ABCMdb

PMID: 11306662

Zhou Z, Hu S, Hwang TC
Voltage-dependent flickery block of an open cystic fibrosis transmembrane conductance regulator (CFTR) channel pore.
J Physiol. 2001 Apr 15;532(Pt 2):435-48., 2001-04-15 [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCC7 p.Lys1250Ala Fast flickery block of the cystic fibrosis transmembrane conductance regulator (CFTR) was studied with cell-attached and whole-cell patch-clamp recordings from mouse NIH3T3 cells stably expressing a mutant CFTR channel, K1250A-CFTR. Login to comment 12 ABCC7 p.Lys1250Ala Flickering block of K1250A-CFTR channels was voltage dependent since the open probability within an opening burst decreased as the membrane was hyperpolarized. Login to comment 22 ABCC7 p.Lys1250Ala Results from macroscopic current noise analysis of both wild-type CFTR and K1250A-CFTR channels further confirm the voltage dependence and Cl_ sensitivity of the fast flickery block observed with single-channel analysis. Login to comment 38 ABCC7 p.Lys1250Ala To overcome these technical difficulties, we characterized the kinetics of the flickery blockade using the CFTR mutant K1250A in which the conserved Walker A lysine (K) at amino acid position 1250 located in NBD2 is replaced by the neutral amino acid alanine (A). Login to comment 40 ABCC7 p.Lys1250Ala Within a long opening of K1250A-CFTR, fast flickers are clearly discernible (Zeltwanger et al. 1999). Login to comment 42 ABCC7 p.Lys1250Ala In the present paper, fast flickery block of CFTR was studied with cell-attached and whole-cell patch-clamp techniques in NIH3T3 cells stably expressing wild-type or K1250A-CFTR. Login to comment 46 ABCC7 p.Lys1250Ala Using spectrum analysis of macroscopic K1250A-CFTR and wild-type CFTR currents, we obtained corner frequencies (fc) at different membrane potentials. Login to comment 47 ABCC7 p.Lys1250Ala The measured fc agreed well with the fc calculated from the single-channel kinetic parameters obtained for K1250A-CFTR channels. Login to comment 50 ABCC7 p.Lys1250Ala METHODS Cell culture NIH3T3 cell lines stably expressing wild-type CFTR (Anderson et al. 1991) or K1250A-CFTR (Zeltwanger et al. 1999) were grown at 37°C and 5 % CO2 in Dulbecco`s Modified Eagle`s Medium (DMEM) supplemented with 10% fetal bovine serum. Login to comment 62 ABCC7 p.Lys1250Ala To obtain single-channel recordings, we activated multiple K1250A-CFTR channels with 10 µM forskolin, then washed out the forskolin and observed current decay. Login to comment 64 ABCC7 p.Lys1250Ala Under this condition, once the K1250A-CFTR channel closes from the activated state, it remains closed unless forskolin is re-applied. Login to comment 97 ABCC7 p.Lys1250Ala Noise analysis Macroscopic K1250A-CFTR or wild-type CFTR currents in cell-attached patches were elicited with 10 µM forskolin. Login to comment 99 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala At least 60 s of K1250A-CFTR current recordings or 180 s of wild-type CFTR current recordings were fast Fourier transformed to generate noise spectra that were further analysed in a bandwidth of 9.7-600 Hz for K1250A-CFTR or 3.9-800 Hz for wild-type CFTR with Igor software. Login to comment 100 ABCC7 p.Lys1250Ala Data from K1250A-CFTR were fitted with a single Lorentzian function to estimate the Lorentzian parameters: S(f) = S0/(1 + (f/fc)2 ) + S1, where fc is the corner frequency, S0 is the zero-frequency asymptote, S1 is non-specific basal noise and S(f) is the spectral density at the frequency f. Data from wild-type CFTR were fitted with the sum of two Lorentzian components: S(f) = S0/(1 + (f/fc1)2 ) + S1/(1 + (f/fc2)2 ) + S2, where S0 and S1 are the zero-frequency asymptotes corresponding to fc1 and fc2, respectively, and S2 is the basal noise. Login to comment 101 ABCC7 p.Lys1250Ala RESULTS Voltage-dependent fast flickery block of the K1250A-CFTR channels It has been shown previously that in cell-attached patches, the inward CFTR current shows more flickering events than the outward current, and that the number of these fast flickers is dramatically reduced upon patch excision (Haws et al. 1992; Fischer & Machen, 1994, 1996). Login to comment 102 ABCC7 p.Lys1250Ala The flickering block of K1250A-CFTR channels shares these two characteristic features with wild-type channels. Login to comment 103 ABCC7 p.Lys1250Ala Figure 1A shows that the fast flickery events present in a cell-attached patch from NIH3T3 cells stably expressing K1250A-CFTR channels were reduced in frequency upon patch excision. Login to comment 109 ABCC7 p.Lys1250Ala The fast flickery block of K1250A-CFTR channels also showed clear voltage dependence when recorded in the cell-attached configuration. Login to comment 112 ABCC7 p.Lys1250Ala Fast flickery block of K1250A-CFTR channels A, the fast flickery events diminished dramatically after patch excision. Login to comment 113 ABCC7 p.Lys1250Ala A cell-attached patch from an NIH3T3 cell stably expressing the K1250A-CFTR channel was held at _70 mV (_Vp). Login to comment 121 ABCC7 p.Lys1250Ala Voltage dependence of the fast flickery block of K1250A-CFTR channels