ABCMdb

PMID: 12407077

Zhou Z, Hu S, Hwang TC
Probing an open CFTR pore with organic anion blockers.
J Gen Physiol. 2002 Nov;120(5):647-62., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Lys1250Ala To simplify the kinetic analysis, a CFTR mutant, K1250A-CFTR, was used because this mutant channel, once opened, can remain open for minutes. Login to comment 51 ABCC7 p.Lys1250Ala M A T E R I A L S A N D M E T H O D S Cell Preparation NIH3T3 cells stably expressing K1250A-CFTR channels (Zeltwanger et al., 1999) were maintained at 37ЊC and 5% CO2 in Dulbecco`s Modified Eagle`s Medium (DMEM) supplemented with 10% fetal bovine serum. Login to comment 81 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala For quantitative analysis of isethionate blockade, net K1250A-CFTR single-channel I-V relationships were obtained by subtracting the I-V relationship of the leak (i.e., basal conductance before the channel is opened) from that of a single locked-open K1250A-CFTR channel. Login to comment 83 ABCC7 p.Lys1250Ala The average of these I-V relationships were then calculated to represent the single-channel I-V curve of K1250A-CFTR. Login to comment 93 ABCC7 p.Lys1250Ala R E S U L T S Glibenclamide Block of K1250A-CFTR Channels Previous studies show that glibenclamide blocks CFTR channels with a time constant in the range of tens of milliseconds that is slow enough to be resolved in single-channel recordings (Schultz et al., 1996; Sheppard and Robinson, 1997). Login to comment 97 ABCC7 p.Lys1250Ala To extract the kinetic parameters of glibenclamide-induced blocking events with minimal contamination of ATP-dependent gating events, we studied glibenclamide block with a CFTR mutant, K1250A-CFTR, instead of wt-CFTR. Login to comment 98 ABCC7 p.Lys1250Ala The advantage of using K1250A-CFTR is that this channel, once opened by ATP, can stay open for minutes even after a complete removal of ATP. Login to comment 100 ABCC7 p.Lys1250Ala Kd 1 Fb-( )Poc Glibenclamide[ ] Fb,/= kon V( ) kon V( )' X[ ] kon 0( )' X[ ] zδonFV RT/( )exp= = koff V( ) koff V( ) z- δoffFV RT/( ),exp= Kd V( ) koff V( ) kon V( )'/ koff 0( ) kon 0( )'/ z- δon δoff+[ ]FV RT/( )exp Kd 0( ) z- δKd FV RT/( ),exp = = = We first tested whether glibenclamide block of K1250A-CFTR channels is similar to that of wt-CFTR channels. Login to comment 101 ABCC7 p.Lys1250Ala Fig. 1 A shows an example of glibenclamide-induced block recorded from an inside-out patch excised from an NIH3T3 cell stably expressing K1250A-CFTR. Login to comment 102 ABCC7 p.Lys1250Ala K1250A-CFTR currents were first activated by PKA and 1mM ATP (not depicted). Login to comment 113 ABCC7 p.Lys1250Ala Therefore, glibenclamide-induced block in K1250A-CFTR channels is completely reversible as seen in wt-CFTR (Schultz et al., 1996; Sheppard and Robinson, 1997; Gupta and Linsdell, 2002; cf. Sheppard and Welsh, 1992). Login to comment 114 ABCC7 p.Lys1250Ala Next, we examined the sensitivity of K1250A-CFTR to glibenclamide block. Login to comment 119 ABCC7 p.Lys1250Ala The resulting dose-response relationship can be fitted with a Michaelis-Menten function with a K1/2 of 49.4 Ϯ 9.5 ␮M at -50 mV, which is similar to that reported for wt-CFTR (Schultz et al., 1996; Sheppard and Robinson, 1997), indicating that the K1250A mutation does not affect the sensitivity of the channel to glibenclamide. Login to comment 120 ABCC7 p.Lys1250Ala We then tested whether glibenclamide blocks K1250A-CFTR channels in a voltage-dependent manner as shown for wt-CFTR channels (Sheppard and Robinson, 1997; Gupta and Linsdell, 2002). Login to comment 126 ABCC7 p.Lys1250Ala We therefore conclude that the mechanisms of voltage dependence of glibenclamide block for K1250A-CFTR channels can also be applied to wt-CFTR channels. Login to comment 128 ABCC7 p.Lys1250Ala Glibenclamide