Runping Wang

Hypercapnic Acidosis Activates KATP Channels in Vascular Smooth Muscles

Abstract —ATP‐sensitive K+ channels (KATP) couple intermediary metabolism to cellular activity, and may play a role in the autoregulation of vascular tones. Such a regulation requires cellular mechanisms for sensing O2, CO2, and pH. Our recent studies have shown that the pancreatic KATP isoform (Kir6.2/SUR1) is regulated by CO2/pH. To identify the vascular KATP isoform(s) and elucidate its response to hypercapnic acidosis, we performed these studies on vascular smooth myocytes (VSMs). Whole‐cell and single‐channel currents were studied on VSMs acutely dissociated from mesenteric arteries and HEK293 cells expressing Kir6.1/SUR2B. Hypercapnic acidosis activated an inward rectifier current that was K+‐selective and sensitive to levcromakalim and glibenclamide with unitary conductance of ≈35pS. The maximal activation occurred at pH 6.5 to 6.8, and the current was inhibited at pH 6.2 to 5.9. The cloned Kir6.1/SUR2B channel responded to hypercapnia and intracellular acidification in an almost identical pattern to the VSM current. In situ hybridization histochemistry revealed expression of Kir6.1/SUR2B mRNAs in mesenteric arteries. Hypercapnia produced vasodilation of the isolated and perfused mesenteric arteries. Pharmacological interference of the KATP channels greatly eliminated the hypercapnic vasodilation. These results thus indicate that the Kir6.1/SUR2B channel is a critical player in the regulation of vascular tones during hypercapnic acidosis. (Circ Res. 2003;92:1225–1232.)

CO2 central chemosensitivity: why are there so many sensing molecules?

CO2 central chemoreceptors (CCRs) play a critical role in respiratory and cardiovascular controls. Although the primary sensory cells and their neuronal networks remain elusive, recent studies have begun to shed insight into the molecular mechanisms of several pH sensitive proteins. These putative CO2/pH-sensing molecules are expressed in the brainstem, detect P(CO2) at physiological levels, and couple the P(CO2) to membrane excitability. Functional analysis suggests that multiple CO2/pH-sensing molecules are needed to achieve high sensitivity and broad bandwidth of the CCRs. In contrast to the diversity of pH sensitive molecules, molecular mechanisms for CO2 sensing are rather general. The sensing molecules detect pH changes rather than molecular CO2. One or a few titratable amino acid residues in these proteins are usually involved. Protonation of these residues may lead to a change in protein conformation that is coupled to a change in channel activity. Depending on the location of the protonation sites, a membrane protein can detect extra- and/or intracellular pH.

Distinct Histidine Residues Control the Acid-induced Activation and Inhibition of the Cloned KATP Channel

The modulation of KATP channels during acidosis has an impact on vascular tone, myocardial rhythmicity, insulin secretion, and neuronal excitability. Our previous studies have shown that the cloned Kir6.2 is activated with mild acidification but inhibited with high acidity. The activation relies on His-175, whereas the molecular basis for the inhibition remains unclear. To elucidate whether the His-175 is indeed the protonation site and what other structures are responsible for the pH-induced inhibition, we performed these studies. Our data showed that the His-175 is the only proton sensor whose protonation is required for the channel activation by acidic pH. In contrast, the channel inhibition at extremely low pH depended on several other histidine residues including His-186, His-193, and His-216. Thus, proton has both stimulatory and inhibitory effects on the Kir6.2 channels, which attribute to two sets of histidine residues in the C terminus.

