The β 2 subunit of the large conductance Ca 2+ - and voltage-activated K + channel (BK Ca ) modulates a number of channel functions, such as the apparent Ca 2+ /voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the β 2 subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BK Ca channels (hSlo), we have investigated the mechanisms of operation of the β 2 subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with β 2 subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the β 2 subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BK Ca channels coexpressed with the β 2 subunit. The experimental observations are consistent with the view that the β 2 subunit of BK Ca channels facilitates channel activation by changing the voltage sensor equilibrium and that the β 2 -induced inactivation process does not follow a typical N-type mechanism.

High conductance, calcium- and voltage-activated potassium (BK, MaxiK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (β) subunits. The most remarkable effects of β1 and β2 subunits are an increase of the calcium sensitivity and the slow down of channel kinetics. However, the detailed characteristics of channels formed by α and β1 or β2 are dissimilar, the most remarkable difference being a reduction of the voltage sensitivity in the presence of β1 but not β2. Here we reveal the molecular regions in these β subunits that determine their differential functional coupling with the pore-forming α-subunit. We made chimeric constructs between β1 and β2 subunits, and BK channels formed by α and chimeric β subunits were expressed in Xenopus laevis oocytes. The electrophysiological characteristics of the resulting channels were determined using the patch clamp technique. Chimeric exchange of the different regions of the β1 and β2 subunits demonstrates that the NH 3 and COOH termini are the most relevant regions in defining the behavior of either subunit. This strongly suggests that the intracellular domains are crucial for the fine tuning of the effects of these β subunits. Moreover, the intracellular domains of β1 are responsible for the reduction of the BK channel voltage dependence. This agrees with previous studies that suggested the intracellular regions of the α-subunit to be the target of the modulation by the β1-subunit.

Neutralization of the aspartate near the selectivity filter in the GYG D pore sequence (D292N) of the voltage- and Ca 2+ -activated K + channel (MaxiK, BKCa) does not prevent conduction like the corresponding mutation in Shaker channel, but profoundly affects major biophysical properties of the channel (Haug, T., D. Sigg, S. Ciani, L. Toro, E. Stefani, and R. Olcese. 2004. J . Gen . Physiol . 124:173–184). Upon depolarizations, the D292N mutant elicited mostly gating current, followed by small or no ionic current, at voltages where the wild-type hSlo channel displayed robust ionic current. In fact, while the voltage dependence of the gating current was not significantly affected by the mutation, the overall activation curve was shifted by ∼20 mV toward more depolarized potentials. Several lines of evidence suggest that the mutation prevents population of certain open states that in the wild type lead to high open probability. The activation curves of WT and D292N can both be fitted to the sum of two Boltzmann distributions with identical slope factors and half activation potentials, just by changing their relative amplitudes. The steeper and more negative component of the activation curve was drastically reduced by the D292N mutation (from 0.65 to 0.30), suggesting that the population of open states that occurs early in the activation pathway is reduced. Furthermore, the slow component of the gating current, which has been suggested to reflect transitions from closed to open states, was greatly reduced in D292N channels. The D292N mutation also affected the limiting open probability: at 0 mV, the limiting open probability dropped from ∼0.5 for the wild-type channel to 0.06 in D292N (in 1 mM [Ca 2+ ] i ). In addition to these effects on gating charge and open probability, as already described in Part I, the D292N mutation introduces a ∼40% reduction of outward single channel conductance, as well as a strong outward rectification.

The pore region of the majority of K + channels contains the highly conserved GYGD sequence, known as the K + channel signature sequence, where the GYG is critical for K + selectivity (Heginbotham, L., T. Abramson, and R. MacKinnon. 1992. Science . 258:1152–1155). Exchanging the aspartate residue with asparagine in this sequence abolishes ionic conductance of the Shaker K + channel (D447N) (Hurst, R.S., L. Toro, and E. Stefani. 1996. FEBS Lett . 388:59–65). In contrast, we found that the corresponding mutation (D292N) in the pore forming α subunit (hSlo) of the voltage- and Ca 2+ -activated K + channel (BK Ca , MaxiK) did not prevent conduction but reduced single channel conductance. We have investigated the role of outer pore negative charges in ion conduction (this paper) and channel gating (Haug, T., R. Olcese, T. Ligia, and E. Stefani. 2004. J . Gen Physiol . 124:185–197). In symmetrical 120 mM [K + ], the D292N mutation reduced the outward single channel conductance by ∼40% and nearly abolished inward K + flow (outward rectification). This rectification was partially relieved by increasing the external K + concentration to 700 mM. Small inward currents were resolved by introducing an additional mutation (R207Q) that greatly increases the open probability of the channel. A four-state multi-ion pore model that incorporates the effects of surface charge was used to simulate the essential properties of channel conduction. The conduction properties of the mutant channel (D292N) could be predicted by a simple ∼8.5-fold reduction of the surface charge density without altering any other parameter. These results indicate that the aspartate residue in the BK Ca pore plays a key role in conduction and suggest that the pore structure is not affected by the mutation. We speculate that the negative charge strongly accumulates K + in the outer vestibule close to the selectivity filter, thus increasing the rate of ion entry into the pore.

Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with changes in the kinetics of gating current known as “charge immobilization” (Armstrong, C.M., and F. Bezanilla. 1977. J. Gen. Physiol. 70:567–590.). Shaker H4 W434F gating currents are very similar to those of the conducting clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at −90 mV return potential changed from a single fast component to at least two components, the slower requiring ∼200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current ( Shaker H4 W434F) and ionic current ( Shaker H4) in 2 mM external potassium have at least two components. Both recoveries are similar at −120 and −90 mV. In contrast, at higher potentials (−70 and −50 mV), the gating charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of “parallel” inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH 2 -terminus region.

Prolonged depolarization induces a slow inactivation process in some K + channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K + channel and in the nonconducting mutant ( Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: ( a ) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and ( b ) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K + channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J . 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.