Dan R. Halm

Activation of the basolateral membrane Cl − conductance essential for electrogenic K + secretion suppresses electrogenic Cl − secretion

Adrenaline activates transient Cl− secretion and sustained K+ secretion across isolated distal colonic mucosa of guinea‐pigs. The Ca2+‐activated Cl− channel inhibitor CaCCinh‐A01 (30 μm) significantly reduced electrogenic K+ secretion, detected as short‐circuit current (Isc). This inhibition supported the cell model for K+ secretion in which basolateral membrane Cl− channels provide an exit pathway for Cl− entering the cell via Na+–K+–2Cl− cotransporters. CaCCinh‐A01 inhibited both Isc and transepithelial conductance in a concentration‐dependent manner (IC50= 6.3 μm). Another Cl− channel inhibitor, GlyH‐101, also reduced sustained adrenaline‐activated Isc (IC50= 9.4 μm). Adrenaline activated whole‐cell Cl− current in isolated intact colonic crypts, confirmed by ion substitution. This adrenaline‐activated whole‐cell Cl− current was also inhibited by CaCCinh‐A01 or GlyH‐101. In contrast to K+ secretion, CaCCinh‐A01 augmented the electrogenic Cl− secretion activated by adrenaline as well as that activated by prostaglandin E2. Synergistic Cl− secretion activated by cholinergic/prostaglandin E2 stimulation was insensitive to CaCCinh‐A01. Colonic expression of the Ca2+‐activated Cl− channel protein Tmem16A was supported by RT‐PCR detection of Tmem16A mRNA, by immunoblot with a Tmem16A antibody, and by detection of immunofluorescence in lateral membranes of epithelial cells. Alternative splices of Tmem16A were detected for exons that are involved in channel activation. Inhibition of K+ secretion and augmentation of Cl− secretion by CaCCinh‐A01 support a common colonic cell model for these two ion secretory processes, such that activation of basolateral membrane Cl− channels contributes to the production of electrogenic K+ secretion and limits the rate of Cl− secretion. Maximal physiological Cl− secretion occurs only for synergistic activation mechanisms that close these basolateral membrane Cl− channels.

Role of the BK channel (KCa1.1) during activation of electrogenic K+ secretion in guinea pig distal colon.

Secretagogues acting at a variety of receptor types activate electrogenic K(+) secretion in guinea pig distal colon, often accompanied by Cl(-) secretion. Distinct blockers of K(Ca)1.1 (BK, Kcnma1), iberiotoxin (IbTx), and paxilline inhibited the negative short-circuit current (I(sc)) associated with K(+) secretion. Mucosal addition of IbTx inhibited epinephrine-activated I(sc) ((epi)I(sc)) and transepithelial conductance ((epi)G(t)) consistent with K(+) secretion occurring via apical membrane K(Ca)1.1. The concentration dependence of IbTx inhibition of (epi)I(sc) yielded an IC(50) of 193 nM, with a maximal inhibition of 51%. Similarly, IbTx inhibited (epi)G(t) with an IC(50) of 220 nM and maximal inhibition of 48%. Mucosally added paxilline (10 μM) inhibited (epi)I(sc) and (epi)G(t) by ∼50%. IbTx and paxilline also inhibited I(sc) activated by mucosal ATP, supporting apical K(Ca)1.1 as a requirement for this K(+) secretagogue. Responses to IbTx and paxilline indicated that a component of K(+) secretion occurred during activation of Cl(-) secretion by prostaglandin-E(2) and cholinergic stimulation. Analysis of K(Ca)1.1α mRNA expression in distal colonic epithelial cells indicated the presence of the ZERO splice variant and three splice variants for the COOH terminus. The presence of the regulatory β-subunits K(Ca)β1 and K(Ca)β4 also was demonstrated. Immunolocalization supported the presence of K(Ca)1.1α in apical and basolateral membranes of surface and crypt cells. Together these results support a cellular mechanism for electrogenic K(+) secretion involving apical membrane K(Ca)1.1 during activation by several secretagogue types, but the observed K(+) secretion likely required the activity of additional K(+) channel types in the apical membrane.