block of K1250A-CFTR is reversible. Login to comment 129 ABCC7 p.Lys1250Ala K1250A-CFTR channel currents were activated by PKA and 1 mM ATP in an excised inside-out patch held at -50 mV with symmetric Cl- (154 mM [Cl-]o/154 mM [Cl-]i). Login to comment 130 ABCC7 p.Lys1250Ala (A) Continuous recording of K1250A-CFTR after the channels were locked open with PKA and ATP. Login to comment 137 ABCC7 p.Lys1250Ala Whole-cell Recordings of Glibenclamide Block of K1250A-CFTR With single-channel recordings in excised inside-out patches, we were only able to investigate glibenclamide block at negative membrane potentials since patches became unstable when held at positive potentials for a long period of time (essential for kinetic analysis). Login to comment 164 ABCC7 p.Lys1250Ala Net K1250A-CFTR current traces were obtained by subtracting the leak from the forskolin-activated current (Fig. 4 A). Login to comment 168 ABCC7 p.Lys1250Ala Net steady-state I-V relationships in the presence or absence of glibenclamide are shown in Fig. 4 C. Clearly, glibenclamide blocks whole-cell K1250A-CFTR currents in a voltage-dependent manner with more block at negative voltages. Login to comment 172 ABCC7 p.Lys1250Ala Glibenclamide block of whole-cell K1250A-CFTR currents. Login to comment 178 ABCC7 p.Lys1250Ala (D) Voltage dependence of glibenclamide block of K1250A-CFTR. Login to comment 182 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala Kinetic Analysis of Voltage-dependent Block of Glibenclamide on K1250A-CFTR For glibenclamide block of K1250A-CFTR, we were able to apply a simple scheme for kinetic analysis (Scheme I). Login to comment 196 ABCC7 p.Lys1250Ala The agreement between this Kd value, obtained from kinetic parameters based on Scheme I, and the model-independent K1/2 further demonstrates that Scheme I is adequate for describing glibenclamide block of K1250A-CFTR channels. Login to comment 236 ABCC7 p.Lys1250Ala Voltage-dependent Block of K1250A-CFTR Channels by Isethionate Glibenclamide is a powerful tool to probe the CFTR pore because it affords direct measurements of both the on and off rate constants. Login to comment 245 ABCC7 p.Lys1250Ala To compare their ability to block K1250A-CFTR channels, we applied voltage ramps on inside-out patches in the presence or absence of various blockers in the perfusion solution. Login to comment 247 ABCC7 p.Lys1250Ala All three anions block K1250A-CFTR channels in a voltage-dependent manner. Login to comment 258 ABCC7 p.Lys1250Ala Voltage-dependent block of K1250A-CFTR by hydrophilic organic anions. Login to comment 280 ABCC7 p.Lys1250Ala Effect of extracellular isethionate on K1250A-CFTR whole-cell current. Login to comment 282 ABCC7 p.Lys1250Ala K1250A-CFTR currents were activated by 10 ␮M Fsk. Login to comment 293 ABCC7 p.Lys1250Ala Block of K1250A-CFTR by Mixture of Glibenclamide and Isethionate If indeed these two blockers share a common binding site, then the kinetics of channel blockade by these two blockers can be described as: SCHEME II where X, XG, and XI represent the binding site, glibenclamide-occupied state and isethionate-occupied state, respectively. Login to comment 322 ABCC7 p.Lys1250Ala With the K1250A-CFTR mutant, we were able to quantify detailed kinetic parameters of glibenclamide block not provided by previous studies (Schultz et al., 1996; Sheppard and Robinson, 1997; Gupta and Linsdell, 2002). Login to comment 332 ABCC7 p.Lys1250Ala Block of K1250A-CFTR current in the presence of both glibenclamide and isethionate. Login to comment 338 ABCC7 p.Lys1250Ala reported that the voltage dependence of the intrinsic flickery blocking events seen in a locked-open K1250A-CFTR channel is mostly determined by the trans-ion effects (Zhou et al., 2001b). 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