- Email: [email protected]
Reemergence of the maxi K+ as a K+ secretory channel
letter to the editor
http://www.kidney-international.org & 2007 International Society of Nephrology
Reemergence of the maxi K þ as a K þ secretory channel Kidney International (2007) 71, 1322–1324; doi:10.1038/sj.ki.5002234
To the Editor: A recent publication by Bailey et al.1 resurrected the micropuncture technique to address a longstanding question regarding the ion channels responsible for K þ secretion in the distal nephron. In this study, the investigators demonstrated that the large, Ca2 þ -activated K þ channel (maxi K þ ) has an important role to secrete K þ when demand is high. The history of the maxi K þ (now genetically termed slowpoke or slo) as a renal K þ secretory channel began in 1984 when the maxi K þ in the rabbit cortical collecting duct was the first renal epithelial channel described at the single channel level by the patch-clamp technique.2 However, the maxi K þ was soon discarded as a K þ secretory channel because of its low open probability at physiological intracellular Ca2 þ concentrations and membrane potentials. The maxi K þ gave way to a small conductance K þ channel (SK), open 80% of the time in physiological conditions.3 The maxi K þ reemerged as a secretory channel on discovering that high flow activated maxi K þ of isolated rabbit connecting tubules (CNTs)4 and collecting ducts.5 A subsequent in vivo study showed that flow-mediated K þ secretion was absent when an accessory subunit of the maxi K þ was genetically deleted.6 The Bailey et al.1 study is the first micropuncture study to provide evidence for the maxi K þ as a secretory channel in the distal nephron of the mouse. The first cloning of a renal K þ channel was from the inner stripe of the rat outer medulla (ROMK). ROMK, classified as part of the inward rectifying family of K þ channels (KIR1.1), is the equivalent of SK.7 The in vivo significance of ROMK was demonstrated after developing the ROMK/ mouse.8 Patch clamping confirmed that SK was absent in the thick ascending limb (TAL) and cortical collecting duct of ROMK/.9 However, instead of a K þ secretory defect, it was surprising that excretion of K þ in the ROMK/ mouse was increased. Bailey et al. offered two explanations for the K þ loss in ROMK/. First, without ROMK, an absence of K þ recycling in the apical membrane of the TAL results in increased distal flow, which activates maxi K þ in distal segments. Second, the absence of ROMK in the TAL resulted in reduced K þ reabsorption in the TAL. The combination of recycling K þ via ROMK and a Na þ selective tight junction creates an efficiently maximized Na þ reabsorption in the TAL (see Figure 1a). In the absence of K þ recycling, the large decrease in Na þ and Cl reabsorption in the TAL results in a large quantity of Na þ , Cl and fluid delivered to the distal neprhon. Thus, the first explanation is consistent with the participation of maxi K þ in flow-induced K secretion. However, the second explanation for increased K þ excretion in ROMK/, namely that the absence of 1322
ROMK resulted in reduced K þ reabsorption in the TAL, is problematic in my opinion. This explanation was offered because the K þ concentration in the earliest micropunctured portion of the distal tubule was higher than in the TAL. On this point, I disagree with Bailey et al. Rather, I believe that there must be another explanation for the increased [K þ ]i in the distal tubule. In my view, K þ secretion by other distal transporters or channels, including the maxi K þ , is responsible for the entire urinary loss of K þ in ROMK/. When the transporters of the TAL are modeled in wild type (Figure 1a) and ROMK/ (Figure 1b), it is clear that K þ reabsorption can continue in the TAL in the absence of ROMK. By inhibiting the NaK2Cl, furosemide would eliminate all Na þ , K þ , and Cl transport in the TAL (Figure 1c). Eliminating ROMK will remove an electrogenic component of NaCl reabsorption that is dependent on K þ recycling in the TAL. However electroneural K þ reabsorption is independent of ROMK. As long as K þ is delivered to the TAL from upstream segments, K þ reabsorption will continue. Indeed, experimentally blocking ROMK with barium inhibited NaCl transport without affecting net K þ transport.10 The additional K þ measured in the distal convoluted tubule (DCT) of ROMK/ by Bailey et al. may have been due to K þ secretion in a later portion of the distal tubule (DCT2). In the rabbit kidney, epithelial sodium channel (ENaC) and maxi K þ are distinctly localized together in the connecting tubule. However, in the