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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.

SK4/IK1 encodes an intermediate conductance, Ca2+-activated K+ channel and fulfills a variety of physiological functions in excitable and nonexcitable cells. Although recent studies have provided evidence for the presence of SK4/IK1 channels in salivary acinar cells, the regulatory mechanisms and the physiological function of the channel remain unknown in these cells. Using molecular and electrophysiological techniques, we examined whether cytosolic ATP-dependent regulation of native SK4/IK1-like channel activity would involve endogenous cAMP-dependent protein kinase (PKA) in rat submandibular acinar (RSA) cells. Electrophysiological properties of tetraethylammonium (TEA) (10 mM)-insensitive, Ca2+-dependent K+ currents in macropatches excised from RSA cells matched those of whole cell currents recorded from human embryonic kidney-293 cells heterologously expressing rat SK4/IK1 (rSK4/IK1) cloned from RSA cells. In outside-out macropatches, activity of native SK4/IK1-like channels, defined as a charybdotoxin (100 nM)-blockable current in the presence of TEA (10 mM) in the bathing solution, ran down unless both ATP and Mg2+ were present in the pipette solution. The nonhydrolyzable ATP analog AMP-PNP failed to support the channel activity as ATP did. The addition of Rp-cAMPS (10 μM), a PKA inhibitor, to the pipette solution containing ATP/Mg2+ induced a rundown of the Ca2+-dependent K+ currents. Inclusion of cAMP (1 mM) into the pipette solution (1 μM free Ca2+) containing ATP/Mg2+ caused a gradual increase in the currents, the effect being pronounced for the currents induced by 0.1 μM free Ca2+. Forskolin (1 μM), an adenylyl cyclase activator, also increased the currents induced by 0.1 μM free Ca2+. In inside-out macropatches, cytosolic ATP/Mg2+ increased both the maximum current (proportional to the maximum channel activity) and Ca2+ sensitivity of current activation. Collectively, these results suggest that ATP-dependent regulation of native SK4/IK1-like channels, at least in part, is mediated by endogenous PKA in RSA cells.

Although Ca2+-activated K+(KCa) channels distinct from maxi-K+channels have been suggested to contribute to muscarinically stimulated K+currents in salivary acinar cells, the molecular nature of the channels is unclear. Using electrophysiological and RT-PCR techniques, we have now investigated the involvement of SK4/IK1-like channels in native KCacurrents in bovine parotid acinar (BPA) cells. Ca2+-dependent K+efflux from perfused bovine parotid tissues was not inhibited by a maxi-K+channel blocker, tetraethylammonium (TEA). Whole cell recordings from BPA cells showed a TEA-insensitive KCaconductance, which was highly permeable to Rb+. In inside-out macropatches, TEA-insensitive Rb+currents were activated by Ca2+with half-maximal values of 0.4 μM. 1-Ethyl-2-benzimidazolinone (1-EBIO) increased the Ca2+sensitivity of the currents. The calmodulin antagonists trifluoperazine, calmidazolium, and W-7 inhibited the Ca2+-activated Rb+currents. In outside-out macropatches, Ca2+-activated Rb+currents were inhibited by Ba2+, quinine, clotrimazole, and charybdotoxin but not by d-tubocrarine or apamin. RT-PCR analysis showed transcripts of SK4/IK1 in BPA cells. These results collectively suggest that SK4/IK1-like channels mediate the native KCacurrents in BPA cells.