Differential Effects of 1 and 2 Subunits on BK Channel Activity

High conductance, calciumand voltage-activated potassium (BK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory ( ) subunits. 1 and 2 subunits increase apparent channel calcium sensitivity. The 1 subunit also decreases the voltage sensitivity of the channel and the 2 subunit produces an N-type inactivation of BK currents. We further characterized the effects of the 1 and 2 subunits on the calcium and voltage sensitivity of the channel, analyzing the data in the context of an allosteric model for BK channel activation by calcium and voltage (Horrigan and Aldrich, 2002). In this study, we used a 2 subunit without its N-type inactivation domain ( 2IR). The results indicate that the 2IR subunit, like the 1 subunit, has a small effect on the calcium binding affinity of the channel. Unlike the 1 subunit, the 2IR subunit also has no effect on the voltage sensitivity of the channel. The limiting voltage dependence for steady-state channel activation, unrelated to voltage sensor movements, is unaffected by any of the studied subunits. The same is observed for the limiting voltage dependence of the deactivation time constant. Thus, the 1 subunit must affect the voltage sensitivity by altering the function of the voltage sensors of the channel. Both subunits reduce the intrinsic equilibrium constant for channel opening ( L 0 ). In the allosteric activation model, the reduction of the voltage dependence for the activation of the voltage sensors accounts for most of the macroscopic steady-state effects of the 1 subunit, including the increase of the apparent calcium sensitivity of the BK channel. All allosteric coupling factors need to be increased in order to explain the observed effects when the subunit is coexpressed with the 2IR subunit. key words: BK channel • subunits • allosteric model • voltage dependence • apparent calcium sensitivity I N T R O D U C T I O N The high conductance, voltageand calcium-activated potassium channel (BK or MaxiK channel) is widely expressed among mammalian tissues (Toro et al., 1998). Its open probability increases on membrane depolarization or an increase in intracellular calcium concentration. As its activation leads to membrane hyperpolarization, it serves as a negative-feedback mechanism for the excitatory events that lead to increases in calcium concentration or raise the membrane potential. In vascular smooth muscle cells, BK channels play a key role in regulating the contractile tone (Nelson and Quayle, 1995; Brenner et al., 2000b); in chromaffin cells these channels help terminate the action potential and thus they modulate secretion (Neely and Lingle, 1992; Solaro et al., 1995), and in neurons BK channels colocalize with voltage-dependent calcium channels and are involved in the control of neurosecretion (Sah and Davies, 2000). The BK channel is a homotetramer of its pore-forming subunit, which is coded by the gene Slo 1 (KNCMA1) and is a member of the voltage-dependent potassium (Kv) channels superfamily. As in all other Kv channels, the S4 transmembrane segment is (or is part of) an intrinsic voltage sensor (Diaz et al., 1998; Cui and Aldrich, 2000). Gating and ionic currents in BK channels can be elicited by membrane depolarization in the absence of calcium, suggesting that this is a voltage-dependent channel (Stefani et al., 1997). The divalent cation acts as a modulator able to decrease the necessary energy to open the channel, promoting a leftward shift in the open probability ( P ( O ) ) vs. voltage relationships. Therefore, as calcium is increased, less voltage is needed to activate the channel. The channel is also activated by intracellular Mg 2 , whose binding sites are also low affinity binding sites for Ca 2 (Shi and Cui, 2001; Zhang et al., 2001). Although only one gene codes for the BK channel ( Slo1 , KCNMA1), a wide variety of channel phenotypes has been found. These phenotypes differ in their calcium and voltage sensitivity as well as in their macroscopic kinetics. This variety is explained by several regulatory factors, including alternative splicing, protein phosphorylation, and, most importantly, coexpression of tissue-specific accessory proteins, called subunits (for review see Orio et al., 2002). Four subunits have been cloned, termed 1– 4 (Knaus et al., 1994; Wallner et al., 1999; Xia et al., 1999; Behrens et al., 2000; Brenner et al., 2000a; Meera et al., 2000; Uebele et al., 2000). The effects of the 1 subunit coexpression include an increase of the apparent calcium sensitivity, a decrease of the voltage dependence of the channel, and a slow Correspondence to Ramon Latorre: rlatorre@cecs.cl Abbreviation used in this paper: RCK, regulatory of K conductance.