Collapse of Conductance Is Prevented by a Glutamate Residue Conserved in Voltage-dependent K 1 Channels

Voltage-dependent K 1 channel gating is influenced by the permeating ions. Extracellular K 1 determines the occupation of sites in the channels where the cation interferes with the motion of the gates. When external [K 1 ] decreases, some K 1 channels open too briefly to allow the conduction of measurable current. Given that extracellular K 1 is normally low, we have studied if negatively charged amino acids in the extracellular loops of Shaker K 1 channels contribute to increase the local [K 1 ]. Surprisingly, neutralization of the charge of most acidic residues has minor effects on gating. However, a glutamate residue (E418) located at the external end of the membrane spanning segment S5 is absolutely required for keeping channels active at the normal external [K 1 ]. E418 is conserved in all families of voltage-dependent K 1 channels. Although the channel mutant E418Q has kinetic properties resembling those produced by removal of K 1 from the pore, it seems that E418 is not simply concentrating cations near the channel mouth, but has a direct and critical role in gating. Our data suggest that E418 contributes to stabilize the S4 voltage sensor in the depolarized position, thus permitting maintenance of the channel open conformation. key words: K 1 -channel gating • extracellular K 1 • acidic residues • open-state stabilization • glutamate mutation I N T R O D U C T I O N Voltage-dependent K 1 channels of the plasmalemma participate in fundamental cellular electrical events such as the genesis of the resting membrane potential, action potential repolarization, and repetitive firing (Hille, 1992). The transmembrane efflux of K 1 ions through the open channels is driven by the chemical gradient existing between the cell’s interior, where [K 1 ] is high ( < 140 mM), and the extracellular milieu with lower [K 1 ] ( < 2.5 mM in most mammalian tissues). However, the low external [K 1 ] imposes a major challenge to many K 1 channels since they do not gate properly, or even become nonfunctional if extracellular K 1 is too scant. Although the channels were classically viewed as pores with gates that move independently of the permeating ions, there are several reports indicating that occupation of the channels by the permeating ions modulates their gating properties (for review, see Yellen, 1997). For example, the rise of extracellular [K 1 ] slows the rates of channel closing (Stanfield et al., 1981; Swenson and Armstrong, 1981; Demo and Yellen, 1992; Molina et al., 1998) and C-type inactivation (López-Barneo et al., 1993; Marom and Levitan, 1994; Baukrowitz and Yellen, 1995; Kiss and Korn, 1998). These observations indicate that the concentration of external permeant cations determines the occupation of sites in the pore, where they interfere with the motion of the inactivation or closing gates. Some K 1 channels have a particularly strong dependence on external K 1 because they can easily loose the resident K 1 ions. In these cases, removal of the cation from the external solution results in immediate loss of the K 1 conductance (Pardo et al., 1992; López-Barneo et al., 1993; Jäger et al., 1998). Given the competition between C-type inactivation and the ions occupying the pore, the channels with fast C-type inactivation rate (such as Kv1.4, HERG, or Shaker mutants T449K and D447E) are, in general, those more exquisitely modulated by the changes in extracellular [K 1 ] (López-Barneo et al., 1993; Schönherr and Heinemann, 1996; Smith et al., 1996; Jäger et al., 1998). Even K 1 channels with slow C-type inactivation kinetics resistant to brief removal of external K 1 become nonfunctional after perfusion of the two sides of the membrane with K 1 free media. This last phenomenon, first observed in squid K 1 channels (Almers and Armstrong, 1980), is a manifestation of the complete depletion of K 1 ions from inside the channels, which results in collapse of the pore and stabilization of the channels in a nonconducting or “defunct” state (Gómez-Lagunas, 1997; Melishchuk et al., 1998). In K 1 channels, both the intraand extracellular entryways contain negatively charged amino acids that presumably contribute to increase the local concentration of cations while lowering the concentration of anAddress correspondence to Dr. J. López-Barneo, Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla, Facultad de Medicina, Avenida Sánchez Pizjuán 4, E-41009, Sevilla, Spain. Fax: (34)954-551769; E-mail: [email protected] on A ril 0, 2017 D ow nladed fom Published July 31, 2000