Mechanistic details of BK channel inhibition by the intermediate conductance, Ca2+-activated K channel

Abstract

Salivary gland acinar cells have two types of Ca(2+)-activated K channels required for fluid secretion: the intermediate conductance (IK1) channel and the large conductance (BK) channel. Activation of IK1 inhibits BK channels including in small, cell-free, excised membrane patches. As a first step toward understanding the mechanism underlying this interaction, we examined its voltage sensitivity. We found that the IK1-induced inhibition of BK channels was only weakly voltage dependent and not accompanied by alteration in BK gating kinetics. These actions of IK1 on BK channels are not consistent with a mechanism whereby activation of IK1 causes a shift of the BK channel's voltage dependence as occurs for many BK modulatory processes. In a search for other clues about the interaction mechanism, we noted that the N-terminus of the IK1 channel shares some chemical features with the N-terminal regions of two BK subunits known to inhibit BK activity by blocking the cytoplasmic end of the BK pore. Thus, we tested the idea that the N-terminus of IK1 channels may act similarly. We found that a peptide derived from the N-terminal region of the IK1 protein blocked BK channels. Significantly, we also found that the activation of IK1 channels competed with block by the N-terminus peptide. Thus, the activation of IK1 channels inhibits BK channels by a mechanism that involves block of the cytoplasmic pore, not an alteration in the voltage dependence of BK gating. The mediator of this cytoplasmic pore block may be the IK1 N-terminus.

Figure 1

Time course of IK1-induced inhibition of BK current. This, and all cells were patched with a solution containing 80 nM free Ca²⁺. (A) Top: currents recorded with the pulse protocol illustrated on the left with the main test potential at +50 mV. Cells were held at −70 mV; a 5 msec step to −120 mV was followed by a 5 msec return to the −70 mV holding potential. This brief return to −70 mV serves to monitor for leak currents since −70 mV is slightly depolarized from the K⁺ equilibrium potential, K channel current will be positive at this voltage and leak current will be negative. The voltage protocol continues with a 70–100 msec pulse to the test potential followed by a step to −30 mV. The currents resulting from this protocol are shown before (Control) during (DCEBIO) and after (Recovery) application of 10 μM DCEBIO. Lines serve only to connect the data points and have no significance. Calib: 0.5 nA/pF, 20 msec. Main: IK1 (○) and BK (■) current components (see Methods) normalized to their maximum average values and shown during application (Onset) and washout of 10 μM DCEBIO. (B) The amount of BK current at the test voltage of +50 mV as a function of the IK1 current at this same potential. The dashed line is from the fit of a simple linear relationship between these two parameters (see text for details).

Figure 2

TRAM-34 reversal of BK current inhibition. (A) Time course of relative BK (■) and IK1 (○) current components at +50 mV recorded every 5 sec during application of, first, 10 μM DCEBIO and then 10 μM DCEBIO + 1 μM TRAM-34 as indicated by the solid bars. (B) The amount of BK current at the test voltage of +50 mV as a function of the IK1 current activated during application of DCEBIO (■) and during inhibition of IK1 current by TRAM-34 (○). The solid line is a linear fit to the data.

Figure 3

Test for voltage-dependence of IK1-induced inhibition of BK current. Shown in the inset are sample patch clamp current records in the absence (Control) and presence (DCEBIO) of 10 μM DCEBIO. Calib: 0.5 nA/pF, 20 msec. Main: total current measured at the end of the pulses to the indicated voltages in the absence (■) and presence (○) of 10 μM DCEBIO. (B) BK current component at the indicated potentials in the absence (■) and presence (○) of 10 μM DCEBIO. Inset: ratio of BK current in the presence of 10 μM DCEBIO to the control BK level at the indicated test voltages. All lines serve only to connect the data points.

Figure 4

Pooled data testing the voltage-dependence of IK1-induced BK inhibition. (A) Time dependent BK current component at the indicated potentials in the absence (■) and presence (○) of 10 μM DCEBIO. Mean values from 15 cells with standard error limits. The data have been normalized to the control BK current value at +50 mV. (B) Ratio of BK current in the presence of 10 μM DCEBIO to the control BK level at the indicated test voltages. Mean values with standard error limits are shown. All lines serve only to connect the data points.

Figure 5

BK current-voltage relation and gating kinetics in 2 μM DCEBIO. (A) Time dependent BK current component at the indicated potentials in the absence (■) and presence (○) of 2 μM DCEBIO. Mean values from 5 cells with standard error limits. The data have been normalized to the control BK current value at +50 mV. The dashed line is the control data scaled by a constant factor of 0.5. (B) Inset: raw current data in response to voltage steps to 30, 50 and 70 mV in the absence (Control) and presence of 2 μM DCEBIO. The red lines are single exponential time function fits to the data. Calib: 0.25 nA/pF, 10 msec. Main: mean values of the fitted time constants with standard error limits at the indicated membrane potentials in the absence (■) and presence (○) of 2 μM DCEBIO.

Figure 6

Time dependent block by the IK1 N-terminal peptide. Currents in response to 250 msec voltage clamp pulses to +50 mV from inside/out patches containing a few BK channels. The vertical panels (left to right) contain current data before (Control), during (NT Peptide), and after removal (Recovery) of 25 μM of the IK1 N-terminal peptide. The first 4 records in each panel are representative examples of current from a single voltage application; the bottom record is the average of several individual records: 7, 16 and 25 for the Control, NT peptide, and Recovery conditions, respectively. The black line in the ensemble average trace with the NT peptide is from the fit of an exponential function to the data-see text for details. Calib: 10 pA/25 msec for singles and 5 pA/25 msec for ensemble averages.

Figure 7

Peptide block rate and IK1 activation. Inset: Sample, whole-cell current records in response to a voltage pulse to 50 mV from an experiment with 250 μM of the IK1 N-terminal peptide in the patch pipette. Data are shown before (Control), during (DCEBIO), after (Washout) application of 10 μM DCEBIO. The red lines are the results of single exponential time function fits to the data with the indicated time constant values. Calib: 0.2 nA/pF, 100 msec. Main: The peptide block rate (1/τ) as a function of the IK1 level during washout of DCEBIO. The dashed line is a linear fit to the data.

Figure 8

Hydropathy analysis of the N-terminus region of several inactivating K channels and accessory, β, peptides. The single letter amino acid code for the various N-termini are shown in colors representing the relative hydrophobic/hydrophilic character of these amino acids according to the scale at the bottom of the figure. This 5-point relative scale was derived from the experimentally determined, whole-residue interface approach described in reference 52. The figure was designed after a similar presentation in reference 51 and includes sequence data from a similar figure in reference 44.