Physiological Roles of the Intermediate Conductance, Ca2+-activated Potassium Channel Kcnn4

Three broad classes of Ca2+-activated potassium channels are defined by their respective single channel conductances, i.e. the small, intermediate, and large conductance channels, often termed the SK, IK, and BK channels, respectively. SK channels are likely encoded by three genes, Kcnn1–3, whereas IK and most BK channels are most likely products of the Kcnn4 and Slo (Kcnma1) genes, respectively. IK channels are prominently expressed in cells of the hematopoietic system and in organs involved in salt and fluid transport, including the colon, lung, and salivary glands. IK channels likely underlie the K+ permeability in red blood cells that is associated with water loss, which is a contributing factor in the pathophysiology of sickle cell disease. IK channels are also involved in the activation of T lymphocytes. The fluid-secreting acinar cells of the parotid gland express both IK and BK channels, raising questions about their particular respective roles. To test the physiological roles of channels encoded by the Kcnn4 gene, we constructed a mouse deficient in its expression. Kcnn4 null mice were of normal appearance and fertility, their parotid acinar cells expressed no IK channels, and their red blood cells lost K+ permeability. The volume regulation of T lymphocytes and erythrocytes was severely impaired in Kcnn4 null mice but was normal in parotid acinar cells. Despite the loss of IK channels, activated fluid secretion from parotid glands was normal. These results confirm that IK channels in red blood cells, T lymphocytes, and parotid acinar cells are indeed encoded by the Kcnn4 gene. The role of these channels in water movement and the subsequent volume changes in red blood cells and T lymphocytes is also confirmed. Surprisingly, Kcnn4 channels appear to play no required role in fluid secretion and regulatory volume decrease in the parotid gland. Three broad classes of Ca2+-activated potassium channels are defined by their respective single channel conductances, i.e. the small, intermediate, and large conductance channels, often termed the SK, IK, and BK channels, respectively. SK channels are likely encoded by three genes, Kcnn1–3, whereas IK and most BK channels are most likely products of the Kcnn4 and Slo (Kcnma1) genes, respectively. IK channels are prominently expressed in cells of the hematopoietic system and in organs involved in salt and fluid transport, including the colon, lung, and salivary glands. IK channels likely underlie the K+ permeability in red blood cells that is associated with water loss, which is a contributing factor in the pathophysiology of sickle cell disease. IK channels are also involved in the activation of T lymphocytes. The fluid-secreting acinar cells of the parotid gland express both IK and BK channels, raising questions about their particular respective roles. To test the physiological roles of channels encoded by the Kcnn4 gene, we constructed a mouse deficient in its expression. Kcnn4 null mice were of normal appearance and fertility, their parotid acinar cells expressed no IK channels, and their red blood cells lost K+ permeability. The volume regulation of T lymphocytes and erythrocytes was severely impaired in Kcnn4 null mice but was normal in parotid acinar cells. Despite the loss of IK channels, activated fluid secretion from parotid glands was normal. These results confirm that IK channels in red blood cells, T lymphocytes, and parotid acinar cells are indeed encoded by the Kcnn4 gene. The role of these channels in water movement and the subsequent volume changes in red blood cells and T lymphocytes is also confirmed. Surprisingly, Kcnn4 channels appear to play no required role in fluid secretion and regulatory volume decrease in the parotid gland. It has become clear that multiple types of Ca2+-activated potassium channels underlie a wide range of distinct physiological processes. Physiological and pharmacological analyses have subdivided Ca2+-activated potassium channels into three groups, i.e. the small, intermediate, and large