Indeed, it has been shown that elevated flow rates to the late distal tubule or the CCD leads to enhanced K+ secretion via activation of the luminal BK channel giving rise to the phenomenon of flow-dependent K+ secretion [24], [28], [29]

Indeed, it has been shown that elevated flow rates to the late distal tubule or the CCD leads to enhanced K+ secretion via activation of the luminal BK channel giving rise to the phenomenon of flow-dependent K+ secretion [24], [28], [29]. and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca2+ influx, leading to elevated intracellular Ca2+ levels, activates this high Ca2+-affinity K+ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca2+-dependent regulation of membrane potential and K+ secretion. Introduction Calcium-activated potassium channels, KCa, are a small group of potassium channels that are widely expressed in numerous tissues ranging from neurons to vascular endothelial cells [1]C[5]. As with other K+ channels, the KCa channels can play a major role in regulating the plasma membrane electrical potential difference, Vm. However, their classical regulation by intracellular Ca2+, [Ca2+]i, leads to a highly dynamic coupling between Vm and [Ca2+]i which appears to underlie their central role in a wide array of functions ranging from neuronal excitability [6], [7], to modulation of vascular smooth muscle tone [8], [9], to cell volume regulation [10], [11]. Indeed, depending on the types of KCa channels expressed by a particular cell type, the hyperpolarization of the cell membrane following Ca2+-induced activation of a given KCa channel can either enhance Ca2+ influx through non-voltage-activated, Ca2+-permeable channels, such as TRP channels, or reduce Ca2+ influx in the case of voltage-activated Ca2+ channels [4], [12]. To date, five subtypes of Ca2+-activated K+ channels have been RHOC identified: the large-conductance channel (BK, KCa1.1), the intermediate-conductance channel (IK1, KCa3.1), and three small-conductance channels (SK1, KCa2.1; SK2, KCa2.2; and SK3, KCa2.3) [1]C[3]. While the channels have similar structure (6C7 transmembrane segments, a pore loop region, and assembly as homo/heterotetramers), the gating mechanisms can differ, especially between BK and the other channels. Indeed, BK is gated by both membrane potential (activates with depoloarization) and intracellular Ca2+. Further, the Ca2+ binding sites in the C-terminus, the Ca2+ bowl, of the channel-forming -subunit of BK are characterized with a low Ca2+ binding affinity requiring high cytoplasmic levels of Ca2+ for activation (EC50?=?1C11 M; [13]C[15]); however, the Ca2+ affinity can be modulated by binding of selective BK subunits. In contrast, IK and SK WAY-600 channels are voltage insensitive. However, the IK/SK Ca2+ binding site is the ubiquitous Ca2+-sensor, calmodulin, constitutively bound to the C-terminus of the channel, which is characterized by a high Ca2+ binding affinity with a Ca2+ EC50 for gating near 300C600 nM [16]C[18]. As a consequence, the SK channels are highly sensitive Ca2+ sensors intimately linking [Ca2+]i to membrane potential and K+ efflux in all cells where these channels are expressed. In the mammalian kidney, K+ channels expressed at the luminal (apical) membrane of the late distal tubule and cortical collecting duct (CCD) are Ba2+-sensitive (blocker) channels that represent the dominant conductance of the apical membrane (see [19], [20]). Hence, the underlying channels serve as key K+ secretory pathways which regulate K+ excretion and, hence, K+ homeostatis [21]C[24]. It has been shown that the ROMK channel (Kir1.1), an inward rectifier K+ channel from the Kir family, WAY-600 is the resting, Ba2+-sensitive, channel responsible for K+ secretion under normal physiological conditions [5], [25]C[27]. Under stimulated states, however, WAY-600 it is becoming apparent that other K+ channels can contribute to K+ secretion. Indeed, it has been shown that elevated flow rates to the late distal tubule or the CCD leads to enhanced K+ secretion via activation of the luminal BK channel giving rise to the phenomenon of flow-dependent K+ secretion [24], [28], [29]. This is a Ca2+-dependent process [28], [30]C[32] that we and others have shown is paralleled by flow-induced Ca2+ influx arising from activation of the Ca2+-permeable TRPV4 channel, a noted mechanotransducer channel [33]C[36], that is highly expressed in the renal collecting duct cells [28], [31], [32]. However, whether the BK channel can fully