Effects of protein tyrosine kinase and protein tyrosine phosphatase on apical K(+) channels in the TAL

We havepreviously demonstrated that the protein level of c-Src, anonreceptor type of protein tyrosine kinase (PTK), was higher in therenal medulla from rats on a K-deficient (KD) diet than that in rats ona high-K (HK) diet (Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165-F171, 2000).We have now used the patch-clamp technique to investigate the role ofPTK in regulating the apical K channels in the medullary thickascending limb (mTAL) of the rat kidney. Inhibition of PTK withherbimycin A increased NP_o, a product of channelnumber (N) and open probability (P_o),of the 70-pS K channel from 0.12 to 0.42 in the mTAL only from rats ona KD diet but had no significant effect in tubules from animals on a HKdiet. In contrast, herbimycin A did not affect the activity of the30-pS K channel in the mTAL from rats on a KD diet. Moreover, additionof N-methylsulfonyl-12,12-dibromododec-11-enamide, an agentthat inhibits the cytochrome P-450-dependent production of20-hydroxyeicosatetraenoic acid, further increasedNP_o of the 70-pS K channel in the presence ofherbimycin A. Furthermore, Western blot detected the presence ofPTP-1D, a membrane-associated protein tyrosine phosphatase (PTP), inthe renal outer medulla. Inhibition of PTP with phenylarsine oxide(PAO) decreased NP_o of the 70-pS K channel inthe mTAL from rats on a HK diet. However, PAO did not inhibit theactivity of the 30-pS K channel in the mTAL. The effect of PAO on the70-pS K channel was due to indirectly stimulating PTK becausepretreatment of the mTAL with herbimycin A abolished the inhibitoryeffect of PAO. Finally, addition of exogenous c-Src reversibly blockedthe activity of the 70-pS K channel in inside-out patches. We concludethat PTK and PTP have no effect on the low-conductance K channels inthe mTAL and that PTK-induced tyrosine phosphorylation inhibits,whereas PTP-induced tyrosine dephosphorylation stimulates, the apical70-pS K channel in the mTAL.

     

Effect of arachidonic acid on activity of the apical K+ channel in the thick ascending limb of the rat kidney

We have used patch-clamp techniques to study the effects of arachidonic acid (AA) on the activity of the 70-pS K+ channel, the predominant type of the two apical K+ channels operating under physiological conditions in the thick ascending limb (TAL) of the rat kidney. Addition of 5-10 microM AA blocked the activity of the 70-pS K+ channel in both cell- attached and inside-out patches. The inhibitory effect of AA was specific, because application of 10 microM linoleic acid, oleic acid, or palmitic acid failed to mimic the effect of AA. The effect of AA could not be blocked by pretreatment of the TAL tubules with either 5 microM indomethacin (inhibitor of cyclooxygenase) or 4 microM cinnamyl- 3,4-dihydroxy-alpha-cyanocinnamate (CDC) (inhibitor of lipooxygenase). In contrast, addition of 5 microM 17-octadecynoic acid (17-ODYA), an inhibitor of P450 monooxygenases, abolised the effect of AA on the channel activity, indicating that the effect was mediated by cytochrome P450 metabolites of AA. Addition of 10 nM 20-hydroxyeicosatetraenoic acid (20-HETE), the main metabolite of the cytochrome P450 metabolic pathway in the medullary TAL, mimicked the inhibitory effect of 10 microM AA. However, addition of 100 nM 19-HETE or 17-HETE had no significant effects and 100 nM 20-carboxy AA (20-COOH) reduced the channel activity by only 20%, indicating that the inhibitory effect of 20-HETE was specific and responsible for the action of AA. Inhibition of the P450 metabolic pathway by either 5 microM 17-ODYA or 12, 12- dibromododec-11-enoic acid (DBDD) dramatically increased the channel activity by 280% in cell-attached patches. The stimulatory effect of 17- ODYA or DBDD was not observed in inside-out patches. The results strongly indicate that 20-HETE is a specific inhibitor for the 70-pS K+ channel and may play an important role in the regulation of the K+ channel activity in the TAL.     

