Expression of tetraspan protein CD63 activates protein-tyrosine kinase (PTK) and enhances the PTK-induced inhibition of ROMK channels.

In the present study, we tested the role of CD63 in regulating ROMK1 channels by protein-tyrosine kinase (PTK). Immunocytochemical staining shows that CD63 and receptor-linked tyrosine phosphatase alpha (RPTPalpha) are expressed in the cortical collecting duct and outer medulla collecting duct. Immunoprecipitation of tissue lysates from renal cortex and outer medulla or 293T cells transfected with CD63 reveals that CD63 was associated with RPTPalpha both in situ and in transfected cells. Expression of CD63 in 293T cells stimulated the phosphorylation of tyrosine residue 416 of c-Src but decreased the phosphorylation of tyrosine residue 527, indicating that expression of CD63 stimulates the activity of c-Src. Furthermore, c-Src was coimmunoprecipitated with RPTPalpha and CD63 both in 293T cells transfected with CD63 and in lysates prepared from native rat kidney. Potassium restriction had no effect on the expression of RPTPalpha, but it increased the association between c-Src and RPTPalpha in the renal cortex and outer medulla. We also used two-electrode voltage clamp to study the effect of CD63 on ROMK channels in Xenopus oocytes. Expression of CD63 had no significant effect on potassium currents in oocytes injected with ROMK1; however, it significantly enhanced the c-Src-induced inhibition of ROMK channels in oocytes injected with ROMK1+c-Src. The effect of CD63 on the c-Src-induced inhibition was not due to a decreased expression of ROMK1 channels, because blocking PTK with herbimycin A abolished the inhibitory effect of c-Src on ROMK channels in oocytes injected with ROMK1+c-Src+CD63. Furthermore, coexpression of CD63 enhanced tyrosine phosphorylation of ROMK1. We conclude that CD63 plays a role in the regulation of ROMK channels through its association with RPTPalpha, which in turn interacts with and activates Src family PTK, thus reducing ROMK activity.

Effect of hydrogen peroxide on ROMK channels in the cortical collecting duct.

We used the patch-clamp technique to study the effect of H(2)O(2) on the apical ROMK-like small-conductance K (SK) channel in the cortical collecting duct (CCD). The addition of H(2)O(2) decreased the activity of the SK channels and the inhibitory effect of H(2)O(2) was larger in the CCD from rats on a K-deficient diet than that from rats on a normal-K or a high-K diet. However, application of H(2)O(2) did not inhibit the SK channels in inside-out patches. This suggests that the H(2)O(2)-mediated inhibition of SK channels was not due to direct oxidation of the SK channel protein. Because a previous study showed that H(2)O(2) stimulated the expression of Src family protein tyrosine kinase (PTK) which inhibited SK channels (3), we explored the role of PTK in mediating the effect of H(2)O(2) on SK channels. The application of H(2)O(2) stimulated the activity of endogenous PTK in M-1 cells and increased tyrosine phosphorylation of ROMK in HEK293 cells transfected with GFP-ROMK1 and c-Src. However, blockade of PTK only attenuated but did not completely abolish the inhibitory effect of H(2)O(2) on SK channels. Since H(2)O(2) has also been demonstrated to activate mitogen-activated protein kinase, P38, and ERK (3), we examined the role of P38 and ERK in mediating the effect of H(2)O(2) on SK channels. Similar to blockade of PTK, suppression of P38 and ERK did not completely abolish the H(2)O(2)-induced inhibition of SK channels. However, combined use of ERK, P38, and PTK inhibitors completely abolished the effect of H(2)O(2) on SK channels. Also, treatment of the CCDs with concanavalin A, an agent which has been shown to inhibit endocytosis (19), abolished the inhibitory effect of H(2)O(2). We conclude that addition of H(2)O(2) inhibited SK channels by stimulating PTK activity, P38, and ERK in the CCD and that H(2)O(2) enhances the internalization of the SK channels.

Mineralocorticoids decrease the activity of the apical small-conductance K channel in the cortical collecting duct.

