Sergey I. Zakharov

Locations of the β1 transmembrane helices in the BK potassium channel

BK channels are composed of α-subunits, which form a voltage- and Ca2+-gated potassium channel, and of modulatory β-subunits. The β1-subunit is expressed in smooth muscle, where it renders the BK channel sensitive to [Ca2+]i in a voltage range near the smooth-muscle resting potential and slows activation and deactivation. BK channel acts thereby as a damped feedback regulator of voltage-dependent Ca2+ channels and of smooth muscle tone. We explored the contacts between α and β1 by determining the extent of endogenous disulfide bond formation between cysteines substituted just extracellular to the two β1 transmembrane (TM) helices, TM1 and TM2, and to the seven α TM helices, consisting of S1–S6, conserved in all voltage-dependent potassium channels, and the unique S0 helix, which we previously concluded was partly surrounded by S1–S4. We now find that the extracellular ends of β1 TM2 and α S0 are in contact and that β1 TM1 is close to both S1 and S2. The extracellular ends of TM1 and TM2 are not close to S3–S6. In almost all cases, cross-linking of TM2 to S0 or of TM1 to S1 or S2 shifted the conductance–voltage curves toward more positive potentials, slowed activation, and speeded deactivation, and in general favored the closed state. TM1 and TM2 are in position to contribute, in concert with the extracellular loop and the intracellular N- and C-terminal tails of β1, to the modulation of BK channel function.

Protein Kinase G Phosphorylates Cav1.2 α1c and β2 Subunits

Voltage-dependent Ca2+ channel function (Cav1.2, L-type Ca2+ channel) is required for cardiac excitation-contraction (E-C) coupling. Cav1.2 plays a key role in modulating cardiac function in response to classic signaling pathways, such as the renin-angiotensin system and sympathetic nervous system. Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with Cav1.2 current through the actions of cAMP and cGMP. Cardiac cGMP, which activates protein kinase G (PKG), is regulated by nitric oxide (NO), and natriuretic peptides. Although PKG has been reported to activate or inhibit Cav1.2 function, it is still unclear whether Cav1.2 subunits are PKG substrates. We have identified phosphorylation sites within the α1c and β2a subunits that are phosphorylated by PKGIα in vitro. We demonstrate that a subset of these phosphorylation sites is modulated, in a cGMP-PKG–specific manner, in intact HEK cells heterologously expressing α1c and β2a subunits. Using phospho-epitope–specific antibodies, we show that the phosphorylation of these residues is enhanced by PKG in intact cardiac myocytes. Activation of PKG in HEK cells transfected with α1c and β2a subunits caused an inhibition of Cav1.2 whole-cell current. PKG-mediated inhibition of Cav1.2 current was significantly reduced by coexpression of an alanine-substituted Cav1.2 β2a subunit (Ser496). Our results identify a molecular mechanism by which cGMP-PKG regulates Cav1.2 phosphorylation and function.

Position and Role of the BK Channel α Subunit S0 Helix Inferred from Disulfide Crosslinking

The position and role of the unique N-terminal transmembrane (TM) helix, S0, in large-conductance, voltage- and calcium-activated potassium (BK) channels are undetermined. From the extents of intra-subunit, endogenous disulfide bond formation between cysteines substituted for the residues just outside the membrane domain, we infer that the extracellular flank of S0 is surrounded on three sides by the extracellular flanks of TM helices S1 and S2 and the four-residue extracellular loop between S3 and S4. Eight different double cysteine–substituted alphas, each with one cysteine in the S0 flank and one in the S3–S4 loop, were at least 90% disulfide cross-linked. Two of these alphas formed channels in which 90% cross-linking had no effect on the V50 or on the activation and deactivation rate constants. This implies that the extracellular ends of S0, S3, and S4 are close in the resting state and move in concert during voltage sensor activation. The association of S0 with the gating charge bearing S3 and S4 could contribute to the considerably larger electrostatic energy required to activate the BK channel compared with typical voltage-gated potassium channels with six TM helices.

Location of the β4 Transmembrane Helices in the BK Potassium Channel

Large-conductance, voltage- and Ca2+-gated potassium (BK) channels control excitability in a number of cell types. BK channels are composed of α subunits, which contain the voltage-sensor domains and the Ca2+- sensor domains and form the pore, and often one of four types of β subunits, which modulate the channel in a cell-specific manner. β4 is expressed in neurons throughout the brain. Deletion of β4 in mice causes temporal lobe epilepsy. Compared with channels composed of α alone, channels composed of α and β4 activate and deactivate more slowly. We inferred the locations of the two β4 transmembrane (TM) helices TM1 and TM2 relative to the seven α TM helices, S0–S6, from the extent of disulfide bond formation between cysteines substituted in the extracellular flanks of these TM helices. We found that β4 TM2 is close to α S0 and that β4 TM1 is close to both α S1 and S2. At least at their extracellular ends, TM1 and TM2 are not close to S3–S6. In six of eight of the most highly crosslinked cysteine pairs, four crosslinks from TM2 to S0 and one each from TM1 to S1 and S2 had small effects on the V50 and on the rates of activation and deactivation. That disulfide crosslinking caused only small functional perturbations is consistent with the proximity of the extracellular ends of TM2 to S0 and of TM1 to S1 and to S2, in both the open and closed states.

Nitric Oxide Synthase Activity in Guinea Pig Ventricular Myocytes Is Not Involved in Muscarinic Inhibition of cAMP-Regulated Ion Channels

It has recently been demonstrated that NO plays an obligatory role in muscarinic inhibition of beta-adrenergically stimulated ion channels in cardiac sinoatrial node cells (J Gen Physiol. 1995;106:45-65). We looked for evidence that NO might play a similar role in ventricular cells by using histochemical staining for NO synthase (NOS) activity and whole-cell patch-clamp recording of cAMP-regulated Cl- currents. Myocytes isolated from guinea pig hearts stained positively for NADPH-diaphorase activity, suggesting that these cells do express NOS. Acetylcholine (ACh) inhibition of the R(-)-isoproterenol bitartrate (Iso)-activated Cl- current was also reversed by the cGMP-lowering agents LY-83583 and methylene blue, consistent with idea that NO activation of guanylate cyclase may contribute to muscarinic responses. However, LY-83583 and methylene blue activated the Cl- current in the presence of subthreshold concentrations of Iso alone, suggesting that their effects may not be due to antagonism of an NO/cGMP-dependent response. Furthermore, ACh inhibition of Iso-activated Cl- currents could not be mimicked by the NO donors sodium nitroprusside,3-morpholinosydnonimine, and spermine-NO. Similarly, ACh inhibition of the Iso-activated Cl- current could not be blocked by the NOS inhibitor NG-monomethyl-L-arginine. These results indicate that even though ventricular myocytes possess NOS activity, NO production does not play an important role in muscarinic inhibition of beta-adrenergically regulated Cl- channels in these cells.

Positions of the cytoplasmic end of BK α S0 helix relative to S1–S6 and of β1 TM1 and TM2 relative to S0–S6

The BK β1 subunit displaces the unique S0 transmembrane helix on the intracellular side of BK α but not on the extracellular side, thereby altering its path through the membrane.