Stimulation of Ca2+-sensing receptor inhibits the basolateral 50-pS K channels in the thick ascending limb of rat kidney

We used the patch-clamp technique to study the effect of changing the external Ca2+ on the basolateral 50-pS K channel in the thick ascending limb (TAL) of rat kidney. Increasing the external Ca2+ concentration from 1 mM to 2 or 3 mM inhibited the basolateral 50-pS K channels while decreasing external Ca2+ to 10 μM ?increased the 50-pS K channel activity. The effect of the external Ca2+ on the 50-pS K channels was observed only in cell-attached patches but not in excised patches. Moreover, the inhibitory effect of increasing external Ca2+ on the 50-pS K channels was absent in the presence of NPS2390, an antagonist of Ca2+-sensing receptor (CaSR), suggesting that the inhibitory effect of the external Ca2+ was the result of stimulation of the CaSR. Application of the membrane-permeable cAMP analog increased the 50-pS K channel activity but did not block the effect of raising the external Ca2+ on the K channels. Neither inhibition of phospholipase A2 (PLA2) nor suppression of cytochrome P450-ω-hydroxylation-dependent metabolism of arachidonic acid was able to abolish the effect of raising the external Ca2+ on the 50-pS K channels. In contrast, inhibition of phospholipase C (PLC) or blocking protein kinase C (PKC) completely abolished the inhibition of the basolateral 50-pS K channels induced by raising the external Ca2+. We conclude that the external Ca2+ concentration plays an important role in the regulation of the basolateral K channel activity in the TAL and that the effect of the external Ca2+ is mediated by the CaSR which stimulates PLC-PKC pathways. The regulation of the basolateral K channels by the CaSR may be the mechanism by which extracellular Ca2+ level modulates the reabsorption of divalent cations.     

Dual effect of insulin-like growth factor on the apical 70-pS K channel in the thick ascending limb of rat kidney

We used the patch-clamp technique to study the effect of insulin-like growth factor I (IGF-I) on the apical 70-pS K channel in the isolated thick ascending limb (TAL) of the rat kidney. The isolated TAL was cut open to gain access to the apical membrane. Addition of 25 nM IGF-I stimulates the apical 70-pS K channel and increases channel activity, defined by the product of channel open probability and channel number, from 0.31 to 1.21. The stimulatory effect of IGF-I is not mediated by nitric oxide- or protein tyrosine phosphatase-dependent mechanisms, because inhibition of nitric oxide synthase or blocking protein tyrosine phosphatase did not abolish the stimulatory effect of IGF-I on the 70-pS K channel. In contrast, inhibition of mitogen-activated protein (MAP) kinase with PD-98059 or U0126 abolished the stimulatory effect of IGF-I. This suggests that MAP kinase is responsible for mediating the effect of IGF-I on the apical K channels. Moreover, the effect of IGF-I on the apical 70-pS K channel is biphasic because high concentrations (>200 nM) inhibit apical 70-pS K channels. Application of 400 nM IGF-I decreased channel activity from 1.45 to 0.2. The inhibitory effect of IGF-I is not blocked by calphostin C (an inhibitor of PKC), but inhibition of protein tyrosine kinase with herbimycin A abolished the IGF-induced inhibition. We conclude that IGF-I has a dual effect on the apical 70-pS K channel in the TAL: low concentrations of IGF-I stimulate, whereas high concentrations inhibit the channel activity. The stimulatory effect of IGF-I is mediated by a MAP kinase-dependent pathway, whereas the inhibitory effect is the result of stimulation of protein tyrosine kinase.     

Role of 20-HETE in the hypoxia-induced activation of Ca2+-activated K+ channel currents in rat cerebral arterial muscle cells

