Cell-free Patches Research Articles

Anesthesia Increases Postoperative Pain

Patients often report a burning sensation when receiving intravenously delivered anesthetics, and respiratory irritation limits the dose of pungent inhalation anesthetics. Matta et al . show that the pungent volatile anesthetics isoflurane and desflurane, as well as the intravenous anesthetics propofol and etomidate (collectively referred to as the noxious general anesthetics), activated TRPA1 channels, which are members of the transient receptor potential family that are present on a large proportion of peripheral nociceptive sensory neurons. The nonpungent volatile anesthetics halothane and sevoflurane did not. Electrophysiology experiments revealed that the noxious general anesthetics activated TRPA1 channels, but not TRPV1 or TRPM8 channels, when the channels were heterologously expressed in cultured cells. Calcium imaging experiments on neurons cultured from dorsal root ganglia indicated that these noxious general anesthetics activated nociceptive neurons containing TRPA1. Cells isolated from TRPA1-null mice failed to respond to the noxious general anesthetics. The noxious general anesthetics activated the channels in cell-free patches, which suggests that these drugs may interact directly with the channel and do not require second messenger signaling for channel regulation. The pain response triggered by propofol administered to mice required TRPA1, and coadministration of a neurogenic irritant with isoflurane triggered more swelling than was observed with the irritant alone. Thus, although these central nervous system depressants are important for induction of surgical anesthesia, choosing a general anesthetic that does not activate TRPA1 may minimize postoperative pain and inflammation. J. A. Matta, P. M. Cornett, R. L. Miyares, K. Abe, N. Sahibzada, G. P. Ahern, General anesthetics activate a nociceptive ion channel to enhance pain and inflammation. Proc. Natl. Acad. Sci. U.S.A. 105 , 8784-8789 (2008). [Abstract] [Full Text]

Calpastatin binds to a calmodulin-binding site of cardiac Cav1.2 Ca 2+ channels

Calpastatin is an endogenous inhibitor of calpain and composed of domain L (CS L), which interacts with the Cav1.2 channels, and four repetitive calpain inhibitory domains. We have previously found that CS L reprimes activity of the Cav1.2 channels in cell-free patches of cardiac myocytes [L.Y. Hao, A. Kameyama, S. Kuroki, J. Takano, E. Takano, M. Maki, M. Kameyama, Calpastatin domain L is involved in the regulation L-type of Ca 2+ channels in guinea pig cardiac myocytes, Biochem. Biophys. Res. Commun. 279 (2000) 756–761; E. Minobe, L.Y. Hao, Z.A. Saud, J.J. Xu, A. Kameyama, M. Maki, K.K. Jewell, T. Parr, R.G. Bardsley, M. Kameyama, A region of calpastatin domain L that reprimes cardiac L-type Ca 2+channels, Biochem. Biophys. Res. Commun. 348 (2006) 288–294]. In this study, we explored the CS L interaction site in the Ca 2+ channel by the pull-down method, using glutathione- S-transferase-fused fragment peptides of the Cav1.2 channel. CS L bound directly to a proximal region of the C-terminal tail of the channel, but not with the N-terminal tail, a distal region of the C-terminal tail or cytoplasmic loops between repeats I–II, II–III or III–IV. Furthermore IQ domain, but not EF-hand-like region or CB domain, in the C-terminal tail was found to bind with CS L in a partially Ca 2+-dependent manner and in a probably competitive manner with calmodulin. These results suggest that CS L modulates Ca 2+-channel activity through interacting with the calmodulin-binding site on the C-terminal tail of the Cav1.2 channel.

Arachidonic acid potentiates acid-sensing ion channels in rat sensory neurons by a direct action

Acid-sensing ion channels (ASICs) are activated by a decrease in extracellular pH. ASICs are expressed in nociceptive sensory neurons, and several lines of evidence suggest that they are responsible for signaling the pain caused by extracellular acidification, but little is understood of the modulation of ASICs by pro-inflammatory factors. Using whole-cell patch clamp we demonstrate that low pH evokes three distinct inward currents in rat dorsal root ganglion neurons: a slowly inactivating transient current, a rapidly inactivating transient current, and a sustained current. All three currents were potentiated by arachidonic acid (AA), to 123%, 171%, and 264% of peak current, respectively. Membrane stretch had no effect on proton-gated currents, implying that AA is unlikely to act via local membrane deformation. The current carried by heterologously expressed ASIC1a and ASIC3 was also potentiated by AA. AA potentiates ASIC activation by a direct mechanism, because inhibition of AA metabolism had no effect on potentiation, and potentiation of single ASIC2a channels could be observed in cell-free patches. Potentiation by lipids with the same chain length as AA increased as the number of double bonds was increased. AA is known to be released in inflammation and the results suggest that AA may be an important pro-inflammatory agent responsible for enhancing acid-mediated pain.

Direct Mechano-Stress Sensitivity of TRPM7 Channel

It has recently been shown that shear stress augments the heterologously expressed TRPM7 channel activity by exocytosis-mediated incorporation of TRPM7 into the plasma membrane. On the other hand, our recent study has shown that the TRPM7-like channel endogenously expressed in HeLa cells is activated by membrane expansion induced by membrane stretch or osmotic cell swelling. Thus, the present study was aimed at exploring the possibility that the heterogously expressed TRPM7 channel is activated directly by membrane expansion in a manner independent