We used the patch-clamp technique to examine the effect of DOCA treatment (2 mg/kg) on the apical small-conductance K (SK) channels, epithelial Na channels (ENaC), and the basolateral 18-pS K channels in the cortical collecting duct (CCD). Treatment of rats with DOCA for 6 days significantly decreased the plasma K from 3.8 to 3.1 meq and reduced the activity of the SK channel, defined as NP(o), from 1.3 in the CCD of control rats to 0.6. In contrast, DOCA treatment significantly increased ENaC activity from 0.01 to 0.53 and the basolateral 18-pS K channel activity from 0.67 to 1.63. Moreover, Western blot analysis revealed that DOCA treatment significantly increased the expression of the nonreceptor type of protein tyrosine kinase (PTK), cSrc, and the tyrosine phosphorylation of ROMK in the renal cortex and outer medulla. The possibility that decreases in apical SK channel activity induced by DOCA treatment were the result of stimulation of PTK activity was further supported by experiments in which inhibition of PTK with herbimycin A significantly increased NP(o) from 0.6 to 2.1 in the CCD from rats receiving DOCA. Also, when rats were fed a high-K (10%) diet, DOCA treatment did not increase the expression of c-Src and decrease the activity of the SK channel in the CCD. We conclude that DOCA treatment decreased the apical SK channel activity in rats on a normal-K diet and that an increase in PTK expression may be responsible for decreased channel activity in the CCD from DOCA-treated rats.

ROMK1 channel activity is regulated by monoubiquitination.

The ubiquitination of proteins can signal their degradation, modify their activity or target them to specific membranes or cellular organelles. Here, we show that monoubiquitination regulates the plasma membrane abundance and function of the potassium channel, ROMK. Immunoprecipitation of proteins obtained from renal cortex and outer medulla with ROMK antibody revealed that this channel was monoubiquitinated. To determine the ubiquitin binding site on ROMK1, all intracellular lysine (Lys) residues of ROMK1 were individually mutated to arginine (Arg), and a two-electrode voltage clamp was used to measure the ROMK1 channel activity in Xenopus oocytes. ROMK1 channel activity increased from 8.1 to 27.2 microA only when Lys-22 was mutated to Arg. Furthermore, Western blotting failed to detect the ubiquitinated ROMK1 in oocytes injected with R1K22R. Patch-clamp experiments showed that biophysical properties of R1K22R were identical to those of wild-type ROMK1. Although total protein expression levels of GFP-ROMK1 and GFP-R1K22R in oocytes were similar, confocal microscopy showed that the surface fluorescence intensity in oocytes injected with GFP-R1K22R was higher than that of GFP-ROMK1. In addition, biotin labeling of ROMK1 and R1K22R proteins expressed in HEK293 cells showed increased surface expression of the Lys-22 mutant channel. Finally, expression of R1K22R in COS7 cells significantly stimulated the surface expression of ROMK1. We conclude that ROMK1 can be monoubiquitinated and that Lys-22 is an ubiquitin-binding site. Thus, monoubiquitination of ROMK1 regulates channel activity by reducing the surface expression of channel protein. This finding implicates the linking of a single ubiquitin molecule to channels as an important posttranslational regulatory signal.

The protein tyrosine kinase-dependent pathway mediates the effect of K intake on renal K secretion.

Dietary K intake plays an important role in the regulation of K secretion: a decrease stimulates and an increase suppresses kidney expression of protein tyrosine kinase (PTK), which plays a role in regulating Kir1.1 (ROMK), which is responsible for K secretion in the cortical collecting duct (CCD) and K recycling in the thick ascending limb. Tyrosine phosphorylation of ROMK channels increases with low dietary K and decreases with high dietary K. Moreover, stimulation of tyrosine phosphorylation of ROMK1 enhances ROMK1 internalization and reduces the K channel number in the cell surface in the CCD.

Superoxide anions are involved in mediating the effect of low K intake on c-Src expression and renal K secretion in the cortical collecting duct.