conductance channels, often termed SK, 1The abbreviations used are: SK, small potassium conductance; IK, intermediate potassium conductance; BK, large potassium conductance; MOPS, 4-morpholinepropanesulfonic acid; NS, normal saline; RVD, regulatory volume decrease. IK, and BK channels, respectively. IK channels, as their designation implies, have a single channel conductance intermediate between the SK and BK channels. These three functional groups have rather distinct pharmacological profiles, and SK, IK, and BK channels can be specifically blocked by apamin, clotrimazole, and paxilline, respectively. SK and IK channels are encoded by four genes of the KCNN gene family. The three SK channels, KCNN1–3, share ∼70–75% amino acid identity. The IK channel KCNN4 is encoded by a protein sharing only ∼40% amino acid identity with each of the three SK channels, KCNN1–3. KCNN4 channels are prominently expressed in cells of the hematopoietic system and in organs involved in salt and fluid transport, including the colon, lung, and salivary glands (1Joiner W.J. Wang L. Tang M.D. Kaczmarek L.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11013-11018Crossref PubMed Scopus (318) Google Scholar, 2Ishii T.M. Silvia C. Hirschberg B. Bond C.T. Adelman J.P. Maylie J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11651-11656Crossref PubMed Scopus (520) Google Scholar, 3Logsdon N.J. Kang J. Togo J.A. Christian E.P. Aiyar J. J. Biol. Chem. 1997; 272: 32723-32726Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 4Vandorpe D.H. Shmukler B.E. Jiang L. Lim B. Maylie J. Adelman J.P. de Franceschi L. Cappellini M.D. Brugnara C. Alper S.L. J. Biol. Chem. 1998; 273: 21542-21553Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 5Warth R. Hamm K. Bleich M. Kunzelmann K. von Hahn T. Schreiber R. Ullrich E. Mengel M. Trautmann N. Kindle P. Schwab A. Greger R. Pflugers Arch. 1999; 438: 437-444Crossref PubMed Scopus (117) Google Scholar). Heterologous expression of KCNN4 produces K+-selective, Ca+2-activated channels without time or voltage dependence (1Joiner W.J. Wang L. Tang M.D. Kaczmarek L.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11013-11018Crossref PubMed Scopus (318) Google Scholar, 2Ishii T.M. Silvia C. Hirschberg B. Bond C.T. Adelman J.P. Maylie J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11651-11656Crossref PubMed Scopus (520) Google Scholar, 3Logsdon N.J. Kang J. Togo J.A. Christian E.P. Aiyar J. J. Biol. Chem. 1997; 272: 32723-32726Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 4Vandorpe D.H. Shmukler B.E. Jiang L. Lim B. Maylie J. Adelman J.P. de Franceschi L. Cappellini M.D. Brugnara C. Alper S.L. J. Biol. Chem. 1998; 273: 21542-21553Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 6Nehrke K. Quinn C.C. Begenisich T. Am. J. Physiol. 2003; 284: C535-C546Crossref PubMed Scopus (56) Google Scholar). The expressed channels are sensitive to clotrimazole, but the reported affinity appears to depend on the expression system used (compare, for example, Refs. 2Ishii T.M. Silvia C. Hirschberg B. Bond C.T. Adelman J.P. Maylie J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11651-11656Crossref PubMed Scopus (520) Google Scholar and 3Logsdon N.J. Kang J. Togo J.A. Christian E.P. Aiyar J. J. Biol. Chem. 1997; 272: 32723-32726Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). These channels are also sensitive to inhibition by charybdotoxin, stichodactyla toxin, and maurotoxin but are insensitive to the SK blocker apamin and the BK blocker iberiotoxin. Red blood cells have a high but latent passive permeability to K+ ions that is mediated by what is often called the Gardos channel (7Gardos G. Biochim. Biophys. Acta. 1958; 30: 653-654Crossref PubMed Scopus (474) Google Scholar). This channel has all the properties of, and is likely encoded by, the KCNN4 gene (4Vandorpe D.H. Shmukler B.E. Jiang L. Lim B. Maylie J. Adelman J.P. de Franceschi L. Cappellini M.D. Brugnara C. Alper S.L. J. Biol. Chem. 1998; 273: 21542-21553Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 8Hoffman J.F. Joiner W. Nehrke K. Potapova O. Foye K. Wickrema