mouse kidney, there is considerable overlap with ENaC in the DCT2,6,11 leaving a very short DCT1 available for micropuncture. The increased negative potential in the micropunctured segment of ROMK/ is evidence that the microelectrode was placed in the ENaC-associated DCT2. The presence of ENaC would give a driving force for K þ secretion by maxi K þ , which may have enhanced expression in the DCT2 of ROMK/. An explanation by Bailey et al. for a possible decrease in K þ reabsorption in the TAL is that [Cl]i would increase in the TAL with the loss of ROMK (Figure 6 of Bailey et al.). High [Cl]i could feedback to inhibit NaK2Cl. The [Cl] has not been measured in the TAL of ROMK/. However, in the rabbit TAL, [Cl]i has been measured at 54 mM,12 yielding an equilibrium potential of 25 mV (140 mM Cl outside). With a membrane potential of 70 mV, there is a large driving force for Cl transport across the basolateral membrane. Without the apical K þ conductance in ROMK/, the basolateral membrane potential will depolarize to between 30 and 40 mV as the cellular conductance is now dominated by the basolateral Cl conductance. However, unless the ionic transport via Na–K–2Cl is increased, [Cl]i would not increase in the absence of ROMK. Not withstanding the argument that K þ absorption is impaired in the TAL of ROMK/, the study by Bailey et al. was a monumental tour de force and a ‘back to the future’ study for two reasons. First, the formerly abandoned Kidney International (2007) 71, 1322–1329
letter to the editor
12 K+, 12 Cl– to distal nephron
12 K+, 6 Na+, 18 Cl–, to distal nephron
TAL (wild type)
6 Na+ 6 K+
–
12 Cl 3 K+
6 Na+ 4 K+ 7 K+
3K
3 Na+ 3 K+ 6 Cl–
3 K+
3 K+
[Cl–]=54 mM
ROMK
12 Cl
3 Na+
+
15 K+, 9 Na+, 24 Cl– from upstream filtrate
Net reabsorption: 9 Na+, 3 K+, 12 Cl–
te
V = +6 mV
15 K+, 9 Na+, 24 Cl– to distal nephron
[Cl–]=54 mM
6 Cl
3 K+ –
Net reabsorption: 3 Na+, 3 K+, 6 Cl–
15 K+, 9 Na+, 24 Cl– from upsteam filtrate
V te = 0 mV
TAL (furosemide) 3 Na+ 3 H+ Na+ K+ 2 Cl– ROMK
15 K+, 9 Na+, 24 Cl– from upsteam filtrate
Vb = –33 mV
3 Na+ 2 K+ 5 K+
–
ROMK
3 Na+
Vb = –70 mV
3 K+
+
TAL (ROMK–/–)
2 K+
3 Na+
Vb = –70 mV
[Cl–]=6 mM Net reabsorption: 0 Na+, 0 K+, 0 Cl–
V te = 0 mV
Figure 1 | Cell models illustrating the effects of eliminating ROMK or adding furosemide on transport of Na þ , K þ , and Cl in the mammalian TAL. (a) Scheme shows how two cycles of the Na–K–ATPase (6Na þ /4K þ ) leads to reabsorption of 9 Na þ , 3 K þ , and 12 Cl across the TAL. Cytoplasmic chloride, which is above electrochemical equilibrium at 54 mM, is transported down an electrochemical gradient through a basolateral Cl channel. An electropositive potential is created by luminal ROMK K þ recycling resulting in a driving force for Na þ absorption through the paracellular pathway. As shown, a net of three out of every 15 K þ (20%) entering the lumen from the filtrate can be reabsorbed by the parallel operation of the apical Na–K–2Cl transporter ( þ 6 K þ ) and ROMK channel (3 K þ ). (b) Illustration of Na þ , K þ , and Cl transport in the absence of ROMK. With the elimination of ROMK, the operation of the Na–K–2Cl transporter is slowed and net Na þ reabsorption reduced to a fraction of the previous capacity. However, net K þ reabsorption is unaffected with 3 Na þ , 3 K þ , and 6 Cl moving electroneutrally across the apical and basolateral membranes. The intracellular Cl concentration likely remains above electrochemical equilibrium (near 54 mM) with continued Cl transport across the basolateral membrane despite a depolarized basolateral membrane potential (33 mV) with the loss of ROMK. (c) Effects of furosemide on Na þ , K þ , and Cl transport in the TAL. Unlike the elimination of ROMK, blocking the Na–K–2Cl transporter eliminates Na þ , Cl, and K þ transport in the TAL. With the blockage of all Cl entering the cell, the intracellular Cl concentration is likely reduced to its equilibrium value of approximately 6 mM across the basolateral membrane. The Na–K pump is slowed to ‘housekeeping’ rates with the Na þ entering the cell through various transporters, such as the Na–Ca exchanger and Na–H exchanger. The numbers of ions transported are given for stoichiometric accounting purposes and are not given as the actual concentrations in the TAL lumen. The amount of Na þ and Cl driven across the TAL in response to ROMK may be much greater than the Na þ and Cl transported electroneutrally with K þ . In other words, for every 9 K þ transported via the Na–K–2Cl, 6 K þ may be recycled and 3 may be reabsorbed. However, removing the recycled component will not affect the net K þ reabsorption.
