Dibasic protein kinase A sites regulate bursting rate and nucleotide sensitivity of the cystic fibrosis transmembrane conductance regulator chloride channel

Abstract

1. The relationship between phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and its gating by nucleotides was examined using the patch clamp technique by comparing strongly phosphorylated wild-type (WT) channels with weakly phosphorylated mutant channels lacking four (4SA) or all ten (10SA) dibasic consensus sequences for phosphorylation by protein kinase A (PKA). 2. The open probability (Po) of strongly phosphorylated WT channels in excised patches was about twice that of 4SA and 10SA channels, after correcting for the number of functional channels per patch by addition of adenylylimidodiphosphate (AMP-PNP). The mean burst durations of WT and mutant channels were similar, and therefore the elevated Po of WT was due to its higher bursting rate. 3. The ATP dependence of the 10SA mutant was shifted to higher nucleotide concentrations compared with WT channels. The relationship between Po and [ATP] was noticeably sigmoid for 10SA channels (Hill coefficient, 1.8), consistent with positive co-operativity between two sites. Increasing ATP concentration to 10 mM caused the Po of both WT and 10SA channels to decline. 4. Wild-type and mutant CFTR channels became locked in open bursts when exposed to mixtures of ATP and the non-hydrolysable analogue AMP-PNP. The rate at which the low phosphorylation mutants became locked open was about half that of WT channels, consistent with Po being the principal determinant of locking rate in WT and mutant channels. 5. We conclude that phosphorylation at 'weak' PKA sites is sufficient to sustain the interactions between the ATP binding domains that mediate locking by AMP-PNP. Phosphorylation of the strong dibasic PKA sites controls the bursting rate and Po of WT channels by increasing the apparent affinity of CFTR for ATP.

Figure 1. Effect of eliminating dibasic PKA consensus sequences on in vivo phosphorylation

A, cartoon showing the locations of dibasic consensus PKA sites altered in the phosphorylation-deficient channels; open circles, 4SA; all circles, 10SA. B, each of the three cell lines (WT, 4SA and 10SA) were grown on 60 mm dishes. Following washes with phosphate-free medium, cells were incubated for 4 h at 37 °C in 1.0 ml of the same medium containing 300 μCi [³²P]orthophosphate (see Methods for details). Agonists were added to plates marked (+) at 15 min before the end of the 4 h labelling period. Vehicle alone (dimethylsulphoxide) was added to the cell controls marked (-). Proteins were immunoprecipitated with monoclonal antibody (M3A7) and analysed on an SDS-polyacrylamide gel. After electrophoresis, the 7% polyacrylamide gel was dried and exposed to X-ray film for 14 h at -80 °C. Lane c indicates host CHO cells not expressing CFTR. Values to the left are in kDa. C, graph of densitometry readings for the autoradiograph shown in B. (-) indicates WT and mutant cells which were exposed to vehicle alone, and (+) indicates cells in which PKA levels were increased by the addition of agonists (10 μM forskolin, 200 μM dibutyryl-cAMP and 1 mM IBMX).

Figure 3. Validation of the method used to calculate kinetics with multichannel patches

A, continuous 32 min recording from one 10SA channel. B, histogram of burst durations for the data shown in A computed using the threshold crossing method after excluding flickery closures (< 9.2 ms). The mean burst duration was 864 ms according to this method and 900 ms when calculated using eqn (2); see Methods for details.

Figure 6. Channel locking after addition of AMP-PNP

A, representative trace showing activity of WT CFTR channels in a patch excised into a recording chamber containing standard buffer, 180 nM PKA and 1 mM MgATP. AMP-PNP (1 mM) was added as indicated and channels were progressively locked in the open state. B, expanded traces of data shown in A before (a), and immediately after (b) addition of AMP-PNP. Some baseline drift was usually observed following addition of AMP-PNP, as indicated in b by the lines to the left and right of the trace. The times at which channels became locked open are indicated by arrows. Expanded traces have been fast Fourier transform (FFT) filtered at 50 Hz (using Microcal Origin 4.1) for presentation purposes.

Figure 7. Comparison of locking rates for wild-type and mutant CFTR channels

A, traces obtained from excised, inside-out patches from cells expressing WT, 4SA or 10SA channels showing the locking open induced by AMP-PNP. PKA (180 nM) and MgATP (1 mM) were present throughout and AMP-PNP (1 mM) was added at the arrows. The segments indicated by a, b, c and d are shown on expanded scale in panel B. Zero current levels are indicated by lines alongside the traces. C, rates of locking were calculated by determining the latency between addition of AMP-PNP and the locking of wild-type (○, n = 47 channels, 6 patches), 4SA (□, n = 38 channels, 8 patches), or 10SA channels (▿, n = 40 channels, 10 patches).

Figure 2. Single-channel activity of WT and mutant CFTR

A, recordings obtained using inside-out patches excised from cells expressing wild-type (WT) or mutant 4SA or 10SA channels. The bath contained 1 mM MgATP and 180 nM PKA catalytic subunit. In this and subsequent figures the closed channel current level is indicated by lines to the left of traces and channel openings are downwards. B, open probability estimated for WT and mutant channels before and after correcting N by locking channels open with 1 mM AMP-PNP. n = 6 patches for WT and 8 patches for each mutant.

Figure 4. Reduced P_o of mutant channels results from altered interburst durations

Mean burst and interburst durations calculated for WT and phosphorylation-deficient mutant channels (4SA, 10SA).

Figure 5. Relationship between ATP concentration and P_o for WT and 10SA mutant channels

Dotted lines show Michaelis-Menten fits to data pooled from 3-9 patches at each concentration between 0 and 5 mM MgATP. WT data were best fitted by an expression containing two K_M (•; continuous line), while 10SA data were best fitted by the Hill equation (▪; continuous line). The values of P_o determined with 10 mM MgATP present are also shown but were not used for fitting. All P_o values were corrected for the maximum number of channels in each patch during exposure to 1 mM AMP-PNP. The number of experiments at each ATP concentration was 3-9 for WT, and 3-7 for 10SA. The bath contained PKA (180 nM) and 2 mM MgCl₂ throughout each experiment, and MgATP was added from a 100 mM stock solution.