A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7366-7371Crossref PubMed Scopus (102) Google Scholar). Increased water loss from red blood cells secondary to increased K+ permeability is a contributing factor in the pathophysiology of sickle cell disease. The resulting increase in the intracellular concentration of hemoglobin S leads to a shortened delay time for the polymerization of deoxyhemoglobin S. Thus, blocking erythrocyte K+ permeability and the consequent dehydration is under evaluation as a potential therapy for sickle cell disease. Indeed, blocking the red blood cell IK channel with clotrimazole prevents erythrocyte dehydration in patients with sickle cell disease (9Brugnara C. Gee B. Armsby C.C. Kurth S. Sakamoto M. Rifai N. Alper S.L. Platt O.S. J. Clin. Investig. 1996; 97: 1227-1234Crossref PubMed Scopus (207) Google Scholar, 10Brugnara C. De Franceschi L. Bennekou P. Alper S.L. Christophersen P. Drug News Perspect. 2001; 14: 208-220Crossref PubMed Scopus (16) Google Scholar) and in a mouse model of sickle cell disease (11De Franceschi L. Saadane N. Trudel M. Alper S.L. Brugnara C. Beuzard Y. J. Clin. Investig. 1994; 93: 1670-1676Crossref PubMed Scopus (127) Google Scholar, 12Stocker J.W. De Franceschi L. McNaughton-Smith G.A. Corrocher R. Beuzard Y. Brugnara C. Blood. 2003; 101: 2412-2418Crossref PubMed Scopus (168) Google Scholar). The KCNN4 gene also likely encodes the Ca2+-activated, intermediate conductance K+ channel expressed in T lymphocytes (3Logsdon N.J. Kang J. Togo J.A. Christian E.P. Aiyar J. J. Biol. Chem. 1997; 272: 32723-32726Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). This channel is expressed at low levels in human resting T cells and is strongly up-regulated during activation by mitogens (13Ghanshani S. Wulff H. Miller M.J. Rohm H. Neben A. Gutman G.A. Cahalan M.D. Chandy K.G. J. Biol. Chem. 2000; 275: 37137-37149Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). The role of the IK channel in lymphocytes appears to be the maintenance of a hyperpolarized membrane potential, thus facilitating and maintaining the intracellular Ca2+ levels required for mitogen activation (14Fanger C.M. Rauer H. Neben A.L. Miller M.J. Wulff H. Rosa J.C. Ganellin C.R. Chandy K.G. Cahalan M.D. J. Biol. Chem. 2001; 276: 12249-12256Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 15Wulff H. Beeton C. Chandy K.G. Curr. Opin. Drug Discovery Devel. 2003; 6: 640-647PubMed Google Scholar). The cell shrinkage that correlates with the initiation of apoptosis in T lymphocytes is specifically blocked by clotrimazole, suggesting that IK1 may also be involved in early events associated with apoptosis in this cell type (16Elliott J.I. Higgins C.F. EMBO Rep. 2003; 4: 189-194Crossref PubMed Scopus (65) Google Scholar). In most fluid-secreting exocrine epithelia, the acinar cell cytoplasmic Cl– concentration is maintained above electrochemical equilibrium by the Na-K-2Cl transporter. The exit of Cl– into the lumen is balanced by the movement of Na+ ions through the interstitial tight junctions into the lumen. This elevated salt concentration draws water into the lumen and, after modification by the duct cells, leads to a secreted fluid rich in NaCl. To maintain fluid secretion, the Cl– efflux into the lumen must be sustained. In the absence of any other active channels, the acinar cell membrane potential would approach the Cl– equilibrium potential and net Cl– movement would cease. Both exocrine and other fluid-secreting epithelia often express K+ channels whose activity can facilitate Cl– efflux by maintaining a hyperpolarized membrane voltage. Rodent (and other) salivary glands express two types of Ca2+-activated potassium channels, a BK channel and an IK channel (17Hayashi T. Young J.A. Cook D.I. J. Membr. Biol. 1996; 151: 19-27Crossref PubMed Scopus (20) Google Scholar, 18Maruyama Y. Nishiyama A. Teshima T. Jpn. J. Physiol. 1986; 36: 219-223Crossref PubMed Scopus (10) Google Scholar, 19Wegman E.A. Ishikawa T. Young J.A. Cook D.I. Am. J. Physiol. 1992; 263: G786-G794PubMed Google Scholar, 20Ishikawa T. M. Y. Pflugers Arch. 1994; PubMed Scopus Google Scholar, K. Arch. Biol. 2001; PubMed Scopus Google Scholar, T. M. Ishikawa T. Am. J. Physiol. 