The mechanism of sensing hypoxia and hypoxia-induced activation of cerebral arterial Ca(2+)-activated K(+) (K(Ca)) channel currents and vasodilation is not known. We investigated the roles of the cytochrome P-450 4A (CYP 4A) omega-hydroxylase metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE), and generation of superoxide in the hypoxia-evoked activation of the K(Ca) channel current in rat cerebral arterial muscle cells (CAMCs) and cerebral vasodilation. Patch-clamp analysis of K(+) channel current identified a voltage- and Ca(2+)-dependent 238 +/- 21-pS unitary K(+) currents that are inhibitable by tetraethylammonium (TEA, 1 mM) or iberiotoxin (100 nM). Hypoxia (<2% O(2)) reversibly enhanced the open-state probability (NP(o)) of the 238-pS unitary K(Ca) current in cell-attached patches. This effect of hypoxia was not observed on unitary K(Ca) currents recorded from either excised inside-out or outside-out membrane patches. Inhibition of CYP 4A omega-hydroxylase activity increased the NP(o) of K(Ca) single-channel current. Hypoxia reduced the basal endogenous level of 20-HETE by 47 +/- 3% as well as catalytic formation of 20-HETE in cerebral arterial muscle homogenates as determined by liquid chromatography-mass spectrometry analysis. The concentration of authentic 20-HETE was reduced when incubated with the superoxide donor KO(2). Exogenous 20-HETE (100 nM) attenuated the hypoxia-induced activation of the K(Ca) current in CAMCs. Hypoxia did not augment the increase in NP(o) of K(Ca) channel current induced by suicide inhibition of endogenous CYP 4A omega-hydroxylase activity with 17-octadecynoic acid. In pressure (80 mmHg)-constricted cerebral arterial segments, hypoxia induced dilation that was partly attenuated by 20-HETE or by the K(Ca) channel blocker TEA. Exposure to hypoxia caused the generation of intracellular superoxide as evidenced by intense staining of arterial muscle with the fluorescent probe hydroethidine, by quantitation using fluorescent HPLC analysis, and by attenuation of the hypoxia-induced activation of the K(Ca) channel current by superoxide dismutation. These results suggest that the exposure of CAMCs to hypoxia results in the generation of superoxide and reduction in endogenous level of 20-HETE that may account for the hypoxia-induced activation of arterial K(Ca) channel currents and cerebral vasodilation.     

Stimulation of A(₂a) adenosine receptor abolishes the inhibitory effect of arachidonic acid on the basolateral 50-pS K channel in the thick ascending limb

The basolateral 50-pS K channels are stimulated by a cAMP-dependent pathway and inhibited by cytochrome P-450-omega-hydroxylase-dependent metabolism of arachidonic acid (AA) in the rat thick ascending limb (TAL). We now used the patch-clamp technique to examine whether stimulation of adenosine A(?a) receptor modulates the inhibitory effect of AA on the basolateral 50-pS K channels in the medullary TAL. Stimulation of adenosine A(?a) receptor with CGS-21680 or inhibition of phospholipase A? (PLA?) with AACOCF3 increased the 50-pS K channel activity in the TAL. Western blot demonstrated that application of CGS-21680 decreased the phosphorylation of PLA(2) at serine residue 505, an indication of inhibiting PLA? activity. In the presence of CGS-21680, inhibition of PLA? had no further effect on the basolateral 50-pS K channels. The possibility that CGS-21680-induced stimulation of the basolateral 50-pS K channels was partially achieved by inhibition of PLA? in the TAL was also supported by the observation that CGS-21680 had no additional effect in the presence of AACOCF3. Moreover, stimulation of adenosine A(?a) receptor with CGS-21680 also abolished the inhibitory effect of AA and 20-hydroxyeicosatetraenoic acid (20-HETE) on the 50-pS K channels. The effect of CGS-21680 on AA and 20-HETE-mediated inhibition of the 50-pS K channels was mediated by cAMP because application of membrane-permeable cAMP analog, dibutyryl-cAMP, not only increased the 50-pS K channel activity but also abolished the inhibitory effect of AA and 20-HETE. We conclude that stimulation of adenosine A(?a) receptor increased the 50-pS K channel activity in the TAL, an effect that is achieved by suppression of PLA? activity and 20-HETE-induced inhibition.     

Mechanism of apical K+ channel modulation in principal renal tubule cells. Effect of inhibition of basolateral Na(+)-K(+)-ATPase

The effects of inhibition of the basolateral Na(+)-K(+)-ATPase (pump) on the apical low-conductance K+ channel of principal cells in rat cortical collecting duct (CCD) were studied with patch-clamp techniques. Inhibition of pump activity by removal of K+ from the bath solution or addition of strophanthidin reversibly reduced K+ channel activity in cell-attached patches to 36% of the control value. The effect of pump inhibition on K+ channel activity was dependent on the presence of extracellular Ca2+, since removal of Ca2+ in the bath solution abolished the inhibitory effect of 0 mM K+ bath. The intracellular [Ca2+] (measured with fura-2) was significantly increased, from 125 nM (control) to 335 nM (0 mM K+ bath) or 408 nM (0.2 mM strophanthidin), during inhibition of pump activity. In contrast, cell pH decreased only moderately, from 7.45 to 7.35. Raising intracellular Ca2+ by addition of 2 microM ionomycin mimicked the effect of pump inhibition on K+ channel activity. 0.1 mM amiloride also significantly reduced the inhibitory effect of the K+ removal. Because the apical low-conductance K channel in inside-out patches is not sensitive to Ca2+ (Wang, W., A. Schwab, and G. Giebisch, 1990. American Journal of Physiology. 259:F494-F502), it is suggested that the inhibitory effect of Ca2+ is mediated by a Ca(2+)-dependent signal transduction pathway. This view was supported in experiments in which application of 200 nM staurosporine, a potent inhibitor of Ca(2+)- dependent protein kinase C (PKC), markedly diminished the effect of the pump inhibition on channel activity. We conclude that a Ca(2+)- dependent protein kinase such as PKC plays a key role in the downregulation of apical low-conductance K+ channel activity during inhibition of the basolateral Na(+)-K(+)-ATPase.     

