Molecular basis of fast inactivation in voltage and Ca ²⁺ -activated K ⁺ channels: A transmembrane β-subunit homolog

Abstract

Voltage-dependent and calcium-sensitive K ؉ (MaxiK) channels are key regulators of neuronal excitability, secretion, and vascular tone because of their ability to sense transmembrane voltage and intracellular Ca 2؉ . In most tissues, their stimulation results in a noninactivating hyperpolarizing K ؉ current that reduces excitability. In addition to noninactivating MaxiK currents, an inactivating MaxiK channel phenotype is found in cells like chromaffin cells and hippocampal neurons. The molecular determinants underlying inactivating MaxiK channels remain unknown. Herein, we report a transmembrane ␤ subunit (␤2) that yields inactivating MaxiK currents on coexpression with the pore-forming ␣ subunit of MaxiK channels. Intracellular application of trypsin as well as deletion of 19 N-terminal amino acids of the ␤2 subunit abolished inactivation of the ␣ subunit. Conversely, fusion of these N-terminal amino acids to the noninactivating smooth muscle ␤1 subunit leads to an inactivating phenotype of MaxiK channels. Furthermore, addition of a synthetic N-terminal peptide of the ␤2 subunit causes inactivation of the MaxiK channel ␣ subunit by occluding its K ؉ -conducting pore resembling the inactivation caused by the ''ball'' peptide in voltage-dependent K ؉ channels. Thus, the inactivating phenotype of MaxiK channels in native tissues can result from the association with different ␤ subunits. Ca 2ϩ-activated K ϩ channels, also known as BK, MaxiK, or slo channels, are key modulators of cellular excitability. They are characterized by their large single-channel conductance, intrinsic voltage dependence, Ca 2ϩ modulation, and blockade by charybdotoxin (CTX) and iberiotoxin (1-5). In most tissues, MaxiK channels produce noninactivating currents when activated by depolarization and͞or an increase in intracellular Ca 2ϩ . However, in chromaffin cells of the adrenal gland (6) and hippocampal neurons (7), inactivating MaxiK currents are also observed that otherwise resemble their noninactivating counterparts in their biophysical and pharmacological properties. The mechanism of inactivation in MaxiK channels has been investigated in detail in rat chromaffin cells, which express both inactivating and noninactivating MaxiK channels (8). Inactivation is removed by trypsin application to the cytosolic face of the membrane, suggesting the presence of an associated cytosolic inactivating particle (6, 7). A model developed to explain the biophysical and pharmacological properties of inactivating channels in chromaffin cells suggests that these channels are formed by a tetrameric assembly of inactivating and noninactivating subunits. Interestingly, inactivating channels in chromaffin cells are less sensitive to CTX, and heteromeric channels consisting of inactivating and noninactivating isoforms seem to have intermediate toxin sensitivities (9). Identification of a MaxiK channel variant or a subunit capable of producing fast inactivating MaxiK channel currents has so far been elusive (10).Here, we report a human ␤ subunit (␤2) ...

Molecular Basis for the Inactivation of Ca2+- and Voltage-Dependent BK Channels in Adrenal Chromaffin Cells and Rat Insulinoma Tumor Cells

“…During the revision of this manuscript, a paper describing the identical human ␤-subunit appeared (termed ␤2 in Wallner et al, 1999). Our results share a number of observations with this other study but differ on some interesting points.…”

Cerebellar ataxia and Purkinje cell dysfunction caused by Ca 2+ -activated K + channel deficiency

“…The BK channel consists of four ␣ subunits and four optional auxiliary ␤ subunits (17). The poreforming ␣ subunit is encoded by only a single gene, KCNMA1 (also called Slo), from which multiple splice isoforms are generated (18,19), whereas there are four different ␤ subunit genes with tissue-specific expression (20)(21)(22)(23). To determine BK channel functions, we generated mice lacking functional BK channels.…”

Mechanism of the modulation of BK potassium channel complexes with different auxiliary subunit compositions by the omega-3 fatty acid DHA

“…Inclusion of the auxiliary subunits in Slo1 BK channel complexes markedly alters their cellular targeting, pharmacological properties, and gating characteristics (2,3,6,9,18,19). For example, activation and deactivation kinetics of Slo1+β1 and Slo1+ β4 channels are markedly slower than those of Slo1 alone without any auxiliary subunit (hereafter referred to as Slo1 channels) (5) and the apparent Ca 2+ sensitivity of Slo1+β1 channels is also greater (20,21).…”