2003; 284: PubMed Google Scholar). These BK and IK channels have and pharmacological properties with of channels encoded by the Slo (Kcnma1) and Kcnn4 genes, K. Quinn C.C. Begenisich T. Am. J. Physiol. 2003; 284: C535-C546Crossref PubMed Scopus (56) Google Scholar, T. M. Ishikawa T. Am. J. Physiol. 2003; 284: PubMed Google Scholar). Both channels are activated by the increase in intracellular Ca2+ by but the that fluid secretion may be of BK channel activity (17Hayashi T. Young J.A. Cook D.I. J. Membr. Biol. 1996; 151: 19-27Crossref PubMed Scopus (20) Google Scholar, 20Ishikawa T. M. Y. Pflugers Arch. 1994; PubMed Scopus Google Scholar, T. P. Young J.A. Cook D.I. J. Membr. Biol. 1996; PubMed Scopus Google IK channels to play this Kcnn4 is to play roles in the volume regulation of blood cells as lymphocytes H. Beeton C. Chandy K.G. Curr. Opin. Drug Discovery Devel. 2003; 6: 640-647PubMed Google Scholar) and erythrocytes C. De Franceschi L. Bennekou P. Alper S.L. Christophersen P. Drug News Perspect. 2001; 14: 208-220Crossref PubMed Scopus (16) Google which the of the In lymphocytes, of cytoplasmic Ca2+ which the of gene expression the of In sickle disease erythrocytes the of Kcnn4 cell shrinkage and the increased concentration of hemoglobin to the of deoxyhemoglobin S in cell and the of To test for the physiological roles of IK channels, we have the Kcnn4 gene in mouse by mice this normal and were the loss of functional IK1 channels in T lymphocytes, and parotid acinar cells, the for this channel the expression of the Kcnn4 gene impaired the volume regulation of T lymphocytes and erythrocytes but that of parotid acinar cells. Despite the loss of IK channels, activated fluid secretion from the parotid glands was normal. for was constructed the which the gene by the mouse IK1 was from an a to and and The was as a by low from the IK1 The was from the as a were for The was into the and the was into the and The between the in the The was with and into cells. of which was and into mice were to from the resulting were used to and null for all of the subsequent were by the of the of and was from of the was on with to the In products were the which both of the in of these products the that the has at the in the mouse IK1 of mice for functional was salivary glands were from and T cells were the under the T the or was used to from all was of or of were on a and The were with a for the Kcnn4 the in and at and the in the are on and S or an cells were from and by as in K. Quinn C.C. Begenisich T. Am. J. Physiol. 2003; 284: C535-C546Crossref PubMed Scopus (56) Google Scholar, C.M. L. T. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google with In mice were by to and by The parotid glands were in a of and at was with in and for were by by with with To single cells, the was in and for and as above in the of a acinar cells were with in with for and were to a on the of a and with salt and with at The was by a volume of The was on an with a system and at were at at and volume changes were expressed as as reported C.M. L. T. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). of the of cell volume were the early of volume were to the In were by of to the from the and parotid glands with the of a The was and to a during the was maintained at a The of the were into was by of was for and at the of the blood was by into and mice were by to glands were and of under a was expressed as of gland and potassium were by activity was an was with a concentration was by a protein were at with an was a by a used an that of potassium and which a Ca2+ concentration of C. R. Biol. 1994; PubMed Scopus Google Scholar) also The of potassium and T lymphocytes were a mice were by to and were in and and through a cell The resulting cell was through a cell and at and for were in and a and for at were for an at the was and T lymphocytes were on were on in the volume was a and volume were from and expressed in into mouse erythrocytes was a of (11De Franceschi L. Saadane N. Trudel M. Alper S.L. Brugnara C. Beuzard Y. J. Clin. Investig. 