account for the flow-induced K+ secretion or during.The first strand cDNA Synthesis kit (Roche) was used to synthesize all cDNAs. both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar degree by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is definitely functionally indicated in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca2+ influx, leading to elevated intracellular Ca2+ levels, activates this high Ca2+-affinity K+ channel. Further, with sites of manifestation localized to the apical cell membrane, especially in the CNT and CCD, SK3 is definitely poised to be a important pathway for Ca2+-dependent rules of membrane potential and K+ secretion. Intro Calcium-activated potassium channels, KCa, are a small group of potassium channels that are widely expressed in numerous tissues ranging from neurons to vascular endothelial cells [1]C[5]. As with additional K+ channels, the KCa channels can play a major part in regulating the plasma membrane electrical potential difference, Vm. However, their classical rules by intracellular Ca2+, [Ca2+]i, prospects to a highly dynamic coupling between Vm and [Ca2+]i which appears to underlie their central part in a WAY-600 wide array of functions ranging from neuronal excitability [6], [7], to modulation of vascular clean muscle firmness [8], [9], to cell volume rules [10], [11]. Indeed, depending on the types of KCa channels expressed by a particular cell type, the hyperpolarization of the cell membrane following Ca2+-induced activation of a given KCa channel can either enhance Ca2+ influx through non-voltage-activated, Ca2+-permeable channels, such as TRP channels, or reduce Ca2+ influx in the case of WAY-600 voltage-activated Ca2+ channels [4], [12]. To day, five subtypes of Ca2+-triggered K+ channels have been recognized: the large-conductance channel (BK, KCa1.1), the intermediate-conductance channel (IK1, KCa3.1), and three small-conductance channels (SK1, KCa2.1; SK2, KCa2.2; and SK3, KCa2.3) [1]C[3]. While the channels have similar structure (6C7 transmembrane segments, a pore loop region, and assembly as homo/heterotetramers), the gating mechanisms can differ, especially between BK and the additional channels. Indeed, BK is definitely gated by both membrane potential (activates with depoloarization) and intracellular Ca2+. Further, the Ca2+ binding sites in the C-terminus, the Ca2+ bowl, of the channel-forming -subunit of BK are characterized with a low Ca2+ binding affinity requiring high cytoplasmic levels of Ca2+ for activation (EC50?=?1C11 M; [13]C[15]); however, the Ca2+ affinity can be modulated by binding of selective BK subunits. In contrast, IK and SK channels are voltage insensitive. However, the IK/SK Ca2+ binding site is the ubiquitous Ca2+-sensor, calmodulin, constitutively bound to the C-terminus of the channel, which is characterized by a high Ca2+ binding affinity having a Ca2+ EC50 for gating near 300C600 nM [16]C[18]. As a consequence, the SK channels are highly sensitive Ca2+ detectors intimately linking [Ca2+]i to membrane potential and K+ efflux in all cells where these channels are indicated. In the mammalian kidney, K+ channels expressed in the luminal (apical) membrane of the late distal tubule and cortical collecting duct (CCD) are Ba2+-sensitive (blocker) channels that represent the dominating conductance of the apical membrane (observe [19], [20]). Hence, the underlying channels serve as important K+ secretory pathways which regulate K+ excretion and, hence, K+ homeostatis [21]C[24]. It has been demonstrated the ROMK channel (Kir1.1), an inward rectifier K+ channel from your Kir family, is the resting, Ba2+-sensitive, channel responsible for K+ secretion less than normal physiological conditions [5], [25]C[27]. Under stimulated states, however, it is becoming apparent that additional K+ channels can contribute to K+ secretion. Indeed, it has been demonstrated that elevated circulation rates to the late distal tubule or the CCD prospects to enhanced K+ secretion via activation of the luminal BK channel giving rise to the trend of flow-dependent K+ secretion [24], [28], [29]. This is a Ca2+-dependent process [28], [30]C[32] that we and others have shown is definitely paralleled by flow-induced Ca2+ influx arising from activation of the Ca2+-permeable TRPV4 channel, a mentioned mechanotransducer channel [33]C[36], that is highly indicated in the renal collecting duct cells [28], [31], [32]. However, whether the BK channel can fully account for the flow-induced.