Effect of angiotensin II on the apical K+ channel in the thick ascending limb of the rat kidney

We have used the patch-clamp technique to study the effect of angiotensin II (AII) on the activity of the apical 70 pS K+ channel and used Na(+)-sensitive fluorescent dye (SBFI) to investigate the effect of AII on intracellular Na+ concentration (Na+i) in the thick ascending limb (TAL) of the rat kidney. Addition of 50 pM AII reversibly reduced NPo, a product of channel open probability (Po) and channel number (N), to 40% of the control value and reduced the Na+i by 26%. The AII (50 pM)-induced decrease in channel activity defined by NPo was partially reversed by addition of 5 microM 17-octadecynoic acid (17-ODYA), an agent which blocks the cytochrome P450 monooxygenase. The notion that P450 metabolites of arachidonic acid (AA) may mediate the inhibitory effect of AII was further suggested by experiments in which addition of 10 nM of 20-hydroxyeicosatetraenoic acid (20-HETE) blocked the channel activity in cell-attached patches in the presence of 17-ODYA. We have used gas chromatography mass spectrometry (GC/MS) to measure the production of 20-HETE, a major AA metabolite of the P450-dependent pathway in the TAL of the rat. Addition of 50 pM AII increased the production of 20-HETE to 260% of the control value, indicating that 20- HETE may be involved in mediating the effect of AII (50 pM). In contrast to the inhibitory effect of 50 pM AII, addition of 50-100 nM AII increased the channel activity to 270% of the control value and elevated the Na+i by 45%. The effect of AII on the activity of the 70 pS K+ channel was also observed in the presence of 5 microM 17-ODYA and 5 microM calphostin C, an inhibitor of protein kinase C. However, addition of 100 microM NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, abolished completely the AII (50- 100 nM)-induced increase in channel activity and addition of an exogenous nitric oxide (NO) donor, S-nitroso-N-acetyl-penicillamine (SNAP), increased channel activity in the presence of L-NAME. These data suggest that the stimulatory effect of AII is mediated by NO. We conclude that AII has dual effects on the activity of the apical 70 pS K+ channel. The inhibitory effect of AII is mediated by P450-dependent metabolites whereas the stimulatory effect may be mediated via NO.     

High dietary salt reduces the contribution of 20-HETE to arteriolar oxygen responsiveness in skeletal muscle

The coupling of tissue blood flow to cellular metabolic demand involves oxygen-dependent adjustments in arteriolar tone, and arteriolar responses to oxygen can be mediated, in part, by changes in local production of 20-HETE. In this study, we examined the long-term effect of dietary salt on arteriolar oxygen responsiveness in the exteriorized, superfused rat spinotrapezius muscle and the role of 20-HETE in this responsiveness. Rats were fed either a normal-salt (NS, 0.45%) or high-salt (HS, 4%) diet for 4-5 wk. There was no difference in steady-state tissue Po(2) between NS and HS rats, and elevation of superfusate oxygen content from 0% to 10% caused tissue Po(2) to increase by the same amount in both groups. However, the resulting reductions in arteriolar diameter and blood flow were less in HS rats than NS rats. Inhibition of 20-HETE formation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or 17-octadecynoic acid (17-ODYA) attenuated oxygen-induced constriction in NS rats but not HS rats. Exogenous 20-HETE elicited arteriolar constriction that was greatly reduced by the large-conductance Ca(2+)-activated potassium (K(Ca)) channel inhibitors tetraethylammonium chloride (TEA) and iberiotoxin (IbTx) in NS rats and a smaller constriction that was less sensitive to TEA or IbTx in HS rats. Arteriolar responses to exogenous angiotensin II were similar in both groups but more sensitive to inhibition with DDMS in NS rats. Norepinephrine-induced arteriolar constriction was similar and insensitive to DDMS in both groups. We conclude that 20-HETE contributes to oxygen-induced constriction of skeletal muscle arterioles via inhibition of K(Ca) channels and that a high-salt diet impairs arteriolar responses to increased oxygen availability due to a reduction in vascular smooth muscle responsiveness to 20-HETE.     

