Intravascular flow stimulates PKD2 (polycystin-2) channels in endothelial cells to reduce blood pressure

PKD2 (polycystin-2, TRPP1), a TRP polycystin channel, is expressed in endothelial cells (ECs), but its physiological functions in this cell type are unclear. Here, we generated inducible, EC-specific Pkd2 knockout mice to examine vascular functions of PKD2. Data show that a broad range of intravascular flow rates stimulate EC PKD2 channels, producing vasodilation. Flow-mediated PKD2 channel activation leads to calcium influx that activates SK/IK channels and eNOS serine 1176 phosphorylation in ECs. These signaling mechanisms produce arterial hyperpolarization and vasodilation. In contrast, EC PKD2 channels do not contribute to acetylcholine-induced vasodilation, suggesting stimulus-specific function. EC-specific PKD2 knockout elevated blood pressure in mice without altering cardiac function or kidney anatomy. These data demonstrate that flow stimulates PKD2 channels in ECs, leading to SK/IK channel and eNOS activation, hyperpolarization, vasodilation and a reduction in systemic blood pressure. Thus, PKD2 channels are a major component of functional flow sensing in the vasculature.

Glucose-mediated inhibition of calcium-activated potassium channels limits α-cell calcium influx and glucagon secretion

Pancreatic α-cells exhibit oscillations in cytosolic Ca2+ (Ca2+c), which control pulsatile glucagon (GCG) secretion. However, the mechanisms that modulate α-cell Ca2+c oscillations have not been elucidated. As β-cell Ca2+c oscillations are regulated in part by Ca2+-activated K+ (Kslow) currents, this work investigated the role of Kslow in α-cell Ca2+ handling and GCG secretion. α-Cells displayed Kslow currents that were dependent on Ca2+ influx through L- and P/Q-type voltage-dependent Ca2+ channels (VDCCs) as well as Ca2+ released from endoplasmic reticulum stores. α-Cell Kslow was decreased by small-conductance Ca2+-activated K+ (SK) channel inhibitors apamin and UCL 1684, large-conductance Ca2+-activated K+ (BK) channel inhibitor iberiotoxin (IbTx), and intermediate-conductance Ca2+-activated K+ (IK) channel inhibitor TRAM 34. Moreover, partial inhibition of α-cell Kslow with apamin depolarized membrane potential ( Vm) (3.8 ± 0.7 mV) and reduced action potential (AP) amplitude (10.4 ± 1.9 mV). Although apamin transiently increased Ca2+ influx into α-cells at low glucose (42.9 ± 10.6%), sustained SK (38.5 ± 10.4%) or BK channel inhibition (31.0 ± 11.7%) decreased α-cell Ca2+ influx. Total α-cell Ca2+c was similarly reduced (28.3 ± 11.1%) following prolonged treatment with high glucose, but it was not decreased further by SK or BK channel inhibition. Consistent with reduced α-cell Ca2+c following prolonged Kslow inhibition, apamin decreased GCG secretion from mouse (20.4 ± 4.2%) and human (27.7 ± 13.1%) islets at low glucose. These data demonstrate that Kslow activation provides a hyperpolarizing influence on α-cell Vm that sustains Ca2+ entry during hypoglycemic conditions, presumably by preventing voltage-dependent inactivation of P/Q-type VDCCs. Thus, when α-cell Ca2+c is elevated during secretagogue stimulation, Kslow activation helps to preserve GCG secretion.

SK but not IK channels regulate human detrusor smooth muscle spontaneous and nerve-evoked contractions

