The cation-conducting channel of the nicotinic acetylcholine (ACh) receptor is lined by the first (M1) and second (M2) membrane-spanning segments of each of its five subunits. Six consecutive residues, alphaS239 to alphaT244, in the alpha subunit M1-M2 loop and at the intracellular end of M2 were mutated to cysteine. The accessibility of the substituted cysteines were probed with small, cationic, sulfhydryl-specific reagents added extracellularly and intracellularly. In the closed state of the channel, there is a barrier to these reagents added from either side between alphaG240 and alphaT244. ACh induces the removal of this barrier, which acts as an activation gate. The residues alphaG240, alphaE241, alphaK242, and alphaT244 line a narrow part of the channel, in which this gate is located.

The nicotinic acetylcholine (ACh) receptors cycle among classes of nonconducting resting states, conducting open states, and nonconducting desensitized states. We previously probed the structure of the mouse-muscle ACh receptor channel in the resting state obtained in the absence of agonist and in the open states obtained after brief exposure to ACh. We now have probed the structure in the stable desensitized state obtained after many minutes of exposure to ACh. Muscle-type receptor has the subunit composition ␣2␤␥␦. Each subunit has four membrane-spanning segments, M1-M4. The channel lumen in the membrane domain is lined largely by M2 and to a lesser extent by M1 from each of the subunits. We determined the rates of reaction of a small, sulfhydryl-specific, charged reagent, 2-aminoethyl methanethiosulfonate with cysteines substituted for residues in ␣M2 and the ␣M1-M2 loop in the desensitized state and compared these rates to rates previously obtained in the resting and open states. The reaction rates of the substituted cysteines are different in the three functional states of the receptor, indicating significant structural differences. By comparing the rates of reaction of extracellularly and intracellularly added 2-aminoethyl methanethiosulfonate, we previously located the closed gate in the resting state between ␣G240 and ␣T244, in the predicted M1-M2 loop at the intracellular end of M2. Now, we have located the closed gate in the stable desensitized state between ␣G240 and ␣L251. The gate in the desensitized state includes the resting state gate and an extension further into M2. T he nicotinic acetylcholine (ACh) receptors cycle among three classes of functional states: resting, open, and desensitized (1). The receptor is conducting in the open state and nonconducting in the resting and desensitized states. The resting state is the most stable state when no agonist is bound, whereas the desensitized state is the most stable state when agonist is bound. Muscle-type ACh receptors have two ACh binding sites corresponding to the two ␣ subunits in the pentameric complex (Fig. 1, refs. 2 and 3). In the prevailing cycle of transitions, receptors in the resting state bind two molecules of ACh, isomerize to the open state, and, in the continued presence of ACh eventually desensitize. After the removal of free ACh and its dissociation from the binding sites, receptors in the desensitized state predominantly isomerize directly to the resting state. This cycle of activation, desensitization, and recovery has been extensively studied electrophysiologically (1, 4-7). The role of desensitization in cholinergic neurotransmission under normal physiological conditions is uncertain but is evident under some pathological conditions and in neurotransmission by other neurotransmitters (8).Desensitization occurs in stages (9). Receptor isomerizes to a transient, fast-onset desensitized state on the 0.1-to 10-s time scale and to a stable, slow-onset desensitized state on the 10-to 100-s time scale (10-17). The ACh affinities of th...

A ring of aligned glutamate residues named the intermediate ring of charge surrounds the intracellular end of the acetylcholine receptor channel and dominates cation conduction (Imoto et al. 1988). Four of the five subunits in mouse-muscle acetylcholine receptor contribute a glutamate to the ring. These glutamates were mutated to glutamine or lysine, and combinations of mutant and native subunits, yielding net ring charges of −1 to −4, were expressed in Xenopus laevis oocytes. In all complexes, the α subunit contained a Cys substituted for αThr244, three residues away from the ring glutamate αGlu241. The rate constants for the reactions of αThr244Cys with the neutral 2-hydroxyethyl-methanethiosulfonate, the positively charged 2-ammonioethyl-methanethiosulfonate, and the doubly positively charged 2-ammonioethyl-2′-ammonioethanethiosulfonate were determined from the rates of irreversible inhibition of the responses to acetylcholine. The reagents were added in the presence and absence of acetylcholine and at various transmembrane potentials, and the rate constants were extrapolated to zero transmembrane potential. The intrinsic electrostatic potential in the channel in the vicinity of the ring of charge was estimated from the ratios of the rate constants of differently charged reagents. In the acetylcholine-induced open state, this potential was −230 mV with four glutamates in the ring and increased linearly towards 0 mV by +57 mV for each negative charge removed from the ring. Thus, the intrinsic electrostatic potential in the narrow, intracellular end of the open channel is almost entirely due to the intermediate ring of charge and is strongly correlated with alkali-metal-ion conductance through the channel. The intrinsic electrostatic potential in the closed state of the channel was more positive than in the open state at all values of the ring charge. These electrostatic properties were simulated by theoretical calculations based on a simplified model of the channel.

