ABCMdb

PMID: 20926782

Li MS, Holstead RG, Wang W, Linsdell P
Regulation of CFTR chloride channel macroscopic conductance by extracellular bicarbonate.
Am J Physiol Cell Physiol. 2011 Jan;300(1):C65-74. Epub 2010 Oct 6., [PubMed]

Sentences

No. Mutations Sentence Comment

9 ABCC7 p.Glu1371Gln Using cell-attached and inside-out patch-clamp recordings from constitutively active mutant E1371Q-CFTR channels, we show that voltage-dependent inhibition of CFTR currents in intact cells is significantly stronger when the extracellular solution contains HCO3 - than when it contains Cl- . Login to comment 10 ABCC7 p.Glu1371Gln Using cell-attached and inside-out patch-clamp recordings from constitutively active mutant E1371Q-CFTR channels, we show that voltage-dependent inhibition of CFTR currents in intact cells is significantly stronger when the extracellular solution contains HCO3 - than when it contains Cl- . Login to comment 29 ABCC7 p.Glu1371Gln In the present study, we have taken advantage of the fact that mutant E1371Q-CFTR channels show constitutive, high levels of activity in baby hamster kidney (BHK) cells (61) to monitor and investigate voltage-dependent channel block in cell-attached patches. Login to comment 30 ABCC7 p.Glu1371Gln In the present study, we have taken advantage of the fact that mutant E1371Q-CFTR channels show constitutive, high levels of activity in baby hamster kidney (BHK) cells (61) to monitor and investigate voltage-dependent channel block in cell-attached patches. Login to comment 37 ABCC7 p.Glu1371Gln All experiments were carried out in an E1371Q-CFTR background, the properties of which were described in detail recently (61). Login to comment 38 ABCC7 p.Glu1371Gln All experiments were carried out in an E1371Q-CFTR background, the properties of which were described in detail recently (61). Login to comment 39 ABCC7 p.Glu1371Gln When expressed in BHK cells, E1371Q-CFTR forms constitutively active channels that are insensitive to stimulation by exogenous protein kinase A, suggesting that the activity of these channels is Fig. 1. Anion-sensitive channel inhibition in intact cells. Login to comment 40 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln When expressed in BHK cells, E1371Q-CFTR forms constitutively active channels that are insensitive to stimulation by exogenous protein kinase A, suggesting that the activity of these channels is Fig. 1. Anion-sensitive channel inhibition in intact cells. Login to comment 41 ABCC7 p.Glu1371Gln A and B: macroscopic currents carried by E1371Q-CFTR during depolarizing voltage ramps from -100 to ϩ60 mV, with Cl- - or gluconate-containing pipette solutions. Login to comment 50 ABCC7 p.Glu1371Gln Consistent with this, E1371Q and other mutations constructed in this background gave stable current amplitudes in cell-attached membrane patches and in excised, inside-out membrane patches under all ionic conditions investigated. Login to comment 51 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln Consistent with this, E1371Q and other mutations constructed in this background gave stable current amplitudes in cell-attached membrane patches and in excised, inside-out membrane patches under all ionic conditions investigated. Login to comment 52 ABCC7 p.Glu1371Gln Patch-clamp recordings of E1371Q-CFTR channel macroscopic currents were as described in detail recently (61). Login to comment 63 ABCC7 p.Glu1371Gln Under these conditions, changes in current amplitude and reversal potential appeared reversible (see Fig. 5), consistent with stable E1371Q channel activity over time. Login to comment 64 ABCC7 p.Glu1371Gln Under these conditions, changes in current amplitude and reversal potential appeared reversible (see Fig. 5), consistent with stable E1371Q channel activity over time. Login to comment 73 ABCC7 p.Glu1371Gln A: macroscopic currents carried by E1371Q-CFTR during depolarizing voltage ramps from -100 to ϩ60 mV with HCO3 - -containing pipette solution. Currents were recorded from cell-attached patches and immediately following patch excision to the inside-out configuration and following addition of 20 ␮M CFTRinh-172 to intracellular solution. Zero current level is indicated by dotted line. B: macroscopic I-V relationships for patch described in A following subtraction of leak current