Emerging Role of the Calcium-Activated, Small Conductance, SK3 K + Channel in Distal Tubule Function: Regulation by TRPV4 Jonathan Berrout 1 , Mykola Mamenko 1 , Oleg L. Zaika 1 , Lihe Chen 2 , Wenzheng Zang 2 , Oleh Pochynyuk 1 , Roger G. O’Neil 1 * 1 Department of Integrative Biology, The University of Texas Health Science Center Medical School, Houston, Texas, United States of America, 2 Department of Internal Medicine-Division of Renal Diseases and Hypertension, The University of Texas Health Science Center Medical School, Houston, Texas, United States of America Abstract The Ca 2+ -activated, maxi-K (BK) K + channel, with low Ca 2+ -binding affinity, is expressed in the distal tubule of the nephron and contributes to flow-dependent K + secretion. In the present study we demonstrate that the Ca 2+ -activated, SK3 (K Ca 2.3) K + channel, with high Ca 2+ -binding affinity, is also expressed in the mouse kidney (RT-PCR, immunoblots). Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K + secretion, the highest levels of expression were along the apical (luminal) cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca 2+ -dependent K + secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca 2+ -permeable TRPV4 channel, thereby inducing Ca 2+ influx and elevating intracellular Ca 2+ levels, activated both the SK3 channel 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 Ca 2+ influx, leading to elevated intracellular Ca 2+ levels, activates this high Ca 2+ -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 Ca 2+ -dependent regulation of membrane potential and K + secretion. Citation: Berrout J, Mamenko M, Zaika OL, Chen L, Zang W, et al. (2014) Emerging Role of the Calcium-Activated, Small Conductance, SK3 K + Channel in Distal Tubule Function: Regulation by TRPV4. PLoS ONE 9(4): e95149. doi:10.1371/journal.pone.0095149 Editor: Michael B. Butterworth, University of Pittsburgh, School of Medicine, United States of America Received October 14, 2013; Accepted March 24, 2014; Published April 24, 2014 Copyright: ß 2014 Berrout et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: NIH Grant R01 DK070950 (to R.G.O.); NIH Grant R01 DK095029 (to O.P.); NIH Grant R01 DK080236 (to W.Z.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: roger.g.oneil@uth.tmc.edu Introduction Calcium-activated potassium channels, K Ca , are a small group of potassium channels that are widely expressed in numerous tissues ranging from neurons to vascular endothelial cells [1–5]. As with other K + channels, the K Ca channels can play a major role in regulating the plasma membrane electrical potential difference, Vm. However, their classical regulation by intracellular Ca 2+ , [Ca 2+ ] i , leads to a highly dynamic coupling between Vm and [Ca 2+ ] i which appears to underlie their central role in a wide array of functions ranging from neuronal excitability [6,7], to modula- tion of vascular smooth muscle tone [8,9], to cell volume regulation [10,11]. Indeed, depending on the types of K Ca channels expressed by a particular cell type, the hyperpolarization of the cell membrane following Ca 2+ -induced activation of a given K Ca channel can either enhance Ca 2+ influx through non-voltage- activated, Ca 2+ -permeable channels, such as TRP channels, or reduce Ca 2+ influx in the case of voltage-activated Ca 2+ channels [4,12]. To date, five subtypes of Ca 2+ -activated K + channels have been identified: the large-conductance channel (BK, K Ca 1.1), the intermediate-conductance channel (IK1, KCa3.1), and three small-conductance channels (SK1, K Ca 2.1; SK2, K Ca 2.2; and SK3, K Ca 2.3) [1–3]. While the channels have similar structure (6– 7 transmembrane segments, a pore loop region, and assembly as homo/heterotetramers), the gating mechanisms can differ, espe- cially between BK and the other channels. Indeed, BK is gated by both membrane potential (activates with depoloarization) and intracellular Ca 2+ . Further, the Ca 2+ binding sites in the C- terminus, the ‘‘Ca 2+ bowl,’’ of the channel-forming a-subunit of BK are characterized with a low Ca 2+ binding affinity requiring high cytoplasmic levels of Ca 2+ for activation (EC 50 = 1–11 mM; [13–15]); however, the Ca 2+ affinity can be modulated by binding of selective BK b subunits. In contrast, IK and SK channels are voltage insensitive. However, the IK/SK Ca 2+ binding site is the ubiquitous Ca 2+ -sensor, calmodulin, constitutively bound to the C- terminus of the channel, which is characterized by a high Ca 2+ binding affinity with a Ca 2+ EC 50 for gating near 300–600 nM PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e95149