Mutations in the pore region of ROMK enhance Ba2+ block

Zhou, Hao; Chepilko, Sergei; Schütt, Wolfgang; Choe, Han; Palmer, Lawrence G.; Sackin, Henry

doi:10.1152/ajpcell.1996.271.6.c1949

Cited by 78 publications

(52 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In their experiments, kon was altered, but koff was not. Our results are also consistent with those of Zhou et al (1996), who showed in Kir1.1 that the mutant V121T − V121 lies in Kir1.1 at the site equivalent to T141 in Kir2.1 − increases Ba 2＋ affinity, with effects on both the blocking and unblocking rates. Similarly, in Shaker K ＋ channels (Hurst et al, 1996;Harris et al, 1998) the mutation of the threonine at position 441, which is located in the corresponding position to T141 in Kir2.1, also affected the Ba 2＋ affinity.…”

Section: ＋supporting

confidence: 93%

“…This characteristic has resulted in Ba 2＋ being used extensively in the separation of Kir currents in native membranes (see Stanfield et al, 2002). Inevitably, more detailed investigations of how Ba 2＋ blocks cloned Kir channels, including the rat epithelial channel Kir 1.1 (ROMK2, Zhou et al, 1996) and mouse macrophage channel Kir 2.1 (IRK1, Shieh et al, 1998), have followed.…”

Section: Introductionmentioning

confidence: 99%

“…Such mutation of the inner glycine in the signature motif GYG decreased binding affinity for Ba 2＋ 6500-fold (Lu et al, 2001). Secondly, Zhou et al (1996) demonstrated that differences in the characteristics of Ba 2＋ blockage of Kir 1.1 and 2.1 could be attributed in part to the presence of a valine residue (Val121) in the P-region of Kir 1.1; this residue aligns with a threonine (Thr141) in Kir 2.1. The mutant channel Kir 1.1 (V121T) exhibited a ten-fold increase in Ba 2＋ affinity.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External Ba2+

Lee

Thompson

Ashmole

et al. 2009

Korean J Physiol Pharmacol

View full text Add to dashboard Cite

for a W T-Y145L dimer, and 267μM for Y145F. Mutant channels T141A and S165L exhibit a reduced affinity together with a large reduction in the rate of blockage. In S165L, blockage proceeds with a double exponential time course, suggestive of more than one blocking site. The double mutation T141A/S165L dramatically reduced affinity for Ba 2＋ , also showing two components with very different time courses. Mutants D172K and D172R (lining the central, aqueous cavity of the channel) showed both a decreased affinity to Ba 2＋ and a decrease in the on transition rate constant (kon). These results imply that residues stabilising the cytoplasmic end of the selectivity filter (T141, S165) and in the central cavity (D172) are major determinants of high affinity Ba 2＋ blockage in Kir 2.1.

show abstract

Section: ＋supporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External Ba2+

Lee

Thompson

Ashmole

et al. 2009

Korean J Physiol Pharmacol

View full text Add to dashboard Cite

show abstract

“…Mutational analysis in KCNQ2, K ir 2.1 and Shaker is consistent with the involvement of T276/A315 in an intracellular ionic block (Yellen et al, 1991;Zhou et al, 1996;Alagem et al, 2001;Slesinger, 2001;Prole and Marrion, 2004). Indeed, the corresponding T623 is critical for anti-arrhythmic drug blockage of human ether-à-go-go related gene channels (Mitcheson et al, 2000), and in KCNQ1 there are important determinants for benzodiazepine channel block in the neighborhood of this residue (Seebohm et al, 2001).…”

Section: Role Of the Pore Residue In Potentiationmentioning

confidence: 71%

Three Mechanisms Underlie KCNQ2/3 Heteromeric Potassium M-Channel Potentiation

Etxeberría¹,

Santana-Castro²,

Regalado³

et al. 2004

J. Neurosci.

117

View full text Add to dashboard Cite

The non-inactivating potassium M-current exerts a strong influence on neuronal excitability. The channels responsible for this current are made up of KCNQ subunits, and mutations in most of these produce human pathologies. Notably, in terms of excitation, mutations in either KCNQ2 or KCNQ3 lead to benign neonatal familial convulsions. Although a mere reduction of 25% in KCNQ2/3 function can increase excitability to epileptogenic levels, the potentiation of these subunits has anti-epileptogenic effects. After KCNQ2/3 heteromerization, current levels can augment as much as 10-fold, and we have discovered that there are three processes underlying this potentiation. First, there is an increase in the number of channels inserted in the membrane after heteromerization of the C-terminal region. Second, the N-terminal domain from KCNQ2 exerts a negative influence on the current level. Finally, Ala 315 of KCNQ3, a residue located in the inner vestibule after the selectivity filter, plays a critical role in preventing current flow in KCNQ3 homomeric channels, whereas it is permissive in heteromers in combination with Thr at the equivalent 276 position of KCNQ2.

