P. R. Stanfield

A single aspartate residue is involved in both intrinsic gating and blockage by Mg2+ of the inward rectifier, IRK1.

1. We describe the effects on channel function of changing an aspartate residue (Asp172) in a membrane‐spanning alpha‐helix of the murine inward rectifier, IRK1, by site‐directed mutagenesis. 2. Alteration of Asp172 to Glu (charged) or to Gln or Asn (polar but uncharged) produced functional channels showing inward rectification, though rectification was weaker with Gln and Asn. 3. Intrinsic gating around the potassium equilibrium potential, EK, was conserved only if the charge on residue 172 was conserved. Currents through channels with Gln or Asn in this position showed no time dependence under hyperpolarization. 4. The change from Asp to Gln also reduced the affinity for internal Mg2+ at least fivefold, indicating that Asp172 also forms part of the site for Mg2+ blockage. 5. The consequences for channel structure of Asp172 lining the pore are discussed.

ATP-dependent potassium channels of muscle cells: Their properties, regulation, and possible functions

ATP-dependent potassium channels are present at high density in the membranes of heart, skeletal, and smooth muscle and have a lowPopen at physiological [ATP]i. The unitary conductance is 15–20 pS at physiological [K+]o, and the channels are highly selective for K+. Certain sulfonylureas are specific blockers, and some K channel openers may also act through these channels. KATP channels are probably regulated through the binding of ATP, which may in turn be regulated through changes in the ADP/ATP ratio or in pHi. There is some evidence for control through G-proteins. The channels have complex kinetics, with multiple open and closed states. The main effect of ATP is to increase occupancy of long-lived closed states. The channels may have a role in the control of excitability and probably act as a route for K+ loss from muscle during activity. In arterial smooth muscle they may act as targets for vasodilators.

THE EFFECT OF INTRACELLULAR ANIONS ON ATP-DEPENDENT POTASSIUM CHANNELS OF RAT SKELETAL MUSCLE

1. We have used excised inside‐out patches to study the effects of anions bathing the cytoplasmic surface of the membrane on ATP‐dependent K+ channels of rat flexor digitorum brevis muscle. Channels were closed by ATP applied to the cytoplasmic face of the patch with a concentration for half‐closure (Ki) of 14 microM, were highly selective for K+ and had unitary conductances of 62 pS in symmetrical 155 mM K+ and 27 pS in 5 mM [K+]o. 2. In 139 mM Cl‐ internal solution channel activity declined rapidly after excision of the patch. Inclusion of 40 mM potassium gluconate (substituted for KCl) in the solution both restored channel activity and greatly slowed its subsequent run‐down. 3. The action of gluconate was concentration dependent. The effect did not involve a change in ATP binding, since the Ki for ATP was not significantly changed by gluconate, and was specific for the cytoplasmic face of the patch. 4. The anions pyruvate, lactate and acetate were all able to restore channel activity after run‐down, though less well than gluconate, while sulphate and methylsulphate were without effect. 5. Analysis of single channel kinetics showed that gluconate did not affect mean open lifetime, but led to a decrease in the number and duration of long closings. 6. Anions are most likely to act by stabilizing the structure of the channel protein. Changes in the intracellular concentration of certain anions may play a role in regulating channel activity.

The intrinsic gating of inward rectifier K+ channels expressed from the murine IRK1 gene depends on voltage, K+ and Mg2+

1. We describe the cloning of the inward rectifier K+ channel IRK1 from genomic DNA of mouse; the gene is intronless. 2. The IRK1 gene can be stably expressed in murine erythroleukaemia (MEL) cells. Such transfected cells show inward rectification under whole‐cell recording. 3. Channels encoded by the IRK1 gene have an intrinsic gating that depends on voltage and [K+]o. Rate constants are reduced e‐fold as the driving force on K+(V‐EK) is reduced by 24.1 mV. 4. Removal of intracellular Mg2+ permits brief outward currents under depolarization. The instantaneous current‐voltage relation may be fitted by an appropriate constant field expression. 5. Removal of intracellular Mg2+ speeds channel closure at positive voltages. In nominally zero [Mg2+]i, rate constants for the opening and closing of channels, processes which are first order, are similar to those of native channels.

A calcium dependent inward current in frog skeletal muscle fibres

SummarySlow inward currents are here reported in frog skeletal muscle. The currents are turned on by depolarising the membrane beyound about −45 mV, and are blocked by replacement of Ca2+ by Mg2+ and in the presence of Ca2+ by Co2+ at 20mM.

Characterisation of Kir2.0 proteins in the rat cerebellum and hippocampus by polyclonal antibodies.

Abstract Rabbit polyclonal antibodies were raised to rat Kir2.0 (Kir2.1, Kir2.2 and Kir2.3) inwardly rectifying potassium ion channel proteins. The antibody specificities were confirmed by immunoprecipitation of [35S]-methionine-labelled in vitro translated channel proteins and western blotting. Immunohistochemistry revealed a different patterns of expression of Kir2.0 subfamily proteins in the rat hind-brain (cerebellum and medulla) and fore-brain (hippocampus). Notably, only Kir2.2 protein was detected in the cerebellum and medulla, Kir2.1, Kir2.2 and Kir2.3 proteins were expressed in the hippocampus and immunostaining was not limited to neuronal cell types. Anti-Kir2.1 (fore-brain only) and anti-Kir2.2 (fore- and hind-brain) antibodies showed positive staining in macroglia, endothelia, ependyma and vascular smooth muscle cells. In contrast, anti-Kir2.3 (fore-brain only) immunostaining was limited to neurons, macroglia and vascular smooth muscle. These results indicate that specific regions within the rat fore- and hind-brain have differential distributions of inwardly rectifying potassium ion channel proteins.