We previously demonstrated that low K intake stimulated the expression of c-Src and that stimulation of protein tyrosine kinase inhibited ROMK channel activity (Wei, Y., Bloom, P., Lin, D. H., Gu, R. M., and Wang, W. H. (2001) Am. J. Physiol. 281, F206-F212). Decreases in dietary K content significantly increased O(2)(-) levels and the phosphorylation of c-Jun, a transcription factor, in renal cortex and outer medulla. The role of O(2)(-) and related products such as H(2)O(2) in stimulating the expression of protein tyrosine kinase is suggested by the observation that addition of 50-200 microm H(2)O(2) increased the phosphorylation of c-Jun and the expression of c-Src in M1 cells, a mouse collecting duct principal cell line. The effect of H(2)O(2) on c-Src expression was completely abolished with cyclohexamide or actinomycin D. The treatment of animals on a K-deficient (KD) diet with tempol for 7 days significantly decreased the production of O(2)(-), c-Jun phosphorylation, and c-Src expression. Moreover, low K intake decreased the activity of ROMK-like small conductance channels from 1.37 (control K diet) to 0.5 in the cortical collecting duct and increased the tyrosine phosphorylation of ROMK in the renal cortex and outer medulla. In contrast, the tempol treatment not only increased channel activity to 1.1 in the cortical collecting duct but also decreased the tyrosine phosphorylation of ROMK from rats on a KD diet. Finally, suppressing O(2)(-) production with tempol significantly increased renal K excretion measured with metabolic cage and lowered the plasma K concentration in comparison with those on a KD diet alone without tempol. We conclude that O(2)(-) and related products play a role in mediating the effect of low K intake on c-Src expression and in suppressing ROMK channel activity and renal K secretion.

PKC expression is regulated by dietary K intake and mediates internalization of SK channels in the CCD.

We have used Western blot analysis and immunocytochemistry to determine the effect of dietary K intake on the expression of protein kinase C (PKC) isoforms in the kidney. Western blot has demonstrated that conventional PKC isoforms (alpha and beta), novel PKC isoforms (delta, epsilon, and eta), and atypical PKC isoforms (zeta) are expressed in the renal cortex and outer medulla. Moreover, a low K intake significantly increases the expression of PKC-epsilon in the renal cortex and outer medulla but does not change the expression of PKC-alpha, PKC-beta, PKC-delta, PKC-eta, and PKC-zeta. Also, immunocytochemistry shows that PKC-epsilon isoform is expressed in the cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) and that the intensity of PKC-epsilon staining is higher in the kidney from rats on a K-deficient diet than those on a control diet. Also, we used the patch-clamp technique to study the role of PKC in mediating internalization of ROMK (Kir 1.1)-like small-conductance K (SK) channels induced by phenylarsine oxide (PAO), an agent that inhibits protein tyrosine phosphatase and has been shown to stimulate the internalization of the SK channel in the CCD (Sterling H, Lin DH, Qu RM, Dong K, Herbert SC, and Wang WH. J Biol Chem 277: 4317-4323, 2002). Inhibition of PKC with calphostin C and GF-109203x had no significant effect on channel activity but abolished the inhibitory effect of PAO on SK channels. In conclusion, a low K intake increases the expression of PKC-epsilon isoform in the renal cortex and outer medulla, and PKC is involved in mediating the internalization of SK channels in the CCD induced by stimulation of protein tyrosine kinase activity.

Protein tyrosine kinase is expressed and regulates ROMK1 location in the cortical collecting duct.

We previously demonstrated that dietary K intake regulates the expression of Src family PTK, which plays an important role in controlling the expression of ROMK1 in plasma membrane (Wei Y, Bloom P, Lin D-H, Gu RM, and Wang WH. Am J Physiol Renal Physiol 281: F206-F212, 2001). In the present study, we used the immunofluorescence staining technique to demonstrate the presence of c-Src, a member of Src family PTK, in the thick ascending limb (TAL) and collecting duct. Confocal microscopy shows that c-Src is highly expressed in the renal cortex and outer medulla. Moreover, c-Src and ROMK are coexpressed in the same nephron segment. Also, the positive staining of c-Src is visible in tubules stained with Tamm-Horsfall glycoprotein or aquaporin-2. This suggests that c-Src is present in the TAL, cortical collecting duct (CCD), and outer medullary collecting duct (OMCD). To study the role of PTK in the regulation of ROMK membrane expression in the TAL and CCD, we carried out immunocytochemical staining with ROMK antibody in the CCD or TAL from rats on either a high-K (HK) or K-deficient (KD) diet. A sharp membrane staining of ROMK can be observed in the TAL from rats on both HK and KD diets. However, a clear plasma membrane staining can be observed only in the CCD from rats on a HK diet but not from those on a KD diet. Treatment of the CCD from rats on a HK diet with phenylarsine oxide (PAO) decreases the positive staining in the plasma/subapical membrane and increases the ROMK staining in the intracellular compartment. However, PAO treatment did not significantly alter the staining pattern of ROMK in the TAL. Moreover, the biotinylation technique has also confirmed that neither herbimycin A nor PAO has significantly changed the biotin-labeled ROMK2 in HEK293 cells transfected with ROMK2 and c-Src. We conclude that c-Src is expressed in the TAL, CCD, and OMCD and that stimulation of PTK increases the ROMK channels in the intracellular compartment but decreases them in the apical/subapical membrane in the CCD.