A Threonine Residue (Thr71) at the Intracellular End of the M1 Helix Plays a Critical Role in the Gating of Kir6.2 Channels by Intracellular ATP and Protons

ATP-sensitive K+ (KATP) channels are known to be gated by several intracellular molecules, but the gating mechanisms remain unclear. To understand the relationship of channel gating to ligand binding, we studied Kir6.2 channel gating by ATP and protons, which inhibit and activate the channel, respectively. We have previously shown that a threonine residue (Thr71) is critical for the pH sensitivity of Kir6.2 channel. If this site is involved in channel gating rather than ligand binding, it should affect channel gating by both ATP and proton. To test this hypothesis we performed a mutation analysis. Site-specific mutations of Thr71 to a bulky residue reduced the ATP sensitivity by >100-fold and eliminated the pH sensitivity. Single-channel activity of these mutants was stabilized at the open state with no detectable rundown. Mutations to a small amino acid had little effect on the ATP and pH sensitivities. Mutations to intermediate amino acids reduced but did not abolish the ATP and pH sensitivities. Hydrophobicity is not critical, as both polar and nonpolar amino acids are found in each group. Mutation to a positively charged lysine markedly exacerbated the pH- but not ATP-sensitivity, whereas mutation to glutamate moderately reduced ATP and pH sensitivities. These results indicate that the residue mass is critical for Kir6.2 channel gating, a mass that should be below 120 daltons with no charge. The existence of such a site as Thr71 involved in channel gating by both ATP and proton suggests that channel gating in the KATP channel likely is separate from ligand binding.

Subunit-stoichiometric evidence for kir6.2 channel gating, ATP binding, and binding-gating coupling.

ATP-sensitive K+ channels are gated by intracellular ATP, allowing them to couple intermediary metabolism to cellular excitability, whereas the gating mechanism remains unclear. To understand subunit stoichiometry for the ATP-dependent channel gating, we constructed tandem-multimeric Kir6.2 channels by selective disruption of the binding or gating mechanism in certain subunits. Stepwise disruptions of channel gating caused graded losses in ATP sensitivity and increases in basal Popen, with no effect on maximum ATP inhibition. Prevention of ATP binding lowered the ATP sensitivity and maximum inhibition without affecting basal Popen. The ATP-dependent gating required a minimum of two functional subunits. Two adjacent subunits are more favorable for ATP binding than two diagonal ones. Subunits showed negative cooperativity in ATP binding and positive cooperativity in channel gating. Joint disruptions of the binding and gating mechanisms in the same or alternate subunits of a concatemer revealed that both intra- and intersubunit couplings contributed to channel gating, although the binding-gating coupling preferred the intrasubunit to intersubunit configuration within the C terminus. No such preference was found between the C and N termini. These phenomena are well-described with the operational model used widely for ligand-receptor interactions.

Critical protein domains and amino acid residues for gating the KIR6.2 channel by intracellular ATP

KATP channels couple intermediary metabolism to cellular excitability. Such a property relies on the inherent ATP‐sensing mechanism known to be located in the Kir6 subunit. However, the molecular basis for the ATP sensitivity remains unclear. Here we showed evidence for protein domains and amino acid residues essential for the channel gating by intracellular ATP. Chimerical channels were constructed using protein domains of Kir6.2 and Kir1.1, expressed in HEK293 cells, and studied in inside‐out patches. The N and C termini, although important, were inadequate for channel gating by intracellular ATP. Full ATP sensitivity also required M1 and M2 helices. Cytosolic portions of the M1 and M2 sequences were crucial, in which six amino acid residues were identified, i.e., Thr76, Met77, Ala161, Iso162, Leu164, and Cys166. Site‐specific mutation of any of them reduced the ATP sensitivity. Construction of these residues together with the N/C termini produced ATP sensitivity identical to the wild‐type channels. The requirement for specific membrane helices suggests that the Kir6.2 gating by ATP is not shared by even two closest relatives in the K+ channel family, although the general gating mechanisms involving membrane helices appear to be conserved in all K+ channels. J. Cell. Physiol. 198: 73–81, 2004.

