J Gen Physiol. 2000 Sep;116(3):477-500.

Severed channels probe regulation of gating of cystic fibrosis transmembrane conductance regulator by its cytoplasmic domains.

Csanády L, Chan KW, Seto-Young D, Kopsco DC, Nairn AC, Gadsby DC.

Source

Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, New York 10021-6399, USA.

Abstract

Opening and closing of a CFTR Cl(-) channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains were probed by characterizing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of oocyte conductance, and detailed functional characteristics of the channels were extracted from kinetic analyses of macroscopic current relaxations and of single-channel gating events in membrane patches excised from the oocytes. The kinetic behavior of wild-type (WT) CFTR channels was compared with that of split CFTR channels bearing a single cut (between residues 633 and 634) just before the R domain, of split channels with a single cut (between residues 835 and 837) just after the R domain, and of split channels from which the entire R domain (residues 634-836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost identical to those of WT channels, except for less than twofold shorter open burst durations in the presence of PKA. Channels cut just after the R domain were characterized by a low level of activity even without phosphorylation, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the locking action of the nonhydrolyzable ATP analog AMPPNP. Split channels with no R domain (from coexpression of CFTR segments 1-633 and 837-1480) were highly active without phosphorylation, but otherwise displayed the characteristics of channels cut after the R domain, including higher apparent ATP affinity, and less tight binding of AMPPNP at the locking site, than for WT. Intriguingly, severed channels with no R domain were still noticeably stimulated by PKA, implying that activation of WT CFTR by PKA likely also includes some component unrelated to the R domain. As the maximal opening rates were the same for WT channels and split channels with no R domain, it seems that the phosphorylated R domain does not stimulate opening of CFTR channels; rather, the dephosphorylated R domain inhibits them.

PMID:: 10962022; [PubMed - indexed for MEDLINE]
PMCID:: PMC2233695

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Scheme S2

The results presented here address the role of the R domain in regulating the interactions of nucleotides with the NBDs that somehow control opening and closing of CFTR channels. Before discussing their implications, an appropriate gating model must be chosen. Our data are consistent with models with two ATP binding sites, one responsible for opening the channel, the other for stabilizing its open (burst) state. Three such gating schemes have been proposed recently (Gadsby and Nairn 1999; Weinreich et al. 1999; Zeltwanger et al. 1999) that, although differing in detail, share common features (Hwang et al. 1994; Gadsby and Nairn 1994), including assigning NBD1 the principal role of channel opening and NBD2 that of channel closing. The latter is supported by the prolonged open bursts that result from mutations of the NBD2 Walker-A lysine, K1250 (Carson et al. 1995; Gunderson and Kopito 1995), known to disrupt ATP hydrolysis there (Ramjeesingh et al. 1999). All three schemes propose that two steps precede channel opening: first, the site responsible for opening (presumed to be NBD1), empty at the start of each interburst closure (state C₁), reversibly binds ATP (state C₂), after which a rate-limiting step (likely related to hydrolysis) leads to channel opening (state O₁). Open channels can then either simply close (back to state C₁) by losing the hydrolysis products from NBD1 (resulting in brief openings), or bind ATP at a second site (presumed to be NBD2), resulting in stabilization of the open state (state O₂; long openings). After hydrolysis of ATP at NBD2, in one model (Zeltwanger et al. 1999), hydrolysis products at NBD2 are lost first, returning the channel to O₁, from which it can either close (to C₁) or rebind ATP at NBD2 (returning to O₂). In the other two models, ATP hydrolysis at NBD2 results in loss of hydrolysis products from both NBDs, and channel closure (back to C₁). The latter models can be simplified to Fig. 1, while that of Zeltwanger et al. 1999 reduces to Fig. 2.

Severed Channels Probe Regulation of Gating of Cystic Fibrosis Transmembrane Conductance Regulator by Its Cytoplasmic Domains

J Gen Physiol. J Gen Physiol;116(3):477-500.

Scheme S1

Severed Channels Probe Regulation of Gating of Cystic Fibrosis Transmembrane Conductance Regulator by Its Cytoplasmic Domains

J Gen Physiol. J Gen Physiol;116(3):477-500.

Figure 11

Model fit, using Fig. 1, to results from WT and severed CFTR channels in the presence of PKA. The set of rate constants (s⁻¹) giving the closest overall fit to the indicated set of observed parameters is printed on Fig. 1 for each construct (rounded to two significant digits); measured and predicted parameters are compared on the right. Parameters a_sh, a_l, τ_sh, and τ_l are fractional amplitudes and time constants of exponential components describing the distributions (Fig. 8) of burst durations (note a_l = 1 − a_sh is not a free parameter); τ_b is mean burst duration, measured in the presence of PKA for WT and 633+634, or pooled from all experiments for 835+837, cut-ΔR (633+837), and Flag-cut-ΔR (F633+837); τ_AMPPNP and a_locked are the time constant and fractional amplitude of the slowly relaxing macroscopic current component after removal of AMPPNP and ATP (see Table ); τ_relax and a_relax, analogous, after just ATP, for cut-ΔR(K1250A) [633+837(K1250A); Fig. 10 B]. For step C₁ ↔ C₂, K_{P_o}is printed instead of K_d. Errors for observed parameters a_sh, a_l, τ_sh, and τ_l are half-widths of 0.5 unit likelihood intervals, calculated separately for each parameter (reasonably symmetric around the optimum, given the numbers of fitted events); for τ_b, τ_AMPPNP, and a_locked errors are ±SEM. The value of k₁ was obtained as the reciprocal of τ_ib in the presence of 2 mM MgATP and PKA (except for cut-ΔR(K1250A) channels, for which all data were obtained in the absence of PKA; apparent affinity was not measured for this construct). *Rate k₂ for WT and 633+634 may be much lower than 200 s⁻¹. From simulations, k₂ > 20 s⁻¹ is large enough to account for the apparently uniform distribution of burst durations of these two constructs in the presence of PKA. **Rate k₋₁ for WT and 633+634 channels was fixed to the reciprocal of the mean burst duration observed after removal of PKA, on the assumption that k₂ << k₋₁ under those conditions. ***Rate k₃, representing a compound step including ATP hydrolysis at NBD2, was set to zero for cut-ΔR(K1250A).

Severed Channels Probe Regulation of Gating of Cystic Fibrosis Transmembrane Conductance Regulator by Its Cytoplasmic Domains

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