Residues beyond the selectivity filter of the K+ channel Kir2.1 regulate permeation and block by external Rb+ and Cs+

1 Kir2.1 channels are blocked by Rb+ and Cs+ in a voltage‐dependent manner, characteristic of many inward rectifier K+ channels. Mutation of Ser165 in the transmembrane domain M2 to Leu (S165L) abolished Rb+ blockage and lowered Cs+ blocking affinity. At negative voltages Rb+ carried large inward currents. 2 A model of the Kir2.1 channel, built by homology with the structure of the Streptomyces lividans K+ channel KcsA, suggested the existence of an intersubunit hydrogen bond between Ser165 and Thr141 in the channel pore‐forming P‐region that helps stabilise the structure of this region. However, mutations of Thr141 and Ser165 did not produce effects consistent with a hydrogen bond between these residues being essential for blockage. 3 An alternative alignment between the M2 regions of Kir2.1 and KcsA suggested that Ser165 is itself a pore‐lining residue, more directly affecting blockage. We were able to replace Ser165 with a variety of polar and non‐polar residues, consistent with this residue being pore lining. Some of these changes affected channel blockage. 4 We tested the hypothesis that Asp172 – a residue implicated in channel gating by polyamines – formed an additional selectivity filter by using the triple mutant T141A/S165L/D172N. Large Rb+ and Cs+ currents were measured in this mutant. 5 We propose that both Thr141 and Ser165 are likely to provide binding sites for monovalent blocking cations in wild‐type channels. These residues lie beyond the carbonyl oxygen tunnel thought to form the channel selectivity filter, which the blocking cations must therefore traverse.

Inward rectification in skeletal muscle: a blocking particle model.

Inwardly rectifying otassium currents were measured in resting frog skeletal muscle in different [K]. A model is presented for inward rectification which supposes that the potassium conductance depends on the K+ concentration within a channel and is reduced by a blocking particle which is driven into the channel by depolarization.

Single inwardly rectifying potassium channels in cultured muscle cells from rat and mouse.

1. Inward unitary currents through inwardly rectifying K+ channels of myotubes derived from newborn rats or from a murine, clonal myoblast cell line were studied in the cell‐attached configuration. Open‐closed transitions of the channel were observed in the absence of blocking ions. 2. The single‐channel conductance was 26.3 +/‐ 2.9 pS (mean + S.D., n = 14) with 150 mM‐K+ pipette solution at room temperature (19‐22 degrees C). The channel showed substates of conductance in addition to the main conductance state. A channel with a smaller conductance (8.9 +/‐ 2.6 pS, n = 4) was also but less frequently observed. 3. The probability of the channel being open is weakly voltage dependent: it decreased from 0.94 to 0.84 as the membrane was hyperpolarized from the resting potential (RP) + 20 mV to RP ‐ 50 mV. 4. The lifetimes of the openings were distributed according to a single exponential. At least three exponentials were required to fit the frequency histogram of the lifetimes of all closed states. The mean open time showed a weak voltage dependence, while the mean closed times had little voltage dependence. 5. In the presence of external Na+, the open probability decreased from 0.89 to 0.43 and the mean open time decreased from 203 to 28 ms (40 mM‐K+, 200 mM‐Na+ pipette solution) when the patch membrane was hyperpolarized from RP ‐ 40 mV to RP ‐ 110 mV. The mean closed times were not different from those with 150 mM‐K+, Na+‐free pipette solution and showed little voltage dependence. 6. It is suggested that inactivation of the macroscopic inward currents during hyperpolarization results mainly from a voltage‐dependent block by Na+ with relatively slow kinetics.

Rubidium ions and the gating of delayed rectifier potassium channels of frog skeletal muscle.

1. Unitary currents were measured through delayed rectifier potassium channels of frog skeletal muscle, under conditions where either potassium or rubidium ions carried current. 2. Unitary currents were reduced in amplitude when Rb+ was the charge carrier, indicating that Rb+ permeated the channel less readily than did K+. On the other hand permeability ratios (PRb/PK) measured from the change in reversal potential upon ionic substitution were 0.92 for the external and 0.67 for the internal mouth of the channel. 3. Ensemble‐averaged currents activated under depolarization along a similarly S‐shaped time course whether K+ or Rb+ carried current, though slightly more slowly in Rb+. However, under repolarization to a negative level, tail currents were prolonged about tenfold in Rb+. 4. The duration of channel opening was substantially prolonged in Rb+. The distribution of open times was fitted by a single exponential whether K+ or Rb+ was the charge carrier, indicating a single open state. But the mean open time, averaged over all voltages investigated, was 2.65 times greater in Rb+. 5. The prolongation in Rb+ of tail currents under repolarization was associated with increases in the number of openings per burst and in the number of bursts during each tail. 6. The implications of these results for channel gating are discussed. It is argued that an early step in channel activation is more voltage dependent than later steps.