Elena N. Makhina

Inward Rectification and Implications for Cardiac Excitability

Since the cloning of the first inwardly rectifying K+ channel in 1993, a family of related clones has been isolated, with many members being expressed in the heart. Exogenous expression of different clones has demonstrated that between them they encode channels with the essential functional properties of classic inward rectifier channels, ATP-sensitive K+ channels, and muscarinic receptor-activated inward rectifier channels. High-level expression of cloned channels has led to the discovery that classic strong inward, or anomalous, rectification is caused by very steeply voltage-dependent block of the channel by polyamines, with an additional contribution by Mg2+ ions. Knowledge of the primary structures of inward rectifying channels and the ability to mutate them have led to the determination of many of the structural requirements of inward rectification. The implications of these advances for basic understanding and pharmacological manipulation of cardiac excitability may be significant. For example, cellular concentrations of polyamines are altered under different conditions and can be manipulated pharmacologically. Simulations predict that changes in polyamine concentrations or changes in the relative proportions of each polyamine species could have profound effects on cardiac excitability.

Structure and dynamics of the pore of inwardly rectifying K(ATP) channels.

Inwardly rectifying K+ currents are generated by a complex of four Kir (Kir1–6) subunits. Pore properties are conferred by the second transmembrane domain (M2) of each subunit. Using cadmium ions as a cysteine-interacting probe, we examined the accessibility of substituted cysteines in M2 of the Kir6.2 subunit of inwardly rectifying KATP channels. The ability of Cd2+ ions to inhibit channels was used as the estimate of accessibility. The distribution of Cd2+ accessibility is consistent with an α-helical structure of M2. The apparent surface of reactivity is broad, and the most reactive residues correspond to the solvent-accessible residues in the bacterial KcsA channel crystal structure. In several mutants, single channel measurements indicated that inhibition occurred by a single transition from the open state to a zero-conductance state. Analysis of currents expressed from mixtures of control and L164C mutant subunits indicated that at least three cysteines are required for coordination of the Cd2+ ion. Application of phosphatidylinositol 4,5-diphosphate to inside-out membrane patches stabilized the open state of all mutants and also reduced cadmium sensitivity. Moreover, the Cd2+ sensitivity of several mutants was greatly reduced in the presence of inhibitory ATP concentrations. Taken together, these results are consistent with state-dependent accessibility of single Cd2+ions to coordination sites within a relatively narrow inner vestibule.

Pancreatic Islet Cells Express a Family of Inwardly Rectifying K+ Channel Subunits Which Interact to Form G-protein-activated Channels

Insulin secretion is associated with changes in pancreatic β-cell K+ permeability. A degenerate polymerase chain reaction strategy based on the conserved features of known inwardly rectifying K+ (KIR) channel genes was used to identify members of this family expressed in human pancreatic islets and insulinoma. Three related human KIR transcript sequences were found: CIR (also known as cardiac KATP-1), GIRK1, and GIRK2 (KATP-2). The pancreatic islet CIR and GIRK2 full-length cDNAs were cloned, and their genes were localized to human chromosomes 11q23-ter and 21, respectively. Northern blot analysis detected CIR mRNA at similar levels in human islets and exocrine pancreas, while the abundance of GIRK2 mRNA in the two tissues was insufficient for detection by this method. Using competitive reverse-transcription polymerase chain reaction, CIR was found to be present at higher levels than GIRK2 mRNA in native purified β-cells. Xenopus oocytes injected with M2 muscarinic receptor (M2) plus either GIRK2 or CIR cRNA expressed only very small carbachol-induced currents, while co-injection of CIR plus GIRK2 along with M2 resulted in expression of carbachol-activated strong inwardly rectifying currents. Activators of KATP channels failed to elicit currents in the presence or absence of co-expressed sulfonylurea receptor. These results show that two components of islet cell KIR channels, CIR and GIRK2, may interact to form heteromeric G-protein-activated inwardly rectifying K+ channels that do not possess the typical properties of KATP channels.