1994; 93: 1670-1676Crossref PubMed Scopus (127) Google Scholar, A. Brugnara C. Am. J. Physiol. 1999; PubMed Google Scholar). blood was in of at and in normal and at with a of in with and in the absence or of The was by the of or to the erythrocyte of cell were at of and These were to of of were at for of the and of the the were and in a was from the from the at and and expressed in of cells was expressed as the of in the of erythrocytes three in were to in by at in the absence or of and for at of cell were to of the by of by of with water of were at and of each were for of at were defined as for The mouse Kcnn4 gene was by in cells. was constructed with and of Kcnn4 were by and a was into was through and analyses of from the of the mice for the of Kcnn4 of were were and were Kcnn4 null and of the of respectively. This is to the normal of and The mice to mice of both were of and from their are as with with with with In the of the parotid glands from null mice was that from with and with was in but with with of any between and IK1 and T shrinkage of T lymphocytes, which can be specifically blocked by clotrimazole (16Elliott J.I. Higgins C.F. EMBO Rep. 2003; 4: 189-194Crossref PubMed Scopus (65) Google Scholar). The in the expression of IK1 in lymphocytes from mice This is in Kcnn4 null of that the Ca2+ T lymphocytes from mice to In the shrinkage of lymphocytes from Kcnn4 null was the channel blocker clotrimazole the cell shrinkage in lymphocytes IK1 and Red red blood cells have a Ca2+-activated K+ permeability to be mediated by the Gardos channel (7Gardos G. Biochim. Biophys. Acta. 1958; 30: 653-654Crossref PubMed Scopus (474) Google Scholar). The in that this permeability is indeed encoded by the Kcnn4 gene. The large into red blood cells from was in the Kcnn4 also the of the Gardos channel to red blood cell volume by the of on for red cells. the of red cell from at the in the absence and of which was used to increase intracellular Ca2+ (11De Franceschi L. Saadane N. Trudel M. Alper S.L. Brugnara C. Beuzard Y. J. Clin. Investig. 1994; 93: 1670-1676Crossref PubMed Scopus (127) Google Scholar, J.A. Brugnara C. A. J. Clin. Investig. PubMed Scopus Google activation of the K+ permeability consequent cell cells from In the absence of of the cells was to of potassium channels to the at which of the cells In the at which red cells from Kcnn4 null was by but a increase IK1 and parotid acinar cells express two types of Ca2+-activated potassium channels K. Quinn C.C. Begenisich T. Am. J. Physiol. 2003; 284: C535-C546Crossref PubMed Scopus (56) Google Scholar). type has time or voltage dependence and is blocked by The type has voltage and time dependence and is blocked by of these from a parotid acinar cell with a Ca2+ are in in the are from to and from a potential of are at all an was activated the of large The of the of the at the of the voltage This is of and These are as by the The is blocked by clotrimazole 6Nehrke K. Quinn C.C. Begenisich T. Am. J. Physiol. 2003; 284: C535-C546Crossref PubMed Scopus (56) Google Scholar). have that the and of the parotid Ca2+-activated K+ is to expression of the Kcnn4 gene K. Quinn C.C. Begenisich T. Am. J. Physiol. 2003; 284: C535-C546Crossref PubMed Scopus (56) Google Scholar). The in this The expression of IK1 in the parotid gland from mice This is in Kcnn4 null and is of a in mice for the null the and of the Ca2+-activated K+ in in to the expression of this in the Kcnn4 null of that this was indeed the The of from a parotid acinar cell from a Kcnn4 null mouse under to for B. The of the of at the of the as in The and in the cell from the in are in the from the Kcnn4 null To a of IK1 channel in and Kcnn4 null we the after the to also cell to for cell in cells from was and was in cells from Kcnn4 null an cell of this is to a of this is the for normal a is that all of the parotid acinar and Ca2+-activated K+ was in the Kcnn4 null of the of the and in an increase in this in the Kcnn4 null as in for the loss of the IK1 channels. of this at this time is as we have an decrease in the of the as the after the of the This is under active in