Secretory Control of Basolateral Membrane Potassium and Chloride Channels in Colonic Crypt Cells

Fluid secretion across epithelia generally is driven by the active secretion of ions and serves to lubricate surfaces and propel macromolecules such as mucus. 2 In the colonic epithelium, secretion of Cl and K is activated to drive water movement into the lumen. Several types of signals act to initiate this secretion, including nerve activity and paracrine release from cells in the intestine. Two potent stimulators of this Cl and K secretion are cholinergic nerves and prostaglandins released from cells in the intestinal mucosa and muscle. K secretion without concurrent Cl secretion also is stimulated through these routes, specifically by epinephrine acting via -adrenergic receptors and by prostaglandin-E2 acting via prostanoid EP2 receptors. 3, 4

Physiologic Influences of Transepithelial K+ Secretion

Cellular ionic balance relies on ion channels and coupled transporters to maintain and use the transmembrane electrochemical gradients of the cations Na+ and K+. High intracellular K+ concentration provides a ready reserve within the body allowing epithelia to secrete K+ into the fluid covering the apical membrane in the service of numerous physiologic activities. A major role for transepithelial K+ secretion concerns the balance of total body K+ such that excretion of excess K+ in the diet safeguards against disturbances to cellular balance. Accomplishing this transepithelial flow involves two archetypical cellular mechanisms, Na+ absorption and Cl− secretion. Ion channels for K+, Na+, and Cl−, as well as cotransporters, exchangers, and pumps contribute to produce transepithelial flow by coupling electrochemical gradients such that K+ flow enters across the basolateral membrane and exits through the apical membrane. Beyond excretion, transepithelial K+ secretion serves to create the high K+ concentration of endolymph in the inner ear that supports sensation of sound and body orientation. For several epithelia such as those in airways and gastric mucosa, the elevated K+ concentration of apical fluid may occur largely as a consequence of supporting the secretion of other ions such as Cl−or H+. Less well-appreciated consequences of K+ secretion may result as in saliva and colonic luminal fluid where a high K+ concentration likely influences interactions with the resident microbiome. Independent control of K+ secretion also allows for specific adjustments in rate that serve the physiology of organs large and small.

Activation of TRPV4 stimulates transepithelial ion flux in a porcine choroid plexus cell line

The choroid plexus (CP) epithelium plays a major role in the production of cerebrospinal fluid (CSF). A polarized cell line, the porcine CP-Riems (PCP-R) line, which exhibits many of the characteristics of the native epithelium, was used to study the effect of activation of the transient receptor potential vanilloid 4 (TRPV4) cation channel found in the PCP-R cells as well as in the native epithelium. Ussing-style electrophysiological experiments showed that activation of TRPV4 with a specific agonist, GSK1016790A, resulted in an immediate increase in both transepithelial ion flux and conductance. These changes were inhibited by either of two distinct antagonists, HC067047 or RN1734. The change in conductance was reversible and did not involve disruption of epithelial junctional complexes. Activation of TRPV4 results in Ca2+ influx, therefore, we examined whether the electrophysiological changes were the result of secondary activation of Ca2+-sensitive channels. PCP-R cells contain two Ca2+-activated K+ channels, the small conductance 2 (SK2) and the intermediate conductance (IK) channels. Based on inhibitor studies, the former is not involved in the TRPV4-mediated electrophysiological changes whereas one of the three isoforms of the IK channel (KCNN4c) may play a role in the apical secretion of K+. Blocking the activity of this IK isoform with TRAM34 inhibited the TRPV4-mediated change in net transepithelial ion flux and the increased conductance. These studies implicate TRPV4 as a hub protein in the control of CSF production through stimulation by multiple effectors resulting in transepithelial ion and subsequent water movement.

Survival and growth of C57BL/6J mice lacking the BK channel, Kcnma1: lower adult body weight occurs together with higher body fat

Big conductance potassium (BK) channels contribute to K+ flow and electrical behavior in many cell types. Mice made null for the gene (Kcnma1) producing the BK channel (BKKO) exhibit numerous deficits in physiological functions. Breeding mice lacking a single allele of Kcnma1 (C57BL/6J background) had litter sizes of approximately eight pups. For the period of maternal care (P0–P21), pup deaths peaked at P1 with a second less severe interval of death peaking near P13. Early deaths were twice as likely during a 20‐month period of building construction compared with the quiescent period after cessation of construction. Births during construction were not consistent with Mendelian predictions indicating the likelihood of a specific disadvantage induced by this environmental stressor. Later BKKO pup deaths (~P13) also were more numerous than Mendelian expectations. After weaning, weight gain was slower for BKKO mice compared with wild‐type littermates: 5 g less for male BKKO mice and 4 g less for female BKKO mice. Body composition determined by quantitative magnetic resonance indicated a higher fat proportion for wild‐type female mice compared with males, as well as a higher hydration ratio. Both male and female BKKO mice showed higher fat proportions than wild‐type, with female BKKO mice exhibiting greater variation. Together, these results indicate that BKKO mice suffered disadvantages that lead to prenatal and perinatal death. A metabolic difference likely related to glucose handling led to the smaller body size and distinct composition for BKKO mice, suggesting a diversion of energy supplies from growth to fat storage.