An endogenous acid-sensitive K+ channel expressed in COS-7 cells

COS-7 cells originally isolated from monkey kidney and used in many transfection studies were found to express a background K+ channel and therefore, its biophysical and pharmacological properties were examined. In cell-attached patches, a 32-pS K+ channel with a linear current-voltage relationship could be recorded. The open probability was highly voltage-dependent, with greater channel activity at depolarized potentials. The channel was markedly sensitive to changes in extracellular pH (pH(o)), showing a 70+/-10% inhibition by changing the pH(o) from 7.3 to 6.3. Arachidonic acid (5 microM) augmented channel activity 12-fold. Applying negative pressure (-40 mmHg) to the membrane patch also increased channel activity by 4-fold. These results show that COS-7 cells express a K+ channel with unique properties that must be considered when using these cells as transfection system.     

Depolarization-Activated K+ Channel in Chara Droplets

A novel potassium channel was characterized in the droplet membrane of Chara gymnophylla. This channel has a conductance of about 90 pS (in symmetrical 0.15 M KCl), which is lower compared to the 170-pS K+ channel predominant in this preparation. In contrast to the large conductance K+ channel, the novel channel opened with a delay at depolarization and closed at hyperpolarization and did not require cytosolic Ca2+ for its opening. It also showed comparatively weak selectivity for K+ over other monovalent cations, although its cation to anion selectivity was high. Externally or internally applied Cs+ blocked the channel in a voltage-dependent manner, similarly to the 170-pS channel. The sensitivity of the 90-pS channel to external tetraethylammonium chloride (half-blocking concentration approximately 1.5 mM) was 20-fold higher compared to the large conductance channel. With respect to its voltage-gating kinetics, the 90-pS channel was identified as a "slow delayed rectifier."     

Protein-tyrosine phosphatase reduces the number of apical small conductance K+ channels in the rat cortical collecting duct

Previous studies have demonstrated that an increase in the activity of protein-tyrosine kinase (PTK) is involved in the down-regulation of the activity of apical small conductance K(+) (SK) channels in the cortical collecting duct (CCD) from rats on a K(+)-deficient diet (). We used the patch clamp technique to investigate the role of protein-tyrosine phosphatase (PTP) in the regulation of the activity of SK channels in the CCD from rats on a high K(+) diet. Western blot analysis indicated that PTP-1D is expressed in the renal cortex. Application of 1 microm phenylarsine oxide (PAO) or 1 mm benzylphosphonic acid, agents that inhibit PTP, reversibly reduced channel activity by 95%. Pretreatment of CCDs with PAO for 30 min decreased the mean NP(o) reversibly from control value 3.20 to 0.40. Addition of 1 microm herbimycin A, an inhibitor of PTK, had no significant effect on channel activity in the CCDs from rats on a high K(+) diet. However, herbimycin A abolished the inhibitory effect of PAO, indicating that the effect of PAO is the result of interaction between PTK and PTP. Addition of brefeldin A, an agent that blocks protein trafficking from Golgi complex to the membrane, had no effect on channel activity. Moreover, application of colchicine, a microtubule inhibitor, or paclitaxel, a microtubule stabilizer, had no effect on channel activity. In contrast, PAO still reduced channel activity in the presence of brefeldin A, colchicine, or paclitaxel. Furthermore, the effect of PAO on channel activity was absent when the tubules were bathed in 16% sucrose-containing bath solution or treated with concanavalin A. We conclude that PTP is involved in the regulation of the activity of SK channels and that inhibition of PTP may facilitate the internalization of the SK channels.     

