Requirement of Multiple Protein Domains and Residues for GatingKATP Channels by Intracellular pH

Abstract

ATP-sensitive K؉ channels (K ATP ) are regulated by pH in addition to ATP, ADP, and phospholipids. In the study we found evidence for the molecular basis of gating the cloned K ATP by intracellular protons. Systematic constructions of chimerical Kir6.2-Kir1.1 channels indicated that full pH sensitivity required the N terminus, C terminus, and M2 region. Three amino acid residues were identified in these protein domains, which are Thr-71 in the N terminus, Cys-166 in the M2 region, and His-175 in the C terminus. Mutation of any of them to their counterpart residues in Kir1.1 was sufficient to completely eliminate the pH sensitivity. Creation of these residues rendered the mutant channels clear pHdependent activation. Thus, critical players in gating K ATP by protons are demonstrated. The pH sensitivity enables the K ATP to regulate cell excitability in a number of physiological and pathophysiological conditions when pH is low but ATP concentration is normal.1 is a unique member in the K ϩ channel family, which directly couples the intermediary metabolism to cellular excitability (1, 2). Such a property enables the K ATP to play an important role in regulating vascular tone, skeletal muscle contractility, insulin secretion, epithelial transport, and neuronal excitability under a variety of physiological and pathophysiological conditions (3-7). Although ATP is the primary regulator of the K ATP , several other cytosolic factors are also involved in the control of channel activity, including ADP, phospholipids, and hydrogen ion (8 -20). Our recent studies have shown that the cloned K ATP also responds to acidic pH (21). These channels are strongly stimulated by hypercapnia and intracellular acidosis. The pH sensitivity is independent of the SUR subunit and other cytosolic factors, suggesting that the pH sensing mechanisms are located in the Kir (inward rectifier K ϩ channel) subunit (21). If the pH sensitivity is an inherent property of Kir6 proteins, there should be special structures responsible for channel gating by protons. These structures are likely to be located in the Kir6 subunit, since the SUR subunit is not required for the pH sensitivity. To identify these structures, we performed these experiments in which we used the Kir6.2 with a truncation of 36 amino acids at the C-terminal end, i.e. Kir6.2⌬C36 that expresses functional channels without the SUR subunit and retains fair ATP sensitivity (22). Several chimerical channels were generated based on peptide sequences of Kir6.2 and Kir1.1, a Kir channel that is inhibited by intracellular acidosis (23-25) and has been shown to express functional channels in its chimeras with Kir6.2 (26). These chimeras were studied in whole-cell recording using hypercapnia, a condition that does not cause channel rundown (21). Our results indicate that there are three separate protein domains in the Kir6.2 protein that are crucial for the pH sensitivity. We have identified critical amino acid residues within each of these protein domains. Replacement of any of them with their...

A Short Motif in Kir6.1 Consisting of Four Phosphorylation Repeats Underlies the Vascular KATP Channel Inhibition by Protein Kinase C

“…The Kir6.2 channels are also activated by intracellular H ϩ (30). The pH-dependent channel gating requires both N and C termini, although there is only one protonation site (His-175) located in the C terminus (21,31). The importance of the proximal N terminus in channel gating has been studied with different ligands, indicating that this region is indeed involved in Kir6.2 channel gating rather than ligand binding (32,33).…”

“…The cDNAs were cloned in the eukaryotic expression vector pcDNA3.1 and used for mammalian cell expression (5, 6). Kir6.1-Kir6.2 chimeras were produced by overlap extension using PCR (Pfu DNA polymerase; Stratagene, La Jolla, CA) (21). Site-specific mutations were made using a site-directed mutagenesis kit (Stratagene).…”

KCNJ11 activating mutations are associated with developmental delay, epilepsy and neonatal diabetes syndrome and other neurological features

“…Functional studies have demonstrated that the mutations identified in patients with syndromic PNDM (V59G, G52R, I296L) result in a larger K ATP current and greater change in ATP sensitivity than those which cause PNDM alone (R201C), suggesting that larger K ATP currents are required to influence extrapancreatic cell function. 9,15 Residue C166 has been extensively mutated 16,17 and functional studies have shown that the C166F mutation results in a channel with a marked increase in open probability (0.77 vs 0.11) and a reduced ATP sensitivity (Ki ATP , 5.3 mM vs wild type 175 mM) where Ki ATP is the ATP concentration at which inhibition is half-maximal. 17 The fact that the channel open probability is increased implies a role for this residue in channel gating.…”

Subunit-Stoichiometric Evidence for Kir6.2 Channel Gating, ATP Binding, and Binding-Gating Coupling

“…In contrast, the Cys166 located in the transmembrane 2 region (Supplemental Fig. S1) is known to participate in the channel gating or the final stage of signal transduction, because the C166S mutation disrupts K ATP channel gating by ATP, proton, and sulfonylurea Piao et al, 2001;Wu et al, 2004). Likewise, the Thr71 at the intracellular end of the transmembrane 1 region is likely to act in channel gating by ATP and protons as well (Cui et al, 2003;Wang et al, 2005b).…”