micropuncture technique was used as a means to determine the in vivo roles of K þ channels in the distal nephron and second, in the process, the initially abandoned maxi K þ was revived as a physiological secretory channel for K þ in the distal nephron. 1. Bailey MA, Cantone A, Yan Q et al. Maxi-K channels contribute to urinary potassium excretion in the ROMK-deficient mouse model of Type II Bartter’s syndrome and in adaptation to a high-K diet. Kidney Int 2006; 70: 51–59. 2. Hunter M, Lopes AG, Boulpaep EL et al. Single channel recordings of calcium-activated potassium channels in the apical membrane of rabbit cortical collecting tubules. Proc Natl Acad Sci 1984; 81: 4237–4239. 3. Frindt G, Palmer LG. Low-conductance K channels in apical membrane of rat cortical collecting tubule. Am J Physiol 1989; 256: F143–F151. Kidney International (2007) 71, 1322–1329
4.
5.
6.
7.
8.
9.
Taniguchi J, Imai M. Flow-dependent activation of maxi K+ channels in apical membrane of rabbit connecting tubule. J Membr Biol 1998; 164: 35–45. Woda CB, Bragin A, Kleyman TR, Satlin LM. Flow-dependent K+ secretion in the cortical collecting duct is mediated by a maxi-K channel. Am J Physiol Renal Physiol 2001; 280: F786–F793. Pluznick JL, Wei P, Grimm PR, Sansom SC. BK-\{beta\}1 subunit: immunolocalization in the mammalian connecting tubule and its role in the kaliure. Am J Physiol Renal Physiol 2005; 288: F846–F854. Ho K, Nichols CG, Lederer WJ et al. 1oning and expression of an inwardly rectifying ATP-regulated potassium channel. Nature 1993; 362: 31–38. Lorenz JN, Baird NR, Judd LM et al. Impaired renal NaCl absorption in mice lacking the ROMK potassium channel, a model for type II Bartter’s syndrome. J Biol Chem 2002; 277: 37871–37880. Lu M, Wang T, Yan Q et al. Absence of small conductance K+ channel (SK) activity in apical membranes of thick ascending limb and cortical
1323
letter to the editor
collecting duct in ROMK (Bartter’s) knockout mice. J Biol Chem 2002; 277: 37881–37887. 10. Walter SJ, Shirley DG, Folkerd EJ, Unwin RJ. Effects of the potassium channel blocker barium on sodium and potassium transport in the rat loop of Henle in vivo. Exp Physiol 2001; 86: 469–474. 11. Biner HL, Arpin-Bott MP, Loffing J et al. Human cortical distal nephron: distribution of electrolyte and water transport pathways. J Am Soc Nephrol 2002; 13: 836–847. 12. Salomonsson M, Gonzalez E, Westerlund P, Persson AE. Chloride concentration in macula densa and cortical thick ascending limb cells. Kidney Int Suppl 1991; 32: S51–S54.
