Voltage-dependent gating of the cystic fibrosis transmembrane conductance regulator Cl- channel

Abstract

When excised inside-out membrane patches are bathed in symmetrical Cl--rich solutions, the current-voltage (I-V) relationship of macroscopic cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents inwardly rectifies at large positive voltages. To investigate the mechanism of inward rectification, we studied CFTR Cl- channels in excised inside-out membrane patches from cells expressing wild-type human and murine CFTR using voltage-ramp and -step protocols. Using a voltage-ramp protocol, the magnitude of human CFTR Cl- current at +100 mV was 74 +/- 2% (n = 10) of that at -100 mV. This rectification of macroscopic CFTR Cl- current was reproduced in full by ensemble currents generated by averaging single-channel currents elicited by an identical voltage-ramp protocol. However, using a voltage-step protocol the single-channel current amplitude (i) of human CFTR at +100 mV was 88 +/- 2% (n = 10) of that at -100 mV. Based on these data, we hypothesized that voltage might alter the gating behavior of human CFTR. Using linear three-state kinetic schemes, we demonstrated that voltage has marked effects on channel gating. Membrane depolarization decreased both the duration of bursts and the interburst interval, but increased the duration of gaps within bursts. However, because the voltage dependencies of the different rate constants were in opposite directions, voltage was without large effect on the open probability (Po) of human CFTR. In contrast, the Po of murine CFTR was decreased markedly at positive voltages, suggesting that the rectification of murine CFTR is stronger than that of human CFTR. We conclude that inward rectification of CFTR is caused by a reduction in i and changes in gating kinetics. We suggest that inward rectification is an intrinsic property of the CFTR Cl- channel and not the result of pore block.

Figure 1.

I-V relationship of CFTR Cl⁻ currents. (A) Current traces from an excised inside-out membrane patch from a C127 cell expressing wild-type human CFTR. The recordings were made in the absence (middle) and presence (bottom) of PKA (75 nM) and ATP (1 mM) in the intracellular solution. The basal recording is the current in response to a single ramp of voltage with no active CFTR Cl⁻ channels, whereas the recording in the presence of PKA + ATP is the average current of 30 ramps of voltage. Holding voltage was −50 mV and the membrane patch was bathed in symmetrical 147 mM NMDGCl solutions. The voltage ramp protocol used is shown and currents were acquired at a sampling rate of 1 kHz as described in the materials and methods. (B) I-V relationship constructed by subtracting the basal trace from the PKA + ATP trace shown in A. The inset shows the I-V relationship of the same data acquired at a sampling rate of 5 kHz (abscissa: −100 to +100 mV; ordinate: −30 to +30 pA). (C) I-V relationship of CFTR Cl⁻ currents. Data are means ± SEM (n = 10) at each voltage calculated by expressing individual current values measured from −100 to +100 mV in 10-mV increments as a percentage of the current value at −100 mV. Error bars are smaller than symbol size. The continuous line is the fit of a second order regression to the data. The dotted line shows the predicted ohmic I-V relationship. Other details as in A.

Figure 2.

Summation of single-channel currents reproduces the rectification of macroscopic CFTR Cl⁻ currents. (A) Representative recordings of a single CFTR Cl⁻ channel in an excised inside-out membrane patch elicited by a depolarizing voltage ramp from −100 to +100 mV. ATP (1 mM) and PKA (75 nM) were continuously present in the intracellular solution. The dotted line indicates the zero current level. For the purpose of illustration, single-channel records were digitally refiltered at 100 Hz. (B) Ensemble current obtained by averaging 70 ramps of voltage from the same experiment as that shown in A. The continuous line is the fit of a second-order regression to the data. (C) I-V relationship expressed as a percentage of the current value at −100 mV. Data are means ± SEM (n = 5) at each voltage. Other details as in A and Fig. 1.

Figure 3.

Effect of voltage on the single-channel activity of CFTR. Representative recordings of a single CFTR Cl⁻ channel at −75 mV (top) and +75 mV (bottom). ATP (1 mM) and PKA (75 nM) were continuously present in the intracellular solution. Dotted lines indicate the closed channel state. Downward and upward deflections correspond to channel openings at −75 mV and +75 mV, respectively. Each trace is 10-s long. For the purpose of illustration, single-channel records were filtered at 500 Hz and digitized at 1 kHz. Other details as in Fig. 1 A.

Figure 4.

Single-channel conductance of CFTR. (A) Single-channel current amplitude histograms of a single CFTR Cl⁻ channel at the indicated voltages recorded using the conditions described in Fig. 3. At negative voltages (top), the closed-channel amplitude is shown on the right, whereas at positive voltages (bottom), the closed channel amplitude is shown on the left. Linear x-axes with 20 bins decade⁻¹ were used for the histograms and the continuous lines represent the fit of Gaussian distributions to the data. The vertical dashed lines indicate the position of the open and closed channel levels at negative voltages. (B) Single-channel I-V relationship of CFTR. (C) Relationship between chord conductance and voltage for the data shown in B. Chord conductance was calculated as described in materials and methods. Other details as in Fig. 1.