Inhibition of heme oxygenase decreases sodium and fluid absorption in the loop of Henle.

We previously demonstrated that carbon monoxide (CO) stimulates the apical 70-pS K+ channel in the thick ascending limb (TAL) of the rat kidney (Liu HJ, Mount DB, Nasjletti A, and Wang WH. J Clin Invest 103: 963-970, 1999). Because the apical K+ channel plays a key role in K+ recycling, we tested the hypothesis that heme oxygenase (HO)-dependent metabolites of heme may affect Na+ transport in the TAL. We used in vivo microperfusion to study the effect of chromium mesoporphyrin (CrMP), an inhibitor of HO, on fluid absorption (Jv) and Na+ absorption (JNa) in the loop of Henle and renal clearance methods to examine the effect of CrMP on renal sodium excretion. Microperfusion experiments demonstrated that addition of CrMP to the loop of Henle decreased Jv by 13% and JNa by 20% in animals on normal rat chow and caused a decrease in Jv (39%) and JNa (40%) in rats on a high-K+ (HK) diet. The effect of CrMP is the result of inhibition of HO because addition of MgPP, an analog of CrMP that does not inhibit HO, had no effect on Jv. Western blot analysis showed that HO-2 is expressed in the kidney and that the level of HO-2 was significantly elevated in animals on a HK diet. Renal clearance studies demonstrated that the infusion of CrMP increased the excretion of urinary Na+ (ENa) and volume (UV) without changes in glomerular filtration rate. The effect of CrMP on ENa and UV was larger in HK rats than those kept on normal chow. We conclude that HK intake increases HO-2 expression in the kidney and that HO-dependent metabolites of heme, presumably CO, play a significant role in the regulation of Na+ transport in the loop of Henle.

Tetanus toxin abolishes exocytosis of ROMK1 induced by inhibition of protein tyrosine kinase.

We used confocal microscopy, patch-clamp, and biotin-labeling techniques to examine the role of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in mediating the effect of inhibition of PTK on ROMK1 trafficking in HEK-293 cells transfected with c-Src and green fluorescent protein (GFP)-ROMK1. Inhibition of c-Src with herbimycin A significantly decreased the tyrosine phosphorylation level of ROMK1. Patch-clamp studies demonstrated that addition of herbimycin A increased the activity of ROMK1 in cell-attached patches. Confocal microscopic imaging showed that herbimycin A decreased the intracellular intensity of GFP-ROMK1. The biotin-labeling technique demonstrated that the inhibition of c-Src increased surface ROMK1 by 110%. In contrast, inhibition of c-Src did not increase the K channel number in HEK cells transfected with R1Y337A, a ROMK1 mutant in which tyrosine residue 337 was mutated to alanine. This suggests that tyrosine residue 337 is essential for the herbimycin A-induced increase in surface ROMK1 channels. To determine whether SNARE proteins are involved in mediating exocytosis of ROMK1 induced by the inhibition of c-Src, we examined the effect of herbimycin A on ROMK1 trafficking in cells treated with tetanus toxin. The incubation of cells in a medium containing tetanus toxin abolished the herbimycin A-induced increase in the number of surface ROMK1. In contrast, inhibition of c-Src still increased the numbers of surface ROMK1 in cells treated with boiled tetanus toxin. We conclude that tyrosine dephosphorylation enhances the exocytosis of ROMK1 and that SNARE proteins are required for exocytosis induced by inhibition of PTK.

Protein kinase C (PKC)-induced phosphorylation of ROMK1 is essential for the surface expression of ROMK1 channels.