SC Sansom1 1 Department of Cellular and Integrative Physiology, University of Nebraska Medical School, Omaha, Nebraska, USA Correspondence: SC Sansom, Department of Cellular and Integrative Physiology, University of Nebraska Medical School, Omaha, Nebraska 681985850, USA. E-mail: [email protected]
Response to ‘Reemergence of the maxi K þ as a K þ secretory channel’ Kidney International (2007) 71, 1324–1325; doi:10.1038/sj.ki.5002236
We certainly appreciate the positive comments by Sansom1 regarding the value of our study2 in demonstrating a clear role for maxi-K channels in renal K transport in the distal convoluted tubule. We obviously agree with Sansom that our study leaves little doubt that maxi-K channels can contribute to distal K secretion and renal K excretion, especially following adaptation to a high K diet. K excretion and hypokalemia in individuals with Type II Bartter’s syndrome associated with ROMK mutations is much less severe than in other types of hyperprostaglandin E syndrome where both ROMK and maxi-K channels can contribute to K wasting. The FEK of 158% stated by Sansom was from the original ROMK null mouse that exhibited marked hydronephrosis.3 The Romk/ mouse used in our study reported in Kidney International2 was developed from intercrossing null males for 45 generations to select for high postnatal survival and less hydronephrosis. Clearance results in our Romk/ mice indicate that FEK is B50% under control diet conditions which is more in line with the less severe K wasting expected in the absence of ROMK (full details will be reported in a future publication). Thus, a highly exaggerated K secretion in the distal tubule is not a prerequisite for the K excretion observed in our Romk/ mice. In our paper, we proposed that decreased K reabsorption by the loop of Henle as well as continued K secretion via maxi-K channels in the distal convoluted tubule (DCT) contributed to the kaluresis in the ROMK knockout animal. The former mechanism for enhanced K excretion in Romk/ mice was based on the experimental observation of an increased free-flow K concentration in early DCT fluid in ROMK knockout, compared to wild-type, mice. The primary issue raised by Sansom was our 1324
interpretation of this elevated K concentration. Our conclusion of diminished K reabsorption in the thick ascending limb (TAL) stems from previous in vivo micropuncture–microperfusion studies by us4 and others.5 in vivo studies reveal no significant K secretion in the early DCT measured under free-flow and perfusion conditions.4,5 Is it possible that the high early distal tubule fluid flow in the Romk/ mice activated maxi-K channels that were quiescent in wild-type animals? We think not because latter in vivo studies showed that K secretion in the early DCT was not clearly changed by adaptation to a high K diet or to increased tubule flow rate. In contrast, both maneuvers greatly increased K secretion in the late DCT, the region where we performed the stationary microperfusion experiments that revealed the iberiotoxinsensitive K secretory pathway in Romk/ mice. Given the results from these in vivo studies, it is problematic to invoke K secretion via maxi-K channels as an alternative mechanism to account for the increased free-flow K concentration observed in the early DCT fluid from Romk/ mice.2 In addition, as we discussed in our paper, the in vivo experiments by Walter et al.6 using 5 mM barium as a K channel blocker should not be used as a strong argument against the role of apical K channel activity as a requirement for TAL function including net K reabsorption. This is based on our previous study in mouse TAL where a much high concentration of barium (about 20 mM) was required to achieve a nearly complete block of the apical K conductance in the absence of luminal potassium.7 In addition, it is virtually impossible to eliminate K completely from the tubule fluid at the TAL in the in vivo setting and the presence of luminal K will reduce the blocking effect of barium. Thus, the ROMK KO animal provides the only model we are aware of for analyzing the effect of the absence of apical K channel activity (both 35 and 70 pS channels) on K reabsorption in the TAL.8,9 Regarding the proposed model by Sansom for potassium transport in the absence of apical K conductance, we would like to make the following comment. Key factors required for any remaining K reabsorption by the TAL are the functional activity of apical Na–K–2Cl cotransporter, the basolateral Cl channel and the intracellular Cl activity. These transporters are under complex regulation and their activities cannot be deduced with certainty in the condition where there is no apical ionic conductance, basolateral membrane is depolarized and the transcellular– paracellular current circuit is disrupted. Ultimately, this issue may only be resolved by measuring tracer rubidium fluxes in the isolated perfused TAL preparation from Romk/ mice. In conclusion, the lack of significant K secretion in the early DCT in vivo, even under circumstances that would highly activate maxi-K channels, suggests that the most likely explanation for the increased K concentration in Kidney International (2007) 71, 1322–1329