A, representative single-channel current traces at different potentials (_Vp) recorded in the cell-attached configuration with 154 mM Cl_ pipette solution. Dotted lines indicate the baseline current level. Login to comment 132 ABCC7 p.Lys1250Ala In the present study, similar observations were made with the K1250A-CFTR channel. Login to comment 168 ABCC7 p.Lys1250Ala Voltage-dependent block of the whole-cell K1250A-CFTR current Our single-channel recordings were filtered at 100 Hz (dead time = 3 ms) in order to obtain a reasonable signal-to-noise ratio for dwell time analysis. Login to comment 172 ABCC7 p.Lys1250Ala Under symmetrical Cl_ conditions with 125 mM Cl_ in both the external and the internal solutions, the whole-cell instantaneous I-V relationship of K1250A-CFTR was linear whereas the steady-state I-V relationship was somewhat outwardly rectifying (data not shown). Login to comment 180 ABCC7 p.Lys1250Ala Flickery block of K1250A-CFTR in the absence of external Cl_ Representative single-channel current traces at different potentials (_Vp) recorded from a cell-attached patch with 0 mM Cl_ pipette solution. Dotted lines indicate the baseline current level. Login to comment 185 ABCC7 p.Lys1250Ala Voltage-dependent block of the whole-cell K1250A-CFTR current A, net CFTR currents were determined by subtracting the current response of the cell in the absence of forskolin from that in the presence of 10 µM forskolin with 125 mM internal Cl_ and 11 mM external Cl_ . Login to comment 186 ABCC7 p.Lys1250Ala The cAMP-activated K1250A-CFTR current density was 55 ± 12 pA pF_1 at 0 mV holding potential (n = 4). Login to comment 209 ABCC7 p.Lys1250Ala Noise analysis of macroscopic K1250A-CFTR and wild-type CFTR currents Ideally, one should analyse single-channel kinetics on data that are lightly filtered in order to obtain more reliable kinetic parameters. Login to comment 212 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala Macroscopic K1250A-CFTR currents were evoked by 10 µM forskolin in cell-attached patches from NIH3T3 cells stably expressing K1250A-CFTR. Login to comment 214 ABCC7 p.Lys1250Ala Figure 6 shows representative noise spectra of K1250A-CFTR macroscopic currents recorded at _50 mV in a cell-attached patch. Login to comment 226 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala K1250A-CFTR current noise spectra of recordings filtered at two different frequencies Macroscopic K1250A-CFTR currents, activated with 10 µM forskolin in a cell-attached patch held at _50 mV, were filtered at 1 kHz (A) or 5 kHz (B). Login to comment 238 ABCC7 p.Lys1250Ala On the other hand, fc2 was in the frequency range of the fast flickery events seen in K1250A-CFTR channels. Login to comment 239 ABCC7 p.Lys1250Ala Furthermore, fc2 showed a similar voltage dependence to that observed in K1250A-CFTR channels (Fig. 9B). Login to comment 241 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala Effects of voltage and external Cl_ on K1250A-CFTR current noise spectra Comparison of normalized K1250A-CFTR current noise spectra at _100 mV (0) and +50 mV (1) with 154 mM external Cl_ (A) and with 0 mM external Cl_ (B). Login to comment 248 ABCC7 p.Lys1250Ala Comparison of fc estimated from noise analysis and fc calculated from kinetic parameters from single-channel analysis Filled circles represent mean fc at _100, _50, _20 and +50 mV estimated from noise analysis of K1250A-CFTR macroscopic currents in the presence of 154 mM external Cl_ . Login to comment 250 ABCC7 p.Lys1250Ala fast flickery block of K1250A-CFTR channels is probably applicable to wild-type CFTR channels. Login to comment 275 ABCC7 p.Lys1250Ala Results from noise analysis of macroscopic K1250A-CFTR current further verify our conclusions Z. Zhou, S. Hu and T.-C. Hwang444 J. Physiol. 532.2 Figure 9. Login to comment 285 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala One concern of using the CFTR mutant K1250A-CFTR to study the mechanism of fast flickery block is whether K1250A-CFTR and wild-type CFTR share the same mechanism of voltage-dependent flickery block. Login to comment 288 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala Since fc2 of wild-type CFTR channels shows a similar voltage dependence to that of K1250A-CFTR (Fig. 9B), we believe that the voltage-dependent mechanism proposed for the fast flickery block of K1250A-CFTR channels is probably also applicable to wild-type CFTR channels. Login to comment 289 ABCC7 p.Lys1250Ala It is worth noting, however, that fc2 of wild-type CFTR channels is slightly higher than the fc of the fast flickers seen in K1250A-CFTR channels. Login to comment 328 ABCC7 p.Lys1250Ala In this study, we took the advantage of the CFTR mutant K1250A-CFTR, which can be 'locked` open for minutes to allow more accurate kinetic analysis of the flickery block of CFTR channels. Login to comment 331 ABCC7 p.Lys1250Ala We believe future studies of channel blockade in K1250A-CFTR channels by exogenously applied blockers could provide useful information on the mechanism of CFTR blockade as well as on the structure of the CFTR pore. Login to comment