Figure 5.

Analysis of the dwell time histograms of a single CFTR Cl⁻ channel. (A and B) Representative open and closed time histograms for a single CFTR Cl⁻ channel recorded at −75 and +75 mV, respectively, using the conditions described in Fig. 3. For open time histograms, the continuous line is the fit of a one-component exponential function. For closed time histograms, the continuous line is the fit of a two-component exponential function. The dotted lines show the individual components of the exponential functions. Logarithmic x-axes with 10 bins decade⁻¹ were used for both open and closed time histograms. (C) Burst duration (top), interburst interval (middle), and P_o (bottom) of CFTR at the indicated voltages. Columns and error bars indicate means ± SEM (n = 10) at each voltage. For the P_o data, filled circles connected by lines represent individual experiments and the open circles are means ± SEM (n = 10). The asterisks indicate values that are significantly different from the −75 mV data (P < 0.05). Burst duration and interburst interval were calculated as described in materials and methods. Other details as in Fig. 1.

Figure 6.

Effect of voltage on the rate constants of the C₁↔C₂↔O kinetic scheme. (A) The C₁↔C₂↔O kinetic scheme that describes CFTR channel gating (Winter et al., 1994). States C₁, C₂, and O represent two closed states and one open state, respectively, while β₁, β₂, α₁, and α₂ represent the rate constants describing transitions between these states. States enclosed within the dashed box represent the bursting state. (B) Rate constants at the indicated voltages determined by the maximum likelihood fit to the model shown in A. Data are means ± SEM (n = 6) at each voltage. The asterisks indicate values that are significantly different from the −75 mV data (P < 0.05). (C) The relationship between the rate constants β₁ (filled circles), β₂ (filled diamonds), α₁ (open circles) and α₂ (open diamonds) and voltage for the C₁↔C₂↔O model. Values are means ± SEM (n = 5–6) at each voltage. The continuous lines represent fits of the single exponential function k=k₀Pexp^k₁V (Eq. 2) as described in the materials and methods. Other details as in Fig. 1 and Table III.

Figure 7.

Effect of voltage on the rate constants of the C₁↔O↔C₂ kinetic scheme. (A) The C₁↔O↔C₂ kinetic scheme that describes CFTR channel gating (Winter et al., 1994). (B) Rate constants at the indicated voltages determined by the maximum likelihood fit to the model shown in A. (C) The relationship between the rate constants β₁ (filled circles), β₂ (filled diamonds), and α₁ (open circles) and voltage for the C₁↔O↔C₂ scheme. The inset shows the relationship between the rate constant α₂ and voltage for the same scheme. Other details as in Fig. 6 and Table IV.

Figure 8.

Pyrophosphate stimulation of CFTR Cl⁻ currents is voltage independent. (A) I-V relationships of CFTR Cl⁻ currents recorded in the absence and presence of pyrophosphate (PP_i; 5 mM) in the intracellular solution. (B) Effect of voltage on the fraction of CFTR Cl⁻ current stimulated by PP_i (5 mM). Values are means ± SEM (n = 4) at each voltage. The continuous line is the fit of a first order regression to the data. (C) I-V relationships of CFTR Cl⁻ currents recorded in the absence (open circles) and presence (filled circles) of PP_i (5 mM) expressed as a percentage of the current value at −100 mV. Data are means ± SEM (n = 4) at each voltage. Other details as in Fig. 1.

Figure 9.

ADP inhibition of CFTR Cl⁻ currents is voltage independent. (A) I-V relationships of CFTR Cl⁻ currents recorded in the absence and presence of ADP (1 mM) in the intracellular solution. (B) Effect of voltage on the fraction of CFTR Cl⁻ current inhibited by ADP (1 mM). (C) I-V relationships of CFTR Cl⁻ currents recorded in the absence (open circles) and presence (filled circles) of ADP (1 mM) expressed as a percentage of the current value at −100 mV. Other details as in Fig. 8.

Figure 10.

Effect of voltage on the murine CFTR Cl⁻ channel. (A) Recordings of a single murine CFTR Cl⁻ channel at −80 mV (top) and +80 mV (bottom) made using the conditions described in Fig. 3. (B) Single-channel I-V relationships of human (open circles) and murine (filled circles) CFTR. Data are means ± SEM (n = 8–10) at each voltage. Other details as in Fig. 1. (C) Relationship between chord conductance and voltage for the data shown in B. (D) Effect of voltage on the P_o of human (left ordinate) and murine (right ordinate) CFTR. Note the change in scale. Columns and error bars indicate means ± SEM (human, n = 6; murine, n = 10). For human CFTR, voltage was −75 and +75 mV, whereas for murine voltage was −80 and +80 mV. The asterisks indicate values that are significantly different from the data at negative voltages (P < 0.05). Other details as in Fig. 4.