http://www.kidney-international.org & 2007 International Society of Nephrology
Reemergence of the maxi K þ as a K þ secretory channel Kidney International (2007) 71, 1322–1324; doi:10.1038/sj.ki.5002234
To the Editor: A recent publication by Bailey et al.1 resurrected the micropuncture technique to address a longstanding question regarding the ion channels responsible for K þ secretion in the distal nephron. In this study, the investigators demonstrated that the large, Ca2 þ -activated K þ channel (maxi K þ ) has an important role to secrete K þ when demand is high. The history of the maxi K þ (now genetically termed slowpoke or slo) as a renal K þ secretory channel began in 1984 when the maxi K þ in the rabbit cortical collecting duct was the first renal epithelial channel described at the single channel level by the patch-clamp technique.2 However, the maxi K þ was soon discarded as a K þ secretory channel because of its low open probability at physiological intracellular Ca2 þ concentrations and membrane potentials. The maxi K þ gave way to a small conductance K þ channel (SK), open 80% of the time in physiological conditions.3 The maxi K þ reemerged as a secretory channel on discovering that high flow activated maxi K þ of isolated rabbit connecting tubules (CNTs)4 and collecting ducts.5 A subsequent in vivo study showed that flow-mediated K þ secretion was absent when an accessory subunit of the maxi K þ was genetically deleted.6 The Bailey et al.1 study is the first micropuncture study to provide evidence for the maxi K þ as a secretory channel in the distal nephron of the mouse. The first cloning of a renal K þ channel was from the inner stripe of the rat outer medulla (ROMK). ROMK, classified as part of the inward rectifying family of K þ channels (KIR1.1), is the equivalent of SK.7 The in vivo significance of ROMK was demonstrated after developing the ROMK/ mouse.8 Patch clamping confirmed that SK was absent in the thick ascending limb (TAL) and cortical collecting duct of ROMK/.9 However, instead of a K þ secretory defect, it was surprising that excretion of K þ in the ROMK/ mouse was increased. Bailey et al. offered two explanations for the K þ loss in ROMK/. First, without ROMK, an absence of K þ recycling in the apical membrane of the TAL results in increased distal flow, which activates maxi K þ in distal segments. Second, the absence of ROMK in the TAL resulted in reduced K þ reabsorption in the TAL. The combination of recycling K þ via ROMK and a Na þ selective tight junction creates an efficiently maximized Na þ reabsorption in the TAL (see Figure 1a). In the absence of K þ recycling, the large decrease in Na þ and Cl reabsorption in the TAL results in a large quantity of Na þ , Cl and fluid delivered to the distal neprhon. Thus, the first explanation is consistent with the participation of maxi K þ in flow-induced K secretion. However, the second explanation for increased K þ excretion in ROMK/, namely that the absence of 1322
ROMK resulted in reduced K þ reabsorption in the TAL, is problematic in my opinion. This explanation was offered because the K þ concentration in the earliest micropunctured portion of the distal tubule was higher than in the TAL. On this point, I disagree with Bailey et al. Rather, I believe that there must be another explanation for the increased [K þ ]i in the distal tubule. In my view, K þ secretion by other distal transporters or channels, including the maxi K þ , is responsible for the entire urinary loss of K þ in ROMK/. When the transporters of the TAL are modeled in wild type (Figure 1a) and ROMK/ (Figure 1b), it is clear that K þ reabsorption can continue in the TAL in the absence of ROMK. By inhibiting the NaK2Cl, furosemide would eliminate all Na þ , K þ , and Cl transport in the TAL (Figure 1c). Eliminating ROMK will remove an electrogenic component of NaCl reabsorption that is dependent on K þ recycling in the TAL. However electroneural K þ reabsorption is independent of ROMK. As long as K þ is delivered to the TAL from upstream segments, K þ reabsorption will continue. Indeed, experimentally blocking ROMK with barium inhibited NaCl transport without affecting net K þ transport.10 The additional K þ measured in the distal convoluted tubule (DCT) of ROMK/ by Bailey et al. may have been due to K þ secretion in a later portion of the distal