Noel W. Davies

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.

Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes: Evidence for tetrameric channel formation

Unitary currents were recorded from insideout membrane patches pulled from Xenopus oocytes that had been injected with RNA transcribed from a cDNA encoding the Drosophila maxi-K channel (Slowpoke). Site-directed mutagenesis was used to make cDNAs encoding channel subunits with single amino acid substitutions (Y308V and C309P). The extracellular side of the patch was exposed to tetraethylammonium (TEA) in the pipette solution; unitary currents in the presence of TEA were compared with currents in the absence of TEA to compute the inhibition. Amplitude distributions were fit by β functions to estimate the blocking and unblocking rate constants. For wild-type channels, TEA blocked with an apparent Kd of 80 μM at 0 mV and sensed 0.18 of the membrane electric field; the voltage dependence lay entirely in the blocking rate constant. TEA blocked currents through C309P channels with a similar affinity to wild-type at 0 mV, but this was not voltage-dependent. Currents through Y308V channels were very insensitive to any block by TEA; the apparent Kd at 0 mV was 26 mM and the blockade sensed 0.18 of the electric field. Oocytes injected with a mixture of RNAs encoding wild-type and Y308V channels showed unitary currents of four discrete amplitudes in the presence of 3 mM TEA; at 40 mV these corresponded to inhibitions of approximately 80%, 55%, 25% and 10%. These values agreed well with those expected for inhibition by TEA of currents through channels containing 3, 2, 1 and 0 tyrosine residues at the channel mouth, assuming that a tyrosine residue from each of four subunits contributes equally to the binding of the TEA ion. This indicates that Slowpoke channels form as tetramers.

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.

Angiotensin II inhibits rat arterial KATP channels by inhibiting steady‐state protein kinase A activity and activating protein kinase Ce

1 We used whole‐cell patch clamp to investigate steady‐state activation of ATP‐sensitive K+ channels (KATP) of rat arterial smooth muscle by protein kinase A (PKA) and the pathway by which angiotensin II (Ang II) inhibits these channels. 2 Rp‐cAMPS, an inhibitor of PKA, did not affect KATP currents activated by pinacidil when the intracellular solution contained 0.1 mM ATP. However, when ATP was increased to 1.0 mM, inhibition of PKA reduced KATP current, while the phosphatase inhibitor calyculin A caused a small increase in current. 3 Ang II (100 nM) inhibited KATP current activated by the K+ channel opener pinacidil. The degree of inhibition was greater with 1.0 mM than with 0.1 mM intracellular ATP. The effect of Ang II was abolished by the AT1 receptor antagonist losartan. 4 The inhibition of KATP currents by Ang II was abolished by a combination of PKA inhibitor peptide 5‐24 (5 μM) and PKC inhibitor peptide 19‐27 (100 μM), while either alone caused only partial block of the effect. 5 In the presence of PKA inhibitor peptide, the inhibitory effect of Ang II was unaffected by the PKC inhibitor Gö 6976, which is selective for Ca2+‐dependent isoforms of PKC, but was abolished by a selective peptide inhibitor of the translocation of the ε isoform of PKC. 6 Our results indicate that KATP channels are activated by steady‐state phosphorylation by PKA at normal intracellular ATP levels, and that Ang II inhibits the channels both through activation of PKCε and inhibition of PKA.

Multiple actions of a molluscan cardioexcitatory neuropeptide and related peptides on identified Helix neurones.

The effects of the molluscan neuropeptide Phe‐Met‐Arg‐Phe‐NH2 (FMRF amide) and related peptides (Price & Greenberg, 1977) were tested on Helix aspersa neurones. Ionophoretic application of FMRFamide depolarized and excited some neurones, but hyperpolarized and inhibited others. In some neurones the sign of the response was dependent on the membrane potential. Two responses resulted from an increase in membrane conductance, a depolarizing response mediated mainly by an increase in Na+ ion permeability, and a hyperpolarizing response mediated by an increase in K+ ion permeability. In the C1 neurone a voltage‐dependent response was observed, which only occurred when the neurone was depolarized from its resting level. This response was recorded as an inward current during voltage clamp and resulted from a decrease in K current(s), possibly Ca‐activated K current. More than one response may occur in a single neurone. In the C1 neurone, the K‐mediated hyperpolarization occurred as well as the voltage‐dependent response, while the depolarization seen in the F2 neurone was a combination of an increase in Na conductance and an increase in K conductance.

Site and mechanism of activation of proton‐induced sodium current in chick dorsal root ganglion neurones.

