channels on ICCs of mouse bladder.4. These down-regulations might be a compensational mechanism of the decreased contractility induced by the knockout of HCN1 channels.