tubule (DCT2). In the rabbit kidney, epithelial sodium channel (ENaC) and maxi K þ are distinctly localized together in the connecting tubule. However, in the mouse kidney, there is considerable overlap with ENaC in the DCT2,6,11 leaving a very short DCT1 available for micropuncture. The increased negative potential in the micropunctured segment of ROMK/ is evidence that the microelectrode was placed in the ENaC-associated DCT2. The presence of ENaC would give a driving force for K þ secretion by maxi K þ , which may have enhanced expression in the DCT2 of ROMK/. An explanation by Bailey et al. for a possible decrease in K þ reabsorption in the TAL is that [Cl]i would increase in the TAL with the loss of ROMK (Figure 6 of Bailey et al.). High [Cl]i could feedback to inhibit NaK2Cl. The [Cl] has not been measured in the TAL of ROMK/. However, in the rabbit TAL, [Cl]i has been measured at 54 mM,12 yielding an equilibrium potential of 25 mV (140 mM Cl outside). With a membrane potential of 70 mV, there is a large driving force for Cl transport across the basolateral membrane. Without the apical K þ conductance in ROMK/, the basolateral membrane potential will depolarize to between 30 and 40 mV as the cellular conductance is now dominated by the basolateral Cl conductance. However, unless the ionic transport via Na–K–2Cl is increased, [Cl]i would not increase in the absence of ROMK. Not withstanding the argument that K þ absorption is impaired in the TAL of ROMK/, the study by Bailey et al. was a monumental tour de force and a ‘back to the future’ study for two reasons. First, the formerly abandoned Kidney International (2007) 71, 1322–1329
letter to the editor
12 K+, 12 Cl– to distal nephron
12 K+, 6 Na+, 18 Cl–, to distal nephron
TAL (wild type)
6 Na+ 6 K+
–
12 Cl 3 K+
6 Na+ 4 K+ 7 K+
3K
3 Na+ 3 K+ 6 Cl–
3 K+
3 K+
[Cl–]=54 mM
ROMK
12 Cl
3 Na+
+
15 K+, 9 Na+, 24 Cl– from upstream filtrate
Net reabsorption: 9 Na+, 3 K+, 12 Cl–
te
V = +6 mV
15 K+, 9 Na+, 24 Cl– to distal nephron
[Cl–]=54 mM
6 Cl
3 K+ –
Net reabsorption: 3 Na+, 3 K+, 6 Cl–
15 K+, 9 Na+, 24 Cl– from upsteam filtrate
V te = 0 mV
TAL (furosemide) 3 Na+ 3 H+ Na+ K+ 2 Cl– ROMK
15 K+, 9 Na+, 24 Cl– from upsteam filtrate
Vb = –33 mV
3 Na+ 2 K+ 5 K+
–
ROMK
3 Na+
Vb = –70 mV
3 K+
+
TAL (ROMK–/–)
2 K+
3 Na+
Vb = –70 mV
[Cl–]=6 mM Net reabsorption: 0 Na+, 0 K+, 0 Cl–
V te = 0 mV
Figure 1 | Cell models illustrating the effects of eliminating ROMK or adding furosemide on transport of Na þ , K þ , and Cl in the mammalian TAL. (a) Scheme shows how two cycles of the Na–K–ATPase (6Na þ /4K þ ) leads to reabsorption of 9 Na þ , 3 K þ , and 12 Cl across the TAL. Cytoplasmic chloride, which is above electrochemical equilibrium at 54 mM, is transported down an electrochemical gradient through a basolateral Cl channel. An electropositive potential is created by luminal ROMK K þ recycling resulting in a driving force for Na þ absorption through the paracellular pathway. As shown, a net of three out of every 15 K þ (20%) entering the lumen from the filtrate can be reabsorbed by the parallel operation of the apical Na–K–2Cl transporter ( þ 6 K þ ) and ROMK channel (3 K þ ). (b) Illustration of Na þ , K þ , and Cl transport in the absence of ROMK. With the elimination of ROMK, the operation of the Na–K–2Cl transporter is slowed and net Na þ reabsorption reduced to a fraction of the previous capacity. However, net K þ reabsorption is unaffected with 3 Na þ , 3 K þ , and 6 Cl moving electroneutrally across the apical and basolateral membranes. The intracellular Cl concentration likely remains above electrochemical equilibrium (near 54 mM) with continued Cl transport across the basolateral membrane despite a depolarized basolateral membrane potential (33 mV) with the loss of ROMK. (c) Effects of furosemide on Na þ , K þ , and Cl transport in the TAL. Unlike the elimination of ROMK, blocking the Na–K–2Cl transporter eliminates Na þ , Cl, and K þ transport in the TAL. With the blockage of all Cl entering the cell, the intracellular Cl concentration is likely reduced to its equilibrium value of approximately 6 mM across the basolateral membrane. The Na–K pump is slowed to ‘housekeeping’ rates with the Na þ entering the cell through various transporters, such as the Na–Ca exchanger and Na–H exchanger. The numbers of ions transported are given for stoichiometric accounting purposes and are not given as the actual concentrations in the TAL lumen. The amount of Na þ and Cl driven across the TAL in response to ROMK may be much greater than the Na þ and Cl transported electroneutrally with K þ . In other words, for every 9 K þ transported via the Na–K–2Cl, 6 K þ may be recycled and 3 may be reabsorbed. However, removing the recycled component will not affect the net K þ reabsorption.