We carried out in vitro phosphorylation assays to determine whether ROMK1 is a substrate of protein kinase C (PKC) and used the two-electrode voltage clamp method to investigate the role of serine residues 4, 183, and 201, the three putative PKC phosphorylation sites, in the regulation of ROMK1 channel activity. Incubation of the purified His-tagged ROMK1 protein with PKC and radiolabeled ATP resulted in (32)P incorporation into ROMK1 detected by autoradiography. Moreover, the in vitro phosphorylation study of three synthesized peptides corresponding to amino acids 1-16, 174-189, and 196-211 of ROMK1 revealed that serine residues 4 and 201 of ROMK1 were the two main PKC phosphorylation sites. In contrast, (32)P incorporation of peptide 174-189 was absent. In vitro phosphorylation studies with ROMK1 mutants, R1S4/201A, R1S4/183A, and R1S183/201A, demonstrated that the phosphorylation levels of R1S4/201A were significantly lower than those of the other two mutants. Also, the Ba(2+)-sensitive K(+) current in oocytes injected with green fluorescent protein (GFP)-R1S4/201A was only 5% of that in oocytes injected with wild type GFP-ROMK1. In contrast, the K(+) current in oocytes injected with GFP-ROMK1 mutants containing either serine residue 4 or 201 was similar to those injected with wild type ROMK1. Confocal microscope imaging shows that the surface expression of the K(+) channels was significantly diminished in oocytes injected with R1S4/201A and completely absent in oocytes injected with R1S4/183/201A. Furthermore, the biotin labeling technique confirmed that the membrane fraction of ROMK channels was almost absent in HEK293 cells transfected with either R1S4/201A or R1S4/183/201A. However, when serine residues 4 and 201 were mutated to aspartate, the K(+) currents and the surface expression were completely restored. Finally, addition of calphostin C in the incubation medium significantly decreased the K(+) current in comparison with that under control conditions. Biotin labeling technique further indicated that inhibition of PKC decreases the surface ROMK1 expression in human embryonic kidney (HEK) cells transfected with ROMK1. We conclude that ROMK1 is a substrate of PKC and that serine residues 4 and 201 are the two main PKC phosphorylation sites that are essential for the expression of ROMK1 in the cell surface.

K depletion increases protein tyrosine kinase-mediated phosphorylation of ROMK.

We purified His-tagged ROMK1 and carried out in vitro phosphorylation assays with (32)P-radiolabeled ATP to determine whether ROMK1 protein is a substrate for PTK. Addition of active c-Src and [(32)P]ATP to the purified ROMK1 protein resulted in the phosphorylation of the ROMK1 protein. However, c-Src did not phosphorylate R1Y337A in which tyrosine residue 337 was mutated to alanine. Furthermore, phosphopeptide mapping identified two phosphopeptides from the trypsin-digested ROMK1 protein. In contrast, no phosphorylated peptide has been found in the trypsin-digested R1Y337A protein. This suggested that two phosphorylated peptides might contain the same tyrosine residue. Also, addition of c-Src and [(32)P]ATP phosphorylated the synthesized peptide corresponding to amino acid sequence 333-362 of the COOH terminus of ROMK1. We then examined the effect of dietary K intake on the tyrosine-phosphorylated ROMK level. Although the ROMK channels pulled down by immunoprecipitation with ROMK antibody were the same from rats on a K-deficient diet or on a high-K diet, more ROMK channels were phosphorylated by PTK in rats on a K-deficient diet than those on a high-K diet. We conclude that ROMK1 can be phosphorylated by PTK and that tyrosine residue 337 is the key site for the phosphorylation. Also, the tyrosine phosphorylation of ROMK is modulated by dietary K intake. This strongly suggests that PTK is an important member of the aldosterone-independent signal transduction pathway for regulating renal K secretion.

Regulation of ROMK channels by protein tyrosine kinase and tyrosine phosphatase.

Renal outer medulla K (ROMK) channels play an important role in K recycling in the thick ascending limb and in K secretion in the cortical collecting duct. ROMK1, a member of the ROMK family, has been shown to be a substrate for protein tyrosine kinase (PTK). The tyrosine phosphorylation of ROMK channels increases with low dietary K intake and decreases with high dietary K intake. Moreover, the stimulation of tyrosine phosphorylation of ROMK1 channels decreases the number of K channels by facilitating endocytosis. In contrast, the stimulation of tyrosine dephosphorylation increases the number of ROMK1 channels in the cell membrane by enhancing membrane insertion. PTK and tyrosine phosphatase-induced regulation of ROMK1 channels play a key role in mediating the effect of the dietary K intake on renal K secretion.

