Molecular and Functional Expression of High Conductance Ca^2+ Activated K^+ Channels in the Eel Intestinal Epithelium

Abstract

Several types of K⁺ channels have been identified in epithelial cells. Among them high conductance Ca²⁺-activated K⁺ channels (BK channels) are of relevant importance for their involvement in regulatory volume decrease (RVD) response following hypotonic stress. The aim of the present work was to investigate the functional and molecular expression of BK in the eel intestine, which is a useful experimental model for cell volume regulation research. In the present paper using rat BK channel-specific primer, a RT-PCR signal of 696 pb cDNA was detected in eel intestine, whole nucleotide sequence showed high similarity (83%) to the alpha subunit of BK channel family. BK channel protein expression was verified by immunoblotting and confocal microscopy, while the functional role of BK channels in epithelial ion transport mechanisms and cell volume regulation was examined by electrophysiological and morphometric analysis on the intact tissue. BK_Ca channels appeared to be localized along all the plasma membrane of the enterocytes; the apical part of the villi showed the most intense immunostaining. These channels were silent in basal condition, but were activated on both membranes (apical and basolateral) by increasing intracellular Ca²⁺ concentration with the Ca²⁺ ionophore ionomycin (1µM). BK_Ca channels were also activated on both membranes by hypotonic swelling of the epithelium and their inhibition by 100 nM iberiotoxin (specific BK_Ca inhibitor) abolished the Regulatory Volume Decrease (RVD) of the intestinal cells after hypotonic swelling. In conclusion, our results demonstrated the molecular and functional expression of high conductance Ca²⁺ -activated K⁺ channels in eel intestine; the physiological role of these channels is mainly related to the RVD response of the epithelial cells following hypotonic swelling.

Cell Volume Regulation and Apoptotic Volume Decrease in Rat Distal Colon Superficial Enterocytes

“…The involvement of other types of Ca 2+ activated K + channels in RVD was ruled out by the use of clotrimazole and apamine, which were ineffective on the RVD response. BK channels appeared expressed on both membranes by confocal immunofluorescence microscopy as already found in rabbit colonic surface epithelium [54], eel intestine [26], and guinea pig distal colon [55]. Interestingly, they showed a major expression in superficial epithelium with respect to crypts, as previously observed in human colon [56].…”

“…In order to define the precise localization of BK channels in rat distal colonic epithelium, colon explants were fixed in paraformaldehyde diluted to 2% in Hepes-Tris solution from a stock solution of 20% paraformaldehyde in TBS containing (in mM): NaCl 150, Tris 10, MgCl 2 1, EGTA 1 adjusted to pH 7.4 with NaOH, as previously described [26]. Sections of 10 μm thickness were cut along planes perpendicular to the luminal epithelium surface through the use of a microtome (MICROM HM 500) and placed on poly-L-lysine coated glass slides.…”

“…The cell volume regulation mechanisms are highly conserved and principally similar in cells from various tissues as well as between evolutionary distant species [22,23,24,25,26,27]. Following a hypotonic cell swelling, they consist in the release of osmolytes, mainly K + and Cl - , followed by loss of osmotically obliged water and are termed Regulatory Volume Decrease (RVD); following a hypertonic shrinkage they consist in the uptake of osmolytes followed by gain of osmotically obliged water and are termed Regulatory Volume Increase (RVI) [28,27].…”

Physiology of Cell Volume Regulation in Vertebrates

“…Expression studies in oocytes indicated that BK channels were only modestly volume sensitive (305) and that the volume sensitivity seems in many cases to be secondary to an increase in [Ca 2ϩ ] i during cell swelling (107,228). In eel intestinal epithelium, it was recently shown that swelling activation of BK channels was dependent on an increase in [Ca 2ϩ ] i , but that the resulting current was larger than accounted for by the increased [Ca 2ϩ ] i (574), ostensibly due to BK modulation by protein phosphorylation events (107). In some cases, BK channels have been found to be directly stretch-activated (450), which in principle could also account for their swelling activation (see, however, sect.…”

“…VIB). Moreover, pharmacological evidence indicates that apical and basolateral BK channels also play impor-tant roles in the RVD response of the eel intestinal epithelium (574). In the killifish opercular epithelium, osmotic control of Cl Ϫ secretion across the operculum epithelium was proposed, albeit based on colocalization and pharmacological evidence only, to involve the following events: 1) hyperosmotic activation of NKCC1 on the basolateral membrane via PKC, MLCK (362), p38 MAPK, OSR1, and SPAK (624); 2) deactivation of NKCC by hypotonic cell swelling and a protein phosphatase (624); and 3) a protein tyrosine kinase acting on FAK, which seems to have an important, not yet defined function in regulating NKCC activity (624).…”