Selectivity filter gating in large-conductance Ca(2+)-activated K+ channels.

Membrane voltage controls the passage of ions through voltage-gated K (K(v)) channels, and many studies have demonstrated that this is accomplished by a physical gate located at the cytoplasmic end of the pore. Critical to this determination were the findings that quaternary ammonium ions and certain peptides have access to their internal pore-blocking sites only when the channel gates are open, and that large blocking ions interfere with channel closing. Although an intracellular location for the physical gate of K(v) channels is well established, it is not clear if such a cytoplasmic gate exists in all K(+) channels.

Apical Ca2+-activated potassium channels in mouse parotid acinar cells.

Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion.

The LRRC26 protein selectively alters the efficacy of BK channel activators.

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.

Ca2+-activated K channels in parotid acinar cells: The functional basis for the hyperpolarized activation of BK channels.

Fluid secretion relies on a close interplay between Ca(2+)-activated Cl and K channels. Salivary acinar cells contain both large conductance, BK, and intermediate conductance, IK1, K channels. Physiological fluid secretion occurs with only modest (<500 nM) increases in intracellular Ca(2+) levels but BK channels in many cell types and in heterologous expression systems require very high concentrations for significant activation.

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

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.

The role of cell cholesterol and the cytoskeleton in the interaction between IK1 and maxi-K channels.

Recently, we demonstrated a novel interaction between large-conductance (maxi-K or K(Ca)1.1) and intermediate-conductance (IK1 or K(Ca)3.1) Ca(2+)-activated K channels: activation of IK1 channels causes the inhibition of maxi-K activity (Thompson J and Begenisich T.

Apical maxi-K (KCa1.1) channels mediate K+ secretion by the mouse submandibular exocrine gland.

The exocrine salivary glands of mammals secrete K+ by an unknown pathway that has been associated with HCO3(-) efflux. However, the present studies found that K+ secretion in the mouse submandibular gland did not require HCO3(-), demonstrating that neither K+/HCO3(-) cotransport nor K+/H+ exchange mechanisms were involved. Because HCO3(-) did not appear to participate in this process, we tested whether a K channel is required.

Regulation of membrane potential and fluid secretion by Ca2+-activated K+ channels in mouse submandibular glands.

We have recently shown that the IK1 and maxi-K channels in parotid salivary gland acinar cells are encoded by the K(Ca)3.1 and K(Ca)1.1 genes, respectively, and in vivo stimulated parotid secretion is severely reduced in double-null mice.

Functional and molecular characterization of the fluid secretion mechanism in human parotid acinar cells.

The strategies available for treating salivary gland hypofunction are limited because relatively little is known about the secretion process in humans. An initial microarray screen detected ion transport proteins generally accepted to be critically involved in salivation. We tested for the activity of some of these proteins, as well as for specific cell properties required to support fluid secretion.

Pharmacology and surface electrostatics of the K channel outer pore vestibule.

In spite of a generally well-conserved outer vestibule and pore structure, there is considerable diversity in the pharmacology of K channels. We have investigated the role of specific outer vestibule charged residues in the pharmacology of K channels using tetraethylammonium (TEA) and a trivalent TEA analog, gallamine. Similar to Shaker K channels, gallamine block of Kv3.

Molecular identification and physiological roles of parotid acinar cell maxi-K channels.

The physiological success of fluid-secreting tissues relies on a regulated interplay between Ca(2+)-activated Cl(-) and K(+) channels. Parotid acinar cells express two types of Ca(2+)-activated K(+) channels: intermediate conductance IK1 channels and maxi-K channels. The IK1 channel is encoded by the K(Ca)3.

Membrane-delimited inhibition of maxi-K channel activity by the intermediate conductance Ca2+-activated K channel.

The complexity of mammalian physiology requires a diverse array of ion channel proteins. This diversity extends even to a single family of channels. For example, the family of Ca2+-activated K channels contains three structural subfamilies characterized by small, intermediate, and large single channel conductances.

Two stable, conducting conformations of the selectivity filter in Shaker K+ channels.

We have examined the voltage dependence of external TEA block of Shaker K(+) channels over a range of internal K(+) concentrations from 2 to 135 mM. We found that the concentration dependence of external TEA block in low internal K(+) solutions could not be described by a single TEA binding affinity. The deviation from a single TEA binding isotherm was increased at more depolarized membrane voltages.

Regulation of fluid and electrolyte secretion in salivary gland acinar cells.

The secretion of fluid and electrolytes by salivary gland acinar cells requires the coordinated regulation of multiple water and ion transporter and channel proteins. Notably, all the key transporter and channel proteins in this process appear to be activated, or are up-regulated, by an increase in the intracellular Ca2+ concentration ([Ca2+]i). Consequently, salivation occurs in response to agonists that generate an increase in [Ca2+]i.

Physiological roles of the intermediate conductance, Ca2+-activated potassium channel Kcnn4.

Three broad classes of Ca(2+)-activated potassium channels are defined by their respective single channel conductances, i.e. the small, intermediate, and large conductance channels, often termed the SK, IK, and BK channels, respectively.

External TEA block of shaker K+ channels is coupled to the movement of K+ ions within the selectivity filter.

Recent molecular dynamic simulations and electrostatic calculations suggested that the external TEA binding site in K+ channels is outside the membrane electric field. However, it has been known for some time that external TEA block of Shaker K+ channels is voltage dependent. To reconcile these two results, we reexamined the voltage dependence of block of Shaker K+ channels by external TEA.

Functional identification of ion binding sites at the internal end of the pore in Shaker K+ channels.

The inner end of the pore in voltage-gated K+ channels is the site of conformational changes related to gating and contains binding sites for permeant ions and pore-blocking molecules including quaternary ammonium ions and drugs. In order to determine the location and affinity of ion binding sites we probed the Shaker K+ channel with the quaternary ammonium analogue, tetrabutyl antimony (TBSb), a compound that is sufficiently electron dense to have been observed to occupy the cavity site in the bacterial K+ channel, KcsA. TBSb has K+ channel blocking properties analogous to those of tetrabutyl ammonium (TBA), and kinetics slow enough to be reliably measured.

Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl- channel gene.

Salivary gland acinar cells shrink when Cl(-) currents are activated following cell swelling induced by exposure to a hypotonic solution or in response to calcium-mobilizing agonists. The molecular identity of the Cl(-) channel(s) in salivary cells involved in these processes is unknown, although ClC-3 has been implicated in several tissues as a cell-volume-sensitive Cl(-) channel. We found that cells isolated from mice with targeted disruption of the Clcn3 gene undergo regulatory volume decrease in a fashion similar to cells from wild-type littermates.

Molecular identification of Ca2+-activated K+ channels in parotid acinar cells.

We used molecular biological and patch-clamp techniques to identify the Ca(2+)-activated K(+) channel genes in mouse parotid acinar cells. Two types of K(+) channels were activated by intracellular Ca(2+) with single-channel conductance values of 22 and 140 pS (in 135 mM external K(+)), consistent with the intermediate and maxi-K classes of Ca(2+)-activated K(+) channels, typified by the mIK1 (Kcnn4) and mSlo (Kcnma1) genes, respectively. The presence of mIK1 mRNA was established in acinar cells by in situ hybridization.

Conformation-dependent regulation of inward rectifier chloride channel gating by extracellular protons.

We have investigated the gating properties of the inward rectifier chloride channel (Cl(ir)) from mouse parotid acinar cells by external protons (H(+)(o)) using the whole-cell patch-clamp technique. Increasing the pH(o) from 7.4 to 8.