Determinant Role of Membrane Helices in KATP Channel Gating

The ATP-sensitive K+ (KATP) channels couple chemical signals to cellular activity, in which the control of channel opening and closure (i.e., channel gating) is crucial. Transmembrane helices play an important role in channel gating. Here we report that the gating of Kir6.2, the core subunit of pancreatic and cardiac KATP channels, can be switched by manipulating the interaction between two residues located in transmembrane domains (TM) 1 and 2 of the channel protein. The Kir6.2 channel is gated by ATP and proton, which inhibit and activate the channel, respectively. The channel gating involves two residues, namely, Thr71 and Cys166, located at the interface of the TM1 and TM2. Creation of electrostatic attraction between these sites reverses the channel gating, which makes the ATP an activator and proton an inhibitor of the channel. Electrostatic repulsion with two acidic residues retains or even enhances the wild-type channel gating. A similar switch of the pH-dependent channel gating was observed in the Kir2.1 channel, which is normally pH- insensitive. Thus, the manner in which the TM1 and TM2 helices interact appears to determine whether the channels are open or closed following ligand binding.

Subunit stoichiometry of the Kir1.1 channel in proton-dependent gating

Kir1.1 channel regulates membrane potential and K+ secretion in renal tubular cells. This channel is gated by intracellular protons, in which a lysine residue (Lys80) plays a critical role. Mutation of the Lys80 to a methionine (K80M) disrupts pH-dependent channel gating. To understand how an individual subunit in a tetrameric channel is involved in pH-dependent channel gating, we performed these studies by introducing K80M-disrupted subunits to tandem tetrameric channels. The pH sensitivity was studied in whole-cell voltage clamp and inside-out patches. Homomeric tetramers of the wild-type (wt) and K80M-disrupted channels showed a pH sensitivity almost identical to that of their monomeric counterparts. In heteromeric tetramers and dimers, pH sensitivity was a function of the number of wt subunits. Recruitment of the first single wt subunit shifts the pKa greatly, whereas additions of any extra wt subunit had smaller effects. Single-channel analysis revealed that the tetrameric channel with two or more wt subunits showed one substate conductance at ∼40% of the full conductance, suggesting that four subunits act as two pairs. However, three and four substates of conductance were seen in the tetrameric wt-3K80M and 4K80M channels. Acidic pH increased long-time closures when there were two or more wt subunits. Disruption of more than two subunits led to flicking activity with appearance of a new opening event and loss of the long period of closures. Interestingly, the channel with two wt subunits at diagonal and adjacent configurations showed the same pH sensitivity, substate conductance, and long-time closure. These results thus suggest that one functional subunit is sufficient to act in the pH-dependent gating of the Kir1.1 channel, the channel sensitivity to pH increases with additional subunits, the full pH sensitivity requires contributions of all four subunits, and two subunits may be coordinated in functional dimers of either trans or cis configuration.

Kir6.2 Channel Gating by Intracellular Protons: Subunit Stoichiometry for Ligand Binding and Channel Gating

The adenosine triphosphate-sensitive K+ (KATP) channels are gated by several metabolites, whereas the gating mechanism remains unclear. Kir6.2, a pore-forming subunit of the KATP channels, has all machineries for ligand binding and channel gating. In Kir6.2, His175 is the protonation site and Thr71 and Cys166 are involved in channel gating. Here, we show how individual subunits act in proton binding and channel gating by selectively disrupting functional subunits using these residues. All homomeric dimers and tetramers showed pH sensitivity similar to the monomeric channels. Concatenated construction of wild type with disrupted subunits revealed that none of these residues had a dominant-negative effect on the proton-dependent channel gating. Subunit action in proton binding was almost identical to that for channel gating involving Cys166, suggesting a one-to-one coupling from the C terminus to the M2 helix. This was significantly different from the effect of T71Y heteromultimers, suggesting distinct contributions of M1 and M2 helices to channel gating. Subunits underwent concerted rather than independent action. Two wild-type subunits appeared to act as a functional dimer in both cis and trans configurations. The understanding of KATP channel gating by intracellular pH has a profound impact on cellular responses to metabolic stress as a significant drop in intracellular pH is more frequently seen under a number of physiological and pathophysiological conditions than a sole decrease in intracellular ATP levels.