K depletion enhances the extracellular Ca2+-induced inhibition of the apical K channels in the mTAL of rat kidney.

We have shown previously that raising extracellular Ca(2)+ inhibited the apical 70-pS K channel in the thick ascending limb (TAL; Wang, W.H., M. Lu, and S.C. Hebert. 1996. Am. J. Physiol. 270:C103-C111). We now used the patch-clamp technique to study the effect of increasing the extracellular Ca(2)+ on the 70-pS K channel in the mTAL from rats on a different K diet. Increasing the extracellular Ca(2)+ from 10 microM to 0.5, 1, and to 1.5 mM in the mTAL from rats on a K-deficient (KD) diet inhibited the channel activity by 30, 65, and 90%, respectively. In contrast, raising the extracellular Ca(2)+ to 1.5 mM had no significant effect on channel activity in the mTAL from animals on a high K (HK) diet and further increasing the extracellular Ca(2)+ to 2.5, 3.5, and 5.5 mM decreased the channel activity by 29, 55, and 90%, respectively. Inhibition of the cytochrome P450 monooxygenase completely abolished the effect of the extracellular Ca(2)+ on channel activity in the mTAL from rats on a different K diet. In contrast, blocking cyclooxygenase did not significantly alter the responsiveness of the 70-pS K channel to the extracellular Ca(2)+. Moreover, addition of sodium nitropruside, a nitric oxide (NO) donor, not only increased the channel activity, but also blunted the inhibitory effect of the extracellular Ca(2)+ on the 70-pS K channel and decreased 20-hydroxyeicosatetraenoic acid (20-HETE) concentration in the mTAL from rats on a KD diet. In contrast, inhibiting NOS with L-NAME enhanced the inhibitory effect of the extracellular Ca(2)+ on the channel activity and increased 20-HETE concentration in the mTAL from rats on a high K diet. Western blot has further shown that the expression of inducible NO synthase (iNOS) is significantly higher in the renal medulla from rats on an HK diet than that on a KD diet. Also, addition of S-nitroso-N-acetylpenicillamine abolished the inhibitory effect of arachidonic acid on channel activity in the mTAL, whereas it did not block the inhibitory effect of 20-HETE. We conclude that a low dietary K intake increases the sensitivity of the 70-pS K channel to the extracellular Ca(2)+, and that a decrease in NOS activity is involved in enhancing the inhibitory effect of the extracellular Ca(2)+ on channel activity in the mTAL during K depletion.

Inhibition of protein-tyrosine phosphatase stimulates the dynamin-dependent endocytosis of ROMK1.

We have previously shown that inhibiting protein-tyrosine kinase increased whereas inhibiting protein-tyrosine phosphatase (PTP) decreased renal outer medullary potassium channel 1 (ROMK1) channel activity (1). We have now used confocal microscopy, the patch clamp technique, and biotin labeling to further examine the role of tyrosine phosphorylation in regulating ROMK1 trafficking. Human embryonic kidney 293 cells were cotransfected with c-Src and green fluorescent protein-ROMK1, which has the same biophysical properties as those of ROMK1. Patch clamp studies have shown that phenylarsine oxide (PAO), an inhibitor of PTP, decreased the activity of ROMK1. Moreover, addition of PAO reduced the cell surface localization of green fluorescent protein-ROMK1 detected by confocal microscopy and diminished the surface ROMK1 density by 65% measured by biotin labeling. Also, PAO treatment significantly increased the phosphorylation of ROMK1. The notion that the effect of PAO is mediated by stimulating tyrosine phosphorylation-induced endocytosis of ROMK1 has also been supported by findings that mutating the tyrosine residue 337 of ROMK1 to alanine abolished the effect of PAO. Finally, the inhibitory effect of PAO on ROMK1 was completely blocked in the cells co-transfected with dominant negative dynamin (dynaminK44A). This indicates that the tyrosine phosphorylation-induced endocytosis of ROMK1 is dynamin-dependent. We conclude that inhibiting PTP increases ROMK1 phosphorylation and results in a dynamin-dependent internalization of the channel.