Inhibition of an Inward Rectifier Potassium Channel (Kir2.3) by G-protein βγ Subunits

The molecular basis of G-protein inhibition of inward rectifier K+ currents was examined by co-expression of G-proteins and cloned Kir2 channel subunits in Xenopus oocytes. Channels encoded by Kir2.3 (HRK1/HIR/BIRK2/BIR11) were completely suppressed by co-expression with G-protein βγ subunits, whereas channels encoded by Kir2.1 (IRK1), which shares 60% amino acid identity with Kir2.3, were unaffected. Co-expression of Gαi1 and Gαq subunits also partially suppressed Kir2.3 currents, but Gαt, Gαs, and a constitutively active mutant of Gαil (Q204L) were ineffective. Gβγ and Kir2.3 subunits were co-immunoprecipitated using an anti-Kir2.3 antibody. Direct binding of G-protein βγ subunits to fusion proteins containing Kir2.3 N terminus, but not to fusion proteins containing Kir2.1 N terminus, was also demonstrated. The results are consistent with suppression of Kir2.3 currents resulting from a direct protein-protein interaction between the channel and G-protein βγ subunits. When Kir2.1 and Kir2.3 subunits were coexpressed, the G-protein inhibitory phenotype of Kir2.3 was dominant, suggesting that co-expression of Kir2.3 with other Kir subunits might give rise to novel G-protein-inhibitable inward rectifier currents.

ATP Interaction with the Open State of the KATP Channel

The mechanism of ATP-sensitive potassium (K(ATP)) channel closure by ATP is unclear, and various kinetic models in which ATP binds to open or to closed states have previously been presented. Effects of phosphatidylinositol bisphosphate (PIP2) and multiple Kir6.2 mutations on ATP inhibition and open probability in the absence of ATP are explainable in kinetic models where ATP stabilizes a closed state and interaction with an open state is not required. Evidence that ATP can in fact interact with the open state of the channel is presented here. The mutant Kir6.2[L164C] is very sensitive to Cd2+ block, but very insensitive to ATP, with no significant inhibition in 1 mM ATP. However, 1 mM ATP fully protects the channel from Cd2+ block. Allosteric kinetic models in which the channel can be in either open or closed states with or without ATP bound are considered. Such models predict a pedestal in the ATP inhibition, i.e., a maximal amount of inhibition at saturating ATP concentrations. This pedestal is predicted to occur at >50 mM ATP in the L164C mutant, but at >1 mM in the double mutant L164C/R176A. As predicted, ATP inhibits Kir6.2[L164C/R176A] to a maximum of approximately 40%, with a clear plateau beyond 2 mM. These results indicate that ATP acts as an allosteric ligand, interacting with both open and closed states of the channel.

Domain Organization of the ATP-sensitive Potassium Channel Complex Examined by Fluorescence Resonance Energy Transfer

Background: We examined FRET between Kir6.2 and SUR1 domains of KATP channels, in various combinations. Results: FRET was detected within and between Kir6.2 subunits and between Kir6.2 and split SUR1 N-terminal constructs. Conclusion: Kir6.2 C termini are centrally located. SUR1 domains can self-associate and are close to Kir6.2 termini in the full complex. Significance: The results indicate domain architecture of this unique channel complex. KATP channels link cell metabolism to excitability in many cells. They are formed as tetramers of Kir6.2 subunits, each associated with a SUR1 subunit. We used mutant GFP-based FRET to assess domain organization in channel complexes. Full-length Kir6.2 subunits were linked to YFP or cyan fluorescent protein (CFP) at N or C termini, and all such constructs, including double-tagged YFP-Kir6.2-CFP (Y6.2C), formed functional KATP channels. In intact COSm6 cells, background emission of YFP excited by 430-nm light was ∼6%, but the Y6.2C construct expressed alone exhibited an apparent FRET efficiency of ∼25%, confirmed by trypsin digestion, with or without SUR1 co-expression. Similar FRET efficiency was detected in mixtures of CFP- and YFP-tagged full-length Kir6.2 subunits and transmembrane domain only constructs, when tagged at the C termini but not at the N termini. The FRET-reported Kir6.2 tetramer domain organization was qualitatively consistent with Kir channel crystal structures: C termini and M2 domains are centrally located relative to N termini and M1 domains, respectively. Additional FRET analyses were performed on cells in which tagged full-length Kir6.2 and tagged SUR1 constructs were co-expressed. These analyses further revealed that 1) NBD1 of SUR1 is closer to the C terminus of Kir6.2 than to the N terminus; 2) the Kir6.2 cytoplasmic domain is not essential for complexation with SUR1; and 3) the N-terminal half of SUR1 can complex with itself in the absence of either the C-terminal half or Kir6.2.

Novel tools for localizing ion channels in living cells

This is an exciting time in the development of techniques to examine ion channel distributions in cells and tissues. As outlined above, novel fluorescent, functional and structural tools are being developed for microscopic and even nanoscopic localization. Following recent discoveries of channel structural components and biophysical mechanisms of permeation and gating, such techniques will be crucial for a new wave of ion channel studies. These will seek to determine exact locations of ion channels and specific patterns of their membrane localization and co-localization with underlying structures as well as the temporal sequence of events of channel biogenesis.