micropuncture technique was used as a means to determine the in vivo roles of K þ channels in the distal nephron and second, in the process, the initially abandoned maxi K þ was revived as a physiological secretory channel for K þ in the distal nephron. 1. Bailey MA, Cantone A, Yan Q et al. Maxi-K channels contribute to urinary potassium excretion in the ROMK-deficient mouse model of Type II Bartter’s syndrome and in adaptation to a high-K diet. Kidney Int 2006; 70: 51–59. 2. Hunter M, Lopes AG, Boulpaep EL et al. Single channel recordings of calcium-activated potassium channels in the apical membrane of rabbit cortical collecting tubules. Proc Natl Acad Sci 1984; 81: 4237–4239. 3. Frindt G, Palmer LG. Low-conductance K channels in apical membrane of rat cortical collecting tubule. Am J Physiol 1989; 256: F143–F151. Kidney International (2007) 71, 1322–1329
4.
5.
6.
7.
8.
9.
Taniguchi J, Imai M. Flow-dependent activation of maxi K+ channels in apical membrane of rabbit connecting tubule. J Membr Biol 1998; 164: 35–45. Woda CB, Bragin A, Kleyman TR, Satlin LM. Flow-dependent K+ secretion in the cortical collecting duct is mediated by a maxi-K channel. Am J Physiol Renal Physiol 2001; 280: F786–F793. Pluznick JL, Wei P, Grimm PR, Sansom SC. BK-\{beta\}1 subunit: immunolocalization in the mammalian connecting tubule and its role in the kaliure. Am J Physiol Renal Physiol 2005; 288: F846–F854. Ho K, Nichols CG, Lederer WJ et al. 1oning and expression of an inwardly rectifying ATP-regulated potassium channel. Nature 1993; 362: 31–38. Lorenz JN, Baird NR, Judd LM et al. Impaired renal NaCl absorption in mice lacking the ROMK potassium channel, a model for type II Bartter’s syndrome. J Biol Chem 2002; 277: 37871–37880. Lu M, Wang T, Yan Q et al. Absence of small conductance K+ channel (SK) activity in apical membranes of thick ascending limb and cortical
1323
letter to the editor
collecting duct in ROMK (Bartter’s) knockout mice. J Biol Chem 2002; 277: 37881–37887. 10. Walter SJ, Shirley DG, Folkerd EJ, Unwin RJ. Effects of the potassium channel blocker barium on sodium and potassium transport in the rat loop of Henle in vivo. Exp Physiol 2001; 86: 469–474. 11. Biner HL, Arpin-Bott MP, Loffing J et al. Human cortical distal nephron: distribution of electrolyte and water transport pathways. J Am Soc Nephrol 2002; 13: 836–847. 12. Salomonsson M, Gonzalez E, Westerlund P, Persson AE. Chloride concentration in macula densa and cortical thick ascending limb cells. Kidney Int Suppl 1991; 32: S51–S54.