the we have these two by the in cells from after and to of the IK1 The was in cells and in cells from Kcnn4 null a Thus, in the Kcnn4 null or the of the between the two types of channels this is at this To test the role of IK1 in salivary fluid secretion in we in parotid The results from are in volume from and Kcnn4 null mice was a the expression of IK1 channels or no in also the of the by with was or no in and of between and Kcnn4 null mice as in as in the was or no in the also the of protein secreted in the protein in from and in from Kcnn4 null the of all increased in the Kcnn4 null of these was cells, including in parotid can their volume in to changes in volume decrease is a in cell volume in to the cell by a in in the of parotid acinar cells in to a in In blocking IK1 channels with clotrimazole a on RVD, but blocking channels with the by a factor of These results or no role for IK1 channels in in parotid acinar cells. This was by of in Kcnn4 null mice the of the Kcnn4 null mice was to the in IK1 channels to the the would be in the Kcnn4 null only was the was in cells from the Kcnn4 null an increase in be the of an of other K+ efflux in the Kcnn4 null The small of the of the volume increase with that in is with this as is the of in the Kcnn4 In these the by a factor of with the in The in red cells of a latent K+ permeability activated by the of intracellular was in (7Gardos G. Biochim. Biophys. Acta. 1958; 30: 653-654Crossref PubMed Scopus (474) Google Scholar) and was to Ca2+-activated K+ channel activity of intermediate with charybdotoxin, clotrimazole, and small of have that the Ca2+-activated K+ conductance of the red cell is mediated by channels. has by in cells B. Bond C. S. A. Maylie J. Adelman J.P. Alper S.L. Biochim. Biophys. Acta. 2001; PubMed Scopus Google Scholar) and in human B. R. J. M. R. 2001; PubMed Scopus Google Scholar). The that the Kcnn4 gene is for all Ca2+-activated K+ permeability in the erythrocyte of the This is by the of and by the absence of to in Kcnn4 null red cells. the of shrinkage of red cells from Kcnn4 null as by and the of K+ channel activity as by and IK1 activity is elevated in red cells from sickle disease patients and in red cells from mouse of sickle disease C. De Franceschi L. Bennekou P. Alper S.L. Christophersen P. Drug News Perspect. 2001; 14: 208-220Crossref PubMed Scopus (16) Google Scholar). and water loss from red cells is by the elevated levels of that Thus, the Kcnn4 null mouse an to test the role of IK1 as a in mouse of sickle disease. It also an to test the of IK1 which are under for the pharmacological of sickle disease J.W. De Franceschi L. McNaughton-Smith G.A. Corrocher R. Beuzard Y. Brugnara C. Blood. 2003; 101: 2412-2418Crossref PubMed Scopus (168) Google Scholar). The of the shrinkage in T lymphocytes to clotrimazole the of IK1 in this (16Elliott J.I. Higgins C.F. EMBO Rep. 2003; 4: 189-194Crossref PubMed Scopus (65) Google Scholar). The of the cell shrinkage in Kcnn4 null that the Ca2+-activated K+ channels in these cells are indeed the of the expression of this gene and are for active volume results also that Kcnn4 encodes the and intermediate Ca2+-activated K+ channel in parotid acinar cells. in the that IK rather BK channel activity would underlie secretion from salivary with the expression of IK1 in these glands K. Quinn C.C. Begenisich T. Am. J. Physiol. 2003; 284: C535-C546Crossref PubMed Scopus (56) Google Scholar). It to or no in the parotid gland fluid secretion between and Kcnn4 null with was also in the of or in and The and associated and the increased of to the BK channel an increased BK channel activity in the Kcnn4 null an increase in BK channel activity for the loss of IK channels in the Kcnn4 null mice and fluid the in IK1 channel activation in the to a of any in expression in Kcnn4 null this is the of The of salivary gland fluid secretion in mice without BK channels A. K. M. M. and R. W. J. Biol. Chem. Scholar) into the of the of BK and IK channel activity in parotid acinar cells. and for and and H. for also and for in the T and for

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