Role of cGMP versus 20-HETE in the vasodilator response to nitric oxide in rat cerebral arteries

This study examined the response to nitric oxide (NO) in rat middle cerebral arteries (MCA). NO donors increased the activity of a 205-pS K(+) channel recorded from vascular smooth muscle (VSM) cells isolated from MCA 10-fold. Blockade of guanylyl cyclase activity with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10(-5) M) did not alter the effect of NO on this channel. In contrast, adding 20-hydroxyeicosatetraenoic acid (20-HETE) to the bath (10(-7) M) abolished the response to NO. NO donors also increased the diameter of serotonin-preconstricted MCA to 85% of control. Blockade of K(+) channels with iberiotoxin or a high-K(+) medium reduced this response by 50%. ODQ (10(-5) M) reduced this response by 47 +/- 3%, whereas preventing the fall of 20-HETE levels reduced the response by 59 +/- 2% (n = 5). Blockade of both pathways eliminated the response to NO donors. These results indicate that activation of K(+) channels contributes 50% to vasodilator response to NO in rat MCA. This is mediated by a fall in 20-HETE levels rather than a rise in cGMP levels or a direct effect of NO.     

The cGMP-dependent protein kinase stimulates the basolateral 18-pS K channel of the rat CCD

We used the patch-clamp technique tostudy the effect of cGMP on the 18-pS K channel in the basolateralmembrane of the rat cortical collecting duct. Addition of 100 µM8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP)increased the activity of the 18-pS K channel, defined byNP_o, by 95%. In contrast, applying 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) hasno effect on channel activity. The effect of 8-Br-cGMP was observed only in cell-attached but not in inside-out patches. Application of 1 µM KT-5823, an inhibitor of the cGMP-dependent protein kinase (PKG),not only reduced the channel activity, but also completely abolishedthe stimulatory effect of 8-Br-cGMP, suggesting that the 18-pS Kchannel is not a cGMP-gated K channel. Addition of H-89, an agent thatalso blocks the PKG, mimicked the effect of KT-5823. To examine thepossibility that the effect of 8-Br-cGMP is the result of inhibitingcGMP-dependent phosphodiesterase (PDE) and, accordingly, increasingcAMP or cGMP levels, we explored the effect on the 18-pS K channel ofIBMX, an agent that inhibits the PDE. The addition of 100 µM IBMX hadno significant effect on channel activity in cell-attached patches.Moreover, in the presence of IBMX, 8-Br-cGMP increased the channelactivity to the same extent as that observed in the absence of IBMX,suggesting that the effect of cGMP is not mediated by inhibiting thecGMP-dependent PDE. That the effect of cGMP is mediated by stimulatingPKG was further indicated by experiments in which application ofexogenous PKG restored the channel activity when it decreased after the excision of the patches. In contrast, adding exogenous cAMP-dependent protein kinase catalytic subunit failed to reactivate therun-down channels. We conclude that cGMP stimulates the 18-pS channel, and the effect of cGMP is mediated by PKG.

     

Single channel studies of the phosphorylation of K+ channels in the squid giant axon. I. Steady-state conditions

Phosphorylation of the delayed rectifier channel of squid potentiates the macroscopic K+ current and slows its activation kinetics. We have studied this phenomenon at the single channel level using the cut-open axon technique under steady-state conditions. In 10 mM external K+/310 mM internal K+ there are predominantly two types of channels present, a 20-pS and a 40-pS channel. In steady state at depolarized potentials, the 40-pS channel was most active, whereas the 20-pS channel tended to disappear due to a slow inactivation process. Two methods were developed to shift the population of channels toward a dephosphorylated state. One method consisted of predialyzing a whole axon with solutions containing no ATP, while recording the currents under axial-wire voltage clamp. A piece of axon was then removed and cut open, and single channel currents were recorded from the cut-open axon. A second method was based on the difference in diffusion coefficients for ATP and proteins such as the endogenous phosphatase. The axon was cut open in a solution that did not contain Ca2+ or Cl- in order to maintain the axoplasm structurally intact and permit endogenous phosphatase to act on the membrane while ATP diffused away, before removing the axoplasm and forming a membrane patch. When dephosphorylating conditions were used, the steady-state open probability of the 40-pS channel at 42 mV was very low (less than 0.0002), and the channel openings appeared as a series of infrequent, short-duration events. The channel activity was increased up to 150-fold by photoreleasing caged ATP inside the patch pipette in the presence of the catalytic subunit of protein kinase A. The sharp increase in open probability could be accounted for by a decrease of the slow component of the closed time distribution from 23 s to 170 ms with little change in the distribution of open times (1-2 ms) and no change in the single channel current amplitude. In voltage-jump experiments the contribution of the 40-pS channel to the delayed rectifier current was often small due to the large values of the latency to the first opening.     