1. In dissociated and cultured 1‐ to 2‐day‐old chick dorsal root ganglion cells, and in isolated outside‐out membrane patches, a large transient current lasting 1‐2 s could be activated upon step increases in [H+]o. The proton‐induced current reversed direction at the Na+ equilibrium potential, was abolished completely in the absence of Na+, and was therefore labelled INa(H). 2. To investigate the activation and deactivation kinetics of INa(H) at the single‐channel level, we employed isolated membrane patches and a method whereby we could change the external solution in less than 1 ms. 3. In outside‐out membrane patches, INa(H) was fully activated within 2 ms between pH 6.7 and 5.7. Half‐times of activation decreased with increasing [H+]o. The calculated association rate constant was 9.5 x 10(9) M‐1 s‐1. 4. Deactivation of INa(H), following a step reduction in [H+]o, occurred with half‐times of within 1.3‐2 ms. 5. In the continued presence of an activating solution (pH 6.7 and 1 mM‐Ca2+), INa(H) inactivated slowly, with a time constant of about 300 ms. 6. Inactivation showed a limited dependence on [Ca2+]o. The time constant of inactivation increased from about 300‐500 ms as [Ca2+]o was decreased from 5 to 0.1 mM. Further decrease in [Ca2+]o did not significantly increase the time course of inactivation. Increases in [Ca2+]i from 10(‐9) to 10(‐3) M had no effect on the activation or inactivation kinetics of INa(H). 7. Conditioning proton concentrations which by themselves failed to activate any channel openings, partially inactivated INa(H). 8. Recovery from inactivation appeared to follow a time course similar to that of inactivation itself. 9. INa(H) could not be activated in inside‐out patches. A step increase in proton concentration outside a cell‐attached patch was also ineffective at producing INa(H) in the patch. Intracellular pH between 7.9 and 6.7 had no effect on the activation or inactivation of INa(H). 10. The activation and inactivation kinetics were not significantly voltage dependent. 11. The single‐channel conductance associated with the activation of INa(H) was 28 pS in symmetrical 120 mM‐NaCl solutions and remained constant throughout the time course of INa(H). 12. During activation of the voltage‐gated calcium current, ICa, a step increase in proton concentration caused a rapid (ca. 2 ms) suppression of ICa which was more than that predicted from the steady‐state effects of H+ on ICa. This effect was independent of [Na+] and the direction of INa(H).(ABSTRACT TRUNCATED AT 400 WORDS)

Properties of BKCa Channels Formed by Bicistronic Expression of hSloα and β1–4 Subunits in HEK293 Cells

Large-conductance Ca2+-activated K+ (BKCa) channels are sensitive to both voltage and internal [Ca2+] and are found in many tissues. Their physiological roles range from causing relaxation of smooth muscle to regulating the frequency of action potential firing. There is considerable variation between different tissues in their Ca2+- and voltage-dependence. Much of this variation results from the association of the pore-forming α subunit (hSloα) with different β subunits leading to altered channel properties. Since hSloα alone produces functional BKCa channels, we have used a bicistronic expression method to ensure that both α and β subunits are expressed, with the β subunit being in excess. Using this method we have investigated the effect of four β subunits (β1 to β4) on cloned BKCa channels. The four β subunits were individually cloned into a vector that had hSloα cDNA inserted downstream of an internal ribosome entry site. The constructs were transiently transfected into HEK293 cells together with a construct that expresses green fluorescent protein, as a marker for transfection. Fluorescent cells expressed BKCa channels whose currents were recorded from inside-out or outside-out patches. The currents we measured using this expression system were similar to those expressed in Xenopus oocytes by Brenner et al. (Brenner, R., Jegla, T.J., Wickenden, A., Liu, Y., Aldrich, R.W. 2000. Cloning and functional expression of novel large-conductance calcium-activated potassium channel β subunits, hKCNMB3 and hKCNMB4. J. Biol. Chem.275:6453-6461.)

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.

Glucose reduces endothelin inhibition of voltage-gated potassium channels in rat arterial smooth muscle cells

Prolonged hyperglycaemia impairs vascular reactivity and inhibits voltage‐activated K+ (Kv) channels. We examined acute effects of altering glucose concentration on the activity and inhibition by endothelin‐1 (ET‐1) of Kv currents of freshly isolated rat arterial myocytes. Peak Kv currents recorded in glucose‐free solution were reversibly reduced within 200 s by increasing extracellular glucose to 4 mm. This inhibitory effect of glucose was abolished by protein kinase C inhibitor peptide (PKC‐IP), and Kv currents were further reduced in 10 mm glucose. In current‐clamped cells, membrane potentials were more negative in 4 than in 10 mm glucose. In 4 mm d‐glucose, 10 nm ET‐1 decreased peak Kv current amplitude at +60 mV from 23.5 ± 3.3 to 12.1 ± 3.1 pA pF−1 (n= 6, P < 0.001) and increased the rate of inactivation, decreasing the time constant around fourfold. Inhibition by ET‐1 was prevented by PKC‐IP. When d‐glucose was increased to 10 mm, ET‐1 no longer inhibited Kv current (n= 6). Glucose metabolism was required for prevention of ET‐1 inhibition of Kv currents, since fructose mimicked the effects of d‐glucose, while l‐glucose, sucrose or mannitol were without effect. Endothelin receptors were still functional in 10 mm d‐glucose, since pinacidil‐activated ATP‐dependent K+ (KATP) currents were reduced by 10 nm ET‐1. This inhibition was nearly abolished by PKC‐IP, indicating that endothelin receptors could still activate PKC in 10 mm d‐glucose. These results indicate that changes in extracellular glucose concentration within the physiological range can reduce Kv current amplitude and can have major effects on Kv channel modulation by vasoconstrictors.