The LRRC26 protein selectively alters the efficacy of BK channel activators

Abstract

Large conductance, Ca(2+)-activated K channel proteins are involved in a wide range of physiological activities, so there is considerable interest in the pharmacology of large conductance calcium-activated K (BK) channels. One potent activator of BK channels is mallotoxin (MTX), which produces a very large hyperpolarizing shift of the voltage gating of heterologously expressed BK channels and causes a dramatic increase in the activity of BK channels in human smooth muscle cells. However, we found that MTX shifted the steady-state activation of BK channels in native parotid acinar cells by only 6 mV. This was not because the parotid BK isoform (parSlo) is inherently insensitive to MTX as MTX shifted the activation of heterologously expressed parSlo channels by 70 mV. Even though MTX had a minimal effect on steady-state activation of parotid BK channels, it produced an approximate 2-fold speeding of the channel-gating kinetics. The BK channels in parotid acinar cells have a much more hyperpolarized voltage activation range than BK channels in most other cell types. We found that this is probably attributable to an accessory protein, LRRC26, which is expressed in parotid glands: expressed parSlo + LRRC26 channels were resistant to the actions of MTX. Another class of BK activators is the benzimidazalones that includes 1,3-dihydro-1-(2-hydroxy-5-(trifluoromethyl)phenyl)-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619). Although the LRRC26 accessory protein strongly inhibited the ability of MTX to activate BK channels, we found that it had only a small effect on the action of NS-1619 on BK channels. Thus, the LRRC26 BK channel accessory protein selectively alters the pharmacology of BK channels.

Fig. 1.

MTX action on BK channels in native parotid cells. A, voltage dependence of relative BK conductance in a representative mouse parotid acinar cell in the absence (○) and presence (■) of 5 μM MTX. Lines, fits of the Boltzmann equation (see Materials and Methods) with the indicated parameters. B, voltage dependence of activation time constant. Pooled data in the absence (○, N = 8–15) and presence (■) of 5 μM MTX (N = 6–7 except at −30 mV with N = 2). Solid lines are fits of eq. 1 (see Materials and Methods) to the data. Insets, raw current data from a cell in the absence and presence of MTX as indicated. Voltages of 0, 20, 40, and 60 mV. Calibration, 1 nA, 20 ms.

Fig. 2.

MTX shifts activation of heterologously expressed parotid BK channels. A, voltage dependence of relative BK conductance in the absence (○) and presence (■) of 5 μM MTX. Solid lines, fits of the Boltzmann equation (see Materials and Methods) with the indicated parameters. Insets, raw BK currents in the absence (voltages, 60, 80, 100, 120, 140, and 160 mV) and presence (voltages, 120, 140, 160, 180, and 200 mV) of 5 μM MTX as indicated. For each condition, the smallest detectable current and the associated voltage are indicated. Calibration, 2.5 nA, 20 ms (Control); 1 nA, 20 ms (MTX). B, voltage dependence of activation time constant. Pooled data in the absence (○, N = 5–19) and presence (■) of 5 μM MTX (N = 4–10). Solid lines are fits of eq. 1 (see Materials and Methods) to the data.

Fig. 3.

Actions of LRRC26 on BK channel gating. A, voltage dependence of expressed parSlo channel conductance without (○, from Fig. 2A) and with (■) association with the LRRC26 protein. Solid lines, fits of the Boltzmann equation with the indicated parameters. B, voltage dependence of the channel activation time constant without (○, N = 5–19, from Fig. 2B) and with (■) the associated LRRC26 protein (N = 5). Solid lines are fits of eq. 1 to the data. Insets, raw currents from parSlo only and parSlo + LRRC26 channels as indicated. Currents from parSlo channels elicited by depolarizations from 140 to 220 mV in 20-mV increments; calibration, 1 nA/25 ms. Currents from parSlo + LRRC26 channels elicited by depolarizations from 20 to 100 mV in 20-mV increments; calibration, 2 nA/25 ms.

Fig. 4.

MTX action on ParSlo + LRRC26 BK channels. A, voltage dependence of relative BK conductance in the absence (○) and presence (■) of 5 μM MTX. Solid lines, fits of the Boltzmann equation (see Materials and Methods) with the indicated parameters. B, voltage dependence of activation time constant in the absence (○, N = 5) and presence (■) of 5 μM MTX (N = 4 except N = 3 at 0 mV). Solid lines are fits of eq. 1 (see Materials and Methods) to the data.

Fig. 5.

MTX shifts expressed parSlo activation in high Ca²⁺. A, voltage dependence of relative BK channel conductance from a cell in the absence (○) and presence (■) of 5 μM MTX. Solid lines, fits of the Boltzmann equation (see Materials and Methods) with the indicated parameters. B, voltage dependence of activation time constant in the absence (○, N = 5–7) and presence (■) of 5 μM MTX (N = 4–5). Solid lines are fits of eq. 1 (see Materials and Methods) to the data. All measurements were made with 10 μM intracellular Ca²⁺.

Fig. 6.

NS-1619 on parSlo and parSlo + LRRC26 BK channels. A, voltage dependence of heterologously expressed parSlo BK channel conductance in the absence (○) and presence (■) of 50 μM NS-1619. B, voltage dependence of parSlo + LRRC26 BK channel conductance in the absence (○) and presence (■) of 50 μM NS-1619. Solid lines, fits of the Boltzmann equation (see Materials and Methods) with the indicated parameters.

Scheme 1.

The voltage sensors in each of the four BK channel subunits have a resting (R) and an activated (A) conformation controlled by a voltage-dependent equilibrium constant, J. The channel pore has a closed (C) and an open conducting (O) conformation governed by the voltage-dependent equilibrium constant L. There is a cooperative interaction between the voltage sensors and the pore represented by the D parameter.

Fig. 7.

H-A model simulations of the activation of expressed parSlo channels in the absence and presence of MTX. Voltage dependence of relative activation of heterologously expressed parSlo BK channels in the absence (○) and presence (■) of 5 μM MTX. Solid lines, simulation of the H-A model with the indicated parameters. Control data are pooled from several cells, N = 4 for V_m ≥140 mV, N = 3 for V_m from 100 to 140 mV; data at +50 mV are single channel measurements from three individual cells. MTX data at large depolarizations are from a single cell with activation properties representative of the mean values of five cells. MTX data at negative voltages are mean values from six cells, with the exception of the value at −120 mV, which is the mean from three cells.