SC Sansom1 1 Department of Cellular and Integrative Physiology, University of Nebraska Medical School, Omaha, Nebraska, USA Correspondence: SC Sansom, Department of Cellular and Integrative Physiology, University of Nebraska Medical School, Omaha, Nebraska 681985850, USA. E-mail: [email protected]
Response to ‘Reemergence of the maxi K þ as a K þ secretory channel’ Kidney International (2007) 71, 1324–1325; doi:10.1038/sj.ki.5002236
We certainly appreciate the positive comments by Sansom1 regarding the value of our study2 in demonstrating a clear role for maxi-K channels in renal K transport in the distal convoluted tubule. We obviously agree with Sansom that our study leaves little doubt that maxi-K channels can contribute to distal K secretion and renal K excretion, especially following adaptation to a high K diet. K excretion and hypokalemia in individuals with Type II Bartter’s syndrome associated with ROMK mutations is much less severe than in other types of hyperprostaglandin E syndrome where both ROMK and maxi-K channels can contribute to K wasting. The FEK of 158% stated by Sansom was from the original ROMK null mouse that exhibited marked hydronephrosis.3 The Romk/ mouse used in our study reported in Kidney International2 was developed from intercrossing null males for 45 generations to select for high postnatal survival and less hydronephrosis. Clearance results in our Romk/ mice indicate that FEK is B50% under control diet conditions which is more in line with the less severe K wasting expected in the absence of ROMK (full details will be reported in a future publication). Thus, a highly exaggerated K secretion in the distal tubule is not a prerequisite for the K excretion observed in our Romk/ mice. In our paper, we proposed that decreased K reabsorption by the loop of Henle as well as continued K secretion via maxi-K channels in the distal convoluted tubule (DCT) contributed to the kaluresis in the ROMK knockout animal. The former mechanism for enhanced K excretion in Romk/ mice was based on the experimental observation of an increased free-flow K concentration in early DCT fluid in ROMK knockout, compared to wild-type, mice. The primary issue raised by Sansom was our 1324
interpretation of this elevated K concentration. Our conclusion of diminished K reabsorption in the thick ascending limb (TAL) stems from previous in vivo micropuncture–microperfusion studies by us4 and others.5 in vivo studies reveal no significant K secretion in the early DCT measured under free-flow and perfusion conditions.4,5 Is it possible that the high early distal tubule fluid flow in the Romk/ mice activated maxi-K channels that were quiescent in wild-type animals? We think not because latter in vivo studies showed that K secretion in the early DCT was not clearly changed by adaptation to a high K diet or to increased tubule flow rate. In contrast, both maneuvers greatly increased K secretion in the late DCT, the region where we performed the stationary microperfusion experiments that revealed the iberiotoxinsensitive K secretory pathway in Romk/ mice. Given the results from these in vivo studies, it is problematic to invoke K secretion via maxi-K channels as an alternative mechanism to account for the increased free-flow K concentration observed in the early DCT fluid from Romk/ mice.2 In addition, as we discussed in our paper, the in vivo experiments by Walter et al.6 using 5 mM barium as a K channel blocker should not be used as a strong argument against the role of apical K channel activity as a requirement for TAL function including net K reabsorption. This is based on our previous study in mouse TAL where a much high concentration of barium (about 20 mM) was required to achieve a nearly complete block of the apical K conductance in the absence of luminal potassium.7 In addition, it is virtually impossible to eliminate K completely from the tubule fluid at the TAL in the in vivo setting and the presence of luminal K will reduce the blocking effect of barium. Thus, the ROMK KO animal provides the only model we are aware of for analyzing the effect of the absence of apical K channel activity (both 35 and 70 pS channels) on K reabsorption in the TAL.8,9 Regarding the proposed model by Sansom for potassium transport in the absence of apical K conductance, we would like to make the following comment. Key factors required for any remaining K reabsorption by the TAL are the functional activity of apical Na–K–2Cl cotransporter, the basolateral Cl channel and the intracellular Cl activity. These transporters are under complex regulation and their activities cannot be deduced with certainty in the condition where there is no apical ionic conductance, basolateral membrane is depolarized and the transcellular– paracellular current circuit is disrupted. Ultimately, this issue may only be resolved by measuring tracer rubidium fluxes in the isolated perfused TAL preparation from Romk/ mice. In conclusion, the lack of significant K secretion in the early DCT in vivo, even under circumstances that would highly activate maxi-K channels, suggests that the most likely explanation for the increased K concentration in Kidney International (2007) 71, 1322–1329