Volume 126, Issue 4, Pages 1104-1114 (April 2004) Basolateral ClC-2 chloride channels in surface colon epithelium: regulation by a direct effect of intracellular chloride  Marcelo Catalán, Marı́a Isabel Niemeyer, L.Pablo Cid, Francisco V. Sepúlveda  Gastroenterology  Volume 126, Issue 4, Pages 1104-1114 (April 2004) DOI: 10.1053/j.gastro.2004.01.010

Figure 1 Cl− and K+ currents shown in guinea pig distal colon by nystatin perforation. The upper panel shows current driven by a 100-mV, serosa-positive potential in symmetric Cl− concentrations. The middle panel shows current driven by a mucosa-to-serosa concentration gradient for K+ at 0 mV. The bottom panel shows transepithelial resistance. All changes indicated were made in the mucosal solution except for addition of serosal CdCl2. Gastroenterology 2004 126, 1104-1114DOI: (10.1053/j.gastro.2004.01.010)

Figure 2 Cl− and K+ currents shown in guinea pig distal colon by nystatin perforation. Panels are as in the legend to Figure 1. Addition of nystatin and replacement of K+ were in the mucosal solution. Alkalinization was of the serosal side. Gastroenterology 2004 126, 1104-1114DOI: (10.1053/j.gastro.2004.01.010)

Figure 3 Effect of serosal alkalinization and Cd2+ on Cl− currents shown in guinea pig distal colon by nystatin perforation at pH 7.4 and 6.5. The upper panels show currents driven by a 100-mV, serosa-positive potential in symmetric Cl− concentrations at pH 7.4 (left-hand graph) or 6.5 and after increasing pH to 8.2. The bottom panels show currents measured under similar conditions before and after addition of serosal CdCl2. All effects shown were significant as judged by paired t test. Gastroenterology 2004 126, 1104-1114DOI: (10.1053/j.gastro.2004.01.010)

Figure 4 Effect of changing mucosal Cl− concentration on epithelial currents before and after apical nystatin perforation. Currents were measured as described in the legend to Figure 1. Mucosal compartment concentrations of Cl− are given in mmol/L ([Cl−]i). The time of nystatin addition is also shown. The bottom panel shows basolateral Cl− currents calculated as described in the text (means ± SEM; n = 4). The line gives a prediction based on the constant field current equation with the PCl value quoted. This was extracted from the current at 41 mmol/L Cl−. Gastroenterology 2004 126, 1104-1114DOI: (10.1053/j.gastro.2004.01.010)

Figure 5 Effect of intracellular Cl− on the voltage dependence of gpClC-2Δ77–86. (A and B) Representative current traces elicited from a VH of 0 mV in response to test pulses ranging from −190 to −15 mV as indicated. These were followed by a pulse to 40 mV. The duration of the main pulses was increased at more positive voltages to approximate full activation of the conductance. For illustration proposes, the beginning of the tail currents at 40 mV were set at the same time. (A ) 135 and (B) 35 mmol/L intracellular Cl−. (C ) Apparent conductance calculated by taking the current at the beginning of the pulses at 40 mV given after various conditioning prepulses to the voltages in the abscissa. Results are means ± SEM of 7 (35 mmol/L Cl−; circles) and 9 (135 mmol/L Cl−; triangles) separate experiments. The relative conductance as a function of voltage was adjusted to a Boltzmann distribution of the form G/Gmax=1/(1+exp[(V−V0.5 )/k]), where G and Gmax are conductance as a function of voltage and maximal conductance at full activation, respectively. V0.5 is the voltage at which 50% activation occurs, and k is the slope factor. Gastroenterology 2004 126, 1104-1114DOI: (10.1053/j.gastro.2004.01.010)

Figure 6 Effect of changing intracellular Cl− concentration on the voltage dependence of gpClC-2Δ77–86 and on currents at physiologic voltage. The upper panel shows the dependence of V0.5 for the activation of gpClC-2Δ77–86 on intracellular Cl−. Values are means ± SEM of 5, 7, 8, and 9 separate experiments for 10, 35, 60, and 135 mmol/L Cl−, respectively. The straight line is a fit with a slope of 54 mV per decade change in Cl− concentration. The bottom panel shows calculated currents at −70 mV (near physiologic) as a function of [Cl−]i. These values were obtained by using parameters from the Boltzmann adjustments (see Figure 5) at a single voltage of −70 mV. The errors have been propagated from the error in measuring the absolute maximal conductance at the appropriate Cl− concentration. The line gives a prediction of the current based on the assumption that the V0.5 measured at 35 mmol/L [Cl−]i holds true at higher concentrations. Gastroenterology 2004 126, 1104-1114DOI: (10.1053/j.gastro.2004.01.010)

Figure 7 Effect of extracellular Na+ on Cl− currents in surface colonocytes. (A ) Representative current traces, elicited by the voltage protocol shown in B, in a surface colonocyte. The currents were obtained by patch-clamp whole-cell recording after perforating the patch with gramicidin. An interval of 60 seconds (not shown) was allowed between pulses. The bars above the traces show changes in extracellular solutions. The composition of intracellular and extracellular solutions used is given in Materials and Methods. (C ) Current vs. voltage relations for the ramps in the traces identified in A. (D) Currents measured at −170 mV and intracellular [Cl−]i values (calculated from the reversal potentials of the currents) are means ± SEM of 4 separate experiments. Gastroenterology 2004 126, 1104-1114DOI: (10.1053/j.gastro.2004.01.010)