Single-channel calcium and barium currents of large and small conductance from sarcoplasmic reticulum.

Two types of divalent cation conducting channels from rabbit skeletal muscle sarcoplasmic reticulum (SR) were incorporated into planar lipid bilayers. A high conductance (100 pS in 53 mM trans Ca2+) Ca2+ channel was incorporated from heavy density SR fractions. The 100-pS channel was activated by adenine nucleotides and Ca2+ and inhibited by Mg2+ and ruthenium red. A 10-pS calcium and barium conducting channel could be incorporated into planar lipid bilayers from light, intermediate, and heavy density SR vesicles. 10-pS channel activity in bilayers was not dependent on cis Ca2+ and was only weakly dependent on adenine nucleotides. Ruthenium red at concentrations up to 1 mM had no effect and Mg2+ was only marginally effective in inhibiting macroscopic Ba2+ currents from this channel. Calcium releasing activity in intermediate and heavy density SR fractions was assayed according to a rapid quench protocol and compared with the results obtained in the bilayer. Results from this comparison indicate that the 10-pS channel is probably not involved in rapid Ca2+- and adenine nucleotide-induced Ca2+ release from isolated SR vesicles.     

Delayed rectifying and calcium-activated K+ channels and their significance for action potential repolarization in mouse pancreatic beta-cells

The contribution of Ca2(+)-activated and delayed rectifying K+ channels to the voltage-dependent outward current involved in spike repolarization in mouse pancreatic beta-cells (Rorsman, P., and G. Trube. 1986. J. Physiol. 374:531-550) was assessed using patch-clamp techniques. A Ca2(+)-dependent component could be identified by its rapid inactivation and sensitivity to the Ca2+ channel blocker Cd2+. This current showed the same voltage dependence as the voltage-activated (Cd2(+)-sensitive) Ca2+ current and contributed 10-20% to the total beta-cell delayed outward current. The single-channel events underlying the Ca2(+)-activated component were investigated in cell-attached patches. Increase of [Ca2+]i invariably induced a dramatic increase in the open state probability of a Ca2(+)-activated K+ channel. This channel had a single-channel conductance of 70 pS [( K+]o = 5.6 mM). The Ca2(+)-independent outward current (constituting greater than 80% of the total) reflected the activation of an 8 pS [( K+]o = 5.6 mM; [K+]i = 155 mM) K+ channel. This channel was the only type observed to be associated with action potentials in cell-attached patches. It is suggested that in mouse beta-cells spike repolarization results mainly from the opening of the 8-pS delayed rectifying K+ channel.     

Ion channels in the thick ascending limb of Henle's loop.

The thick ascending limb of Henle's loop (TAL) is polarized with respect to its conductances. The luminal membrane contains a K+ conductance which is made up by the synchronous operation of 60- to 80-pS K+ channels. The basolateral membrane contains a chloride conductance. This conductance corresponds most likely to a 30- to 60-pS Cl- channel present in this membrane. Our knowledge on the properties of the K+ channels of these cells has been increased rapidly by patch clamp studies: these K+ channels are inwardly rectifying. They are highly selective for K+ over Na+, Li+ and many other cations. They do not conduct Rb+, Cs+, NH+4 or other larger cations. In fact, all these three cations as well as choline, tetraethylammonium, lidocaine, verapamil, diltiazem, quinine, quinidine and Ba2+ inhibit these K+ channels. As apparent from kinetic studies the mechanisms of inhibition are different for the various blockers. The TAL K+ channels are downregulated by increasing cytosolic Ca2+ activity. Cytosolic adenosine trisphosphate (ATP) has a similar effect. This ATP inhibition is Ca2+ dependent. The affinity to ATP is augmented by increasing Ca2+. Cytosolic alkalinity increases the open probability of these channels, and cytosolic acidification has the opposite effect. This pH dependence is very marked. A change by 0.2 pH units leads to a more than twofold change in the open-channel probability. The basolateral chloride conductance reflects the properties of an outwardly rectifying 30- to 60-pS Cl- channel. This channel behaves, in many respects, like the Cl- channels of a multitude of Cl- transporting epithelia. It is characterized by two open and two closed states. It is highly selective for Cl- as compared with larger anions, and it is inhibited reversibly by Cl- channel blockers such as 5-nitro-2-(3-phenylpropylamino)-benzoate.