Animal studies suggest that the small (SK) and intermediate (IK) conductance Ca2+-activated K+channels may contribute to detrusor smooth muscle (DSM) excitability and contractility. However, the ability of SK and IK channels to control DSM spontaneous phasic and nerve-evoked contractions in human DSM remains unclear. We first investigated SK and IK channels molecular expression in native human DSM and further assessed their functional role using isometric DSM tension recordings and SK/IK channel-selective inhibitors. Quantitative PCR experiments revealed that SK3 channel mRNA expression in isolated DSM single cells was ∼12- to 44-fold higher than SK1, SK2, and IK channels. RT-PCR studies at the single-cell level detected mRNA messages for SK3 channels but not SK1, SK2, and IK channels. Western blot and immunohistochemistry analysis further confirmed protein expression for the SK3 channel and lack of detectable protein expression for IK channel in whole DSM tissue. Apamin (1 μM), a selective SK channel inhibitor, significantly increased the spontaneous phasic contraction amplitude, muscle force integral, phasic contraction duration, and muscle tone of human DSM isolated strips. Apamin (1 μM) also increased the amplitude of human DSM electrical field stimulation (EFS)-induced contractions. However, TRAM-34 (1 μM), a selective IK channel inhibitor, had no effect on the spontaneous phasic and EFS-induced DSM contractions suggesting a lack of IK channel functional role in human DSM. In summary, our molecular and functional studies revealed that the SK, particularly the SK3 subtype, but not IK channels are expressed and regulate the spontaneous and nerve-evoked contractions in human DSM.

An intermediate-conductance Ca2+-activated K+ channel is important for secretion in pancreatic duct cells

Potassium channels play a vital role in maintaining the membrane potential and the driving force for anion secretion in epithelia. In pancreatic ducts, which secrete bicarbonate-rich fluid, the identity of K+ channels has not been extensively investigated. In this study, we investigated the molecular basis of functional K+ channels in rodent and human pancreatic ducts (Capan-1, PANC-1, and CFPAC-1) using molecular and electrophysiological techniques. RT-PCR analysis revealed mRNAs for KCNQ1, KCNH2, KCNH5, KCNT1, and KCNT2, as well as KCNN4 coding for the following channels: KVLQT1; HERG; EAG2; Slack; Slick; and an intermediate-conductance Ca2+-activated K+ (IK) channel (KCa3.1). The following functional studies were focused on the IK channel. 5,6-Dichloro-1-ethyl-1,3-dihydro-2 H-benzimidazole-2-one (DC-EBIO), an activator of IK channel, increased equivalent short-circuit current ( Isc) in Capan-1 monolayer, consistent with a secretory response. Clotrimazole, a blocker of IK channel, inhibited Isc. IK channel blockers depolarized the membrane potential of cells in microperfused ducts dissected from rodent pancreas. Cell-attached patch-clamp single-channel recordings revealed IK channels with an average conductance of 80 pS in freshly isolated rodent duct cells. These results indicated that the IK channels may, at least in part, be involved in setting the resting membrane potential. Furthermore, the IK channels are involved in anion and potassium transport in stimulated pancreatic ducts.

Effects of Compounds That Influence IK (KCNN4) Channels on Afterhyperpolarizing Potentials, and Determination of IK Channel Sequence, in Guinea Pig Enteric Neurons

The late afterhyperpolarizing potential (AHP) that follows the action potential in intrinsic primary afferent neurons of the gastrointestinal tract has a profound influence on their firing patterns. There has been uncertainty about the identity of the channels that carry the late AHP current, especially in guinea pigs, where the majority of the physiological studies have been made. In the present work, the late AHP was recorded with intracellular microelectrodes from myenteric neurons in the guinea pig small intestine. mRNA was extracted from the ganglia to determine the identity of the guinea pig intermediate conductance potassium ( IK) channel gene transcript. The late AHP was inhibited by two blockers of IK channels, TRAM34 (0.1–1 μM) and clotrimazole (10 μM), and was enhanced by the potentiator of the opening of these channels, DC-EBIO (100 nM). Action potential characteristics were unchanged by TRAM34 or DC-EBIO. The full sequence of the gene transcript and the deduced amino acid sequence were determined from extracts including myenteric ganglia and from bladder urothelium, which is a rich source of IK channel mRNA. This showed that the guinea pig sequence has a high degree of homology with other mammalian sequences but that the guinea pig channel lacks a phosphorylation site that was thought to be critical for channel regulation. It is concluded that the channels that carry the current of the late afterhyperpolarizing potential in guinea pig enteric neurons are IK channels.