A potassium channel from rat brain was expressed in Xenopus oocytes in order to study modulation of channel function by phosphorylation via protein kinase A. Application of 8-Br-cAMP to oocytes expressing the drk1 channel (with the first 139 amino acids of the N terminus deleted, delta Ndrk1) caused a voltage-independent elevation of current amplitude, which was not seen for endogenous currents or for wild-type full-length drk1 channel. This effect on delta Ndrk1 was blocked by pre-injection of oocytes with Walsh-peptide protein kinase A inhibitor, suggesting mediation via protein kinase A. The protein kinase inhibitor also reduced both delta Ndrk1 and full-length drk1 currents. Substitution of the serine residues by alanine at one or both of the two consensus protein kinase A phosphorylation sites on the C terminus (residues 440 and 492) of delta Ndrk1 resulted in a loss of function of the expressed channels. These results indicate that phosphorylation via protein kinase A modulates drk1 channel function and that both consensus phosphorylation sites seems to be essential for channels to function.

Expression of the rat kidney IsK protein in Xenopus oocytes produces slowly-activating potassium channel currents. We have investigated the relationship between structure and function of the single putative membrane-spanning domain using site-directed mutagenesis. Six mutants were constructed in which consecutive individual amino acids (53 to 58) of the transmembrane region were substituted by cysteine. Expression of four of these mutants in Xenopus oocytes resulted in currents which were similar to wild-type. However, for one mutant (position 55) activation curves were shifted in a hyperpolarising direction and for another mutant (position 58) activation curve were shifted in a depolarising direction. This suggests that the hydrophobic phenylalanine residues at positions 55 and 58 may play a critical role in I,K activation gating. This spacing of functional amino acids at every third residue may indicate an or-helical conformation for the membrane-spanning domain of I,K. Furthermore, these results also indicate that one face of the helix may represent a region of subunit association. Key words." Potassium channel; Delayed rectifier l.~u~onPotassium channels play an essential role in the generation of electrical responses of all excitable cells [1]. Different classes of potassium channel protein have been categorised by their molecular structure. One type, exemplified by the delayed rectifier potassium channel, consist of proteins which have a relatively large molecular weight (up to around 100 kDa) and possess six putative transmembrane segments with a characteristic H5 pore-forming region [2]. Recently, several inwardly rectifying potassium channels have also been sequenced which appear to be rather similar to this type in structure except that they only seem to possess two putative transmembrane segments [3]. Much attention has been focused on the above channel types which has widened our insight into their structurefunction relationships [4]. The subject of this study, however, is the further type of potassium channel known as IsK or minK which has a smaller molecular weight (around 15 kDa), possessing only around 130 amino acids [5]. Much less has been reported concerning the relationship between structure and function for this channel type.Hydropathy analysis of the amino acid sequence of Is~¢ from rat kidney suggests a single putative transmembrane sequence and current models predict an extracellular amino-terminal domain and an intracellular carboxyl-terminal domain [5]. Expression of I~K in Xenopus oocytes [5] and in HEK293 cells [6] has been shown to induce a very slowly-activating voltagedependent potassium current with no inactivation. It is unclear, however, how the protein is able to evoke potassium currents. Two mechanisms have been proposed. It has been suggested that several I~K subunits may aggregate together to directly form a pore-containing structure [7]. An alternative proposal is that IsK itself is not a channel forming protein but acts as a modulator of endogenous oocyte channels...