show abstract

“…Recently, we showed that several H5 loci of ROMK1 could be glycosylated and that one of them, Q139N, in both its glycosylated and unglycosylated forms was nonselective between K ϩ and Na ϩ (14). That H5 is a hotspot for residues that affect the pore is supported by the report that Leu 117 and Val 121 in ROMK2, corresponding to 136 and 140 in ROMK1, retained K ϩ selectivity but increased single channel conductance and Ba 2ϩ sensitivity (15). However H5 is not the sole pore-determining segment, since most of the important determinants of pore blockade by Mg 2ϩ and polyamines are negatively charged residues located in the carboxyl terminus (16 -19) and M2 (20 -24).…”

Section: /Namentioning

confidence: 83%

Mapping the Kidney Potassium Channel ROMK1

Schwalbe

Bianchi

Brown

1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

ROMK1, also known as Kir 1.1, is an inwardly rectifying K ؉ channel and is the prototypical member of the large Kir gene family. The accepted model of Kir topology predicts intracellular NH 2 and COOH termini, and two membrane-spanning segments, M1 and M2, connected by an intramembranous pore-forming segment, H5. The sequence of H5 is similar in voltage-dependent K ؉ channels and features a strictly conserved GY/FG in its mid-region, which has been proposed as the selectivity filter of the pore. We have been using N-glycosylation substitution mutants to map the extracellular topology of ROMK1 biochemically and have described several loci in H5 that were glycosylated. We now report glycosylation at loci Tyr 144 and Phe 146 , which indicates that the signature GYG sequence (143-145) rather than being intramembranous is extracellular. The COOH terminus was predicted to begin at position 178, but contrary to the model, we observed that position 257 was glycosylated and surrounding positions at 199, 222, and 298 were unglycosylated. N-Glycosylation sequon substitution at the latter three positions abolished K ؉ /Na ؉ selectivity. Our results suggest a major revision of the topology of ROMK1 with H5 and the pore signature sequence now completely extracellular. The COOH terminus appears to form two additional membrane-spanning segments and to contribute to the ion conduction pathway.ROMK1 is a weak, inwardly rectifying ATP-regulated K ϩ channel that was cloned from rat kidney outer medulla (1). There are three isoforms, which differ in their NH 2 terminus as a result of alternative splicing (2, 3). The isoforms are differentially expressed along the loop of Henle and distal nephron (4). In the thick ascending loop of Henle, ROMK provides K ϩ to the Na-K-2Cl cotransporter and thereby enhances Na ϩ uptake (5, 6). This physiological role is supported by mutations in ROMK that have been linked to the antenatal form of Bartter's syndrome, characterized by hypokalemic metabolic alkalosis, hypotension, renal salt wasting, and hypercalciuria (7, 8). Furthermore, characterization of some of these Bartter's ROMK1 mutations revealed disruption of function by phosphorylation, proteolytic processing, and protein transport. The topological model of ROMK1 and related members of the Kir gene family was predicted from hydropathy plots and, in the case of H5, from sequence similarity to Kv channels 2 (Fig. 1A). The NH 2 and COOH termini were cytoplasmic, based on the absence of a signal sequence in the open reading frame, and the ␣-helical transmembrane segments M1 and M2 were separated by a linker containing a 17-residue stretch called H5 (9 -13). In Kv channels, H5 is thought to form an intramembranous hairpin structure with the selectivity filter at the highly conserved GYG, which is at loci 143-145 of ROMK1. Recently, we showed that several H5 loci of ROMK1 could be glycosylated and that one of them, Q139N, in both its glycosylated and unglycosylated forms was nonselective between K ϩ and Na ϩ (14). That H5 is a hotspot for residue...

show abstract

Mutations in the pore region of ROMK enhance Ba2+ block

Cited by 78 publications

References 16 publications

Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External Ba2+

Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External Ba2+

Three Mechanisms Underlie KCNQ2/3 Heteromeric Potassium M-Channel Potentiation

Mapping the Kidney Potassium Channel ROMK1

Contact Info

Product

Resources

About