recorded in the presence of CFTRinh-172 in on-cell and inside-out patch configurations. Login to comment 74 ABCC7 p.Glu1371Gln A: macroscopic currents carried by E1371Q-CFTR during depolarizing voltage ramps from -100 to ϩ60 mV with HCO3 - -containing pipette solution. Currents were recorded from cell-attached patches and immediately following patch excision to the inside-out configuration and following addition of 20 ␮M CFTRinh-172 to intracellular solution. Zero current level is indicated by dotted line. B: macroscopic I-V relationships for patch described in A following subtraction of leak current recorded in the presence of CFTRinh-172 in on-cell and inside-out patch configurations. Login to comment 83 ABCC7 p.Glu1371Gln Recently, we used constitutively active E1371Q-CFTR channels expressed in BHK cells to quantify the degree of channel inhibition that underlies this outward rectification in intact cells (61). Login to comment 84 ABCC7 p.Glu1371Gln Recently, we used constitutively active E1371Q-CFTR channels expressed in BHK cells to quantify the degree of channel inhibition that underlies this outward rectification in intact cells (61). Login to comment 90 ABCC7 p.Glu1371Gln A: leak-subtracted E1371Q-CFTR macroscopic I-V relationships recorded with Cl- -, gluconate-, or HCO3 - -containing pipette solutions. Login to comment 91 ABCC7 p.Glu1371Gln A: leak-subtracted E1371Q-CFTR macroscopic I-V relationships recorded with Cl- -, gluconate-, or HCO3 - -containing pipette solutions. Login to comment 104 ABCC7 p.Glu1371Gln This confirms that gluconate and HCO3 - are less able to generate knock off of intracellular open channel blockers in E1371Q-CFTR than is Cl- . Login to comment 105 ABCC7 p.Glu1371Gln This confirms that gluconate and HCO3 - are less able to generate knock off of intracellular open channel blockers in E1371Q-CFTR than is Cl- . Login to comment 111 ABCC7 p.Glu1371Gln Interestingly, PHCO3/PCl for E1371Q-CFTR estimated using a high concentration of extracellular HCO3 - (0.12 Ϯ 0.01, n ϭ 12; Fig. 4D) was significantly lower (P Ͻ 0.02) than that under the reversed ionic gradient (150 mM internal HCO3 - , 154 mM external Cl- ; Fig. 5). Login to comment 112 ABCC7 p.Glu1371Gln Interestingly, PHCO3/PCl for E1371Q-CFTR estimated using a high concentration of extracellular HCO3 - (0.12 Ϯ 0.01, n ϭ 12; Fig. 4D) was significantly lower (P Ͻ 0.02) than that under the reversed ionic gradient (150 mM internal HCO3 - , 154 mM external Cl- ; Fig. 5). Login to comment 117 ABCC7 p.Glu1371Gln To investigate whether these positively charged residues influence the strength of channel block by cytosolic anions in intact cells, we neutralized these charges in an E1371Q background. Login to comment 118 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Arg303Gln ABCC7 p.Lys978Gln To investigate whether these positively charged residues influence the strength of channel block by cytosolic anions in intact cells, we neutralized these charges in an E1371Q background. Login to comment 119 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Arg303Gln ABCC7 p.Lys978Gln Figure 6A shows macroscopic currents carried by K95Q/ E1371Q-, R303Q/E1371Q-, and K978Q/E1371Q-CFTR in Fig. 4. Login to comment 129 ABCC7 p.Glu1371Gln Comparison of the fractional current in cell-attached patches seen in these mutants with those for E1371Q under the same ionic conditions (Fig. 1) suggests that block of each of these mutants is weakened under cell-attached conditions, particularly at hyperpolarized voltages, where block is usually strongest (Fig. 6B). Login to comment 130 ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Arg303Gln ABCC7 p.Arg303Gln Comparison of the fractional current in cell-attached patches seen in these mutants with those for E1371Q under the same ionic conditions (Fig. 1) suggests that block of each of these mutants is weakened under cell-attached conditions, particularly at hyperpolarized voltages, where block is usually strongest (Fig. 6B). Login to comment 131 ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Arg303Gln ABCC7 p.Arg303Gln Interestingly, the effects of the K95Q and R303Q mutations did not appear additive, with the K95Q/R303Q/E1371Q mutant showing relief of block following patch excision similar to that shown by K95Q/E1371Q (Fig. 6). Login to comment 132 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln Although channels bearing the K95Q mutation did give smaller currents in cell-attached patches than those seen following patch excision, this effect appeared voltage-independent, in contrast to the clearly voltage-dependent inhibition of E1371Q (Fig. 6B). Login to comment 133 ABCC7 p.Lys95Gln In fact, because of its low single-channel conductance (16) and weakened interactions with intracellular Cl- , as well as blocking anions (31), K95Q could, in theory, show increased susceptibility to block by uncharged substances. Login to comment 134 ABCC7 p.Lys95Gln In fact, because of its low single-channel conductance (16) and weakened interactions with intracellular Cl- , as well as blocking anions (31), K95Q could, in theory, show increased susceptibility to block by uncharged substances. Login to comment 143 ABCC7 p.Glu1371Gln Permeability of intracellular HCO3 - in E1371Q-CFTR. Login to comment 144 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln Permeability of intracellular HCO3 - in E1371Q-CFTR. Login to comment 145 ABCC7 p.Glu1371Gln A: macroscopic currents carried by E1371Q in an inside-out patch during depolarizing voltage ramps from -100 to ϩ60 mV with Cl- -containing pipette solution. Currents were recorded after they had attained a stable amplitude in inside-out patches, during perfusion with Cl- - or HCO3 - -containing intracellular (bath) solutions and following addition of 20 ␮M CFTRinh-172 to intracellular solution. Zero current level is indicated by dotted line. B: macroscopic I-V relationships for patch in A following subtraction of leak current recorded in the presence of CFTRinh-172, when intracellular solution contained Cl- or HCO3 - . Login to comment 150 ABCC7 p.Glu1371Gln In native epithelial cells, we would expect changes in external [Cl- ] and [HCO3 - ] to alter open channel conductance (present study) and channel open probability (38, 58); the use of the constitutively active, gating-defective E1371Q-CFTR mutant in the present study was therefore important to isolate and characterize anion effects on open channels. Login to comment 151 ABCC7 p.Glu1371Gln In native epithelial cells, we would expect changes in external [Cl- ] and [HCO3 - ] to alter open channel conductance (present study) and channel open probability (38, 58); the use of the constitutively active, gating-defective E1371Q-CFTR mutant in the present study was therefore important to isolate and characterize anion effects on open channels. Login to comment 153 ABCC7 p.Glu1371Gln A: macroscopic I-V relationships for channel mutants (in an E1371Q background) following subtraction of leak currents recorded in the presence of CFTRinh-172 in on-cell and inside-out patch configurations with gluconate-containing pipette solutions. Login to comment 154 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln A: macroscopic I-V relationships for channel mutants (in an E1371Q background) following subtraction of leak currents recorded in the presence of CFTRinh-172 in on-cell and inside-out patch configurations with gluconate-containing pipette solutions. Login to comment 155 ABCC7 p.Glu1371Gln B: quantification of current inhibition in intact cells. Macroscopic current amplitude in cell-attached patches is plotted as a fraction of current in the same patch immediately after excision to inside-out patch configuration for the named mutant () and for E1371Q as control (Œ, see Fig. 1). Login to comment 156 ABCC7 p.Glu1371Gln Values are means for 18 (for E1371Q) and 5-7 (for other mutants) patches. Login to comment 157 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln Values are means for 18 (for E1371Q) and 5-7 (for other mutants) patches. Login to comment 158 ABCC7 p.Glu1371Gln Significant difference from E1371Q: *P Ͻ 0.02; **P Ͻ 0.00001. Login to comment 172 ABCC7 p.Glu1371Gln The HCO3 - permeability of E1371Q-CFTR (Fig. 4) was comparable to that reported many times previously for wild-type CFTR in epithelial cells and heterologous expression systems (25, 55). Login to comment 173 ABCC7 p.Glu1371Gln The HCO3 - permeability of E1371Q-CFTR (Fig. 4) was comparable to that reported many times previously for wild-type CFTR in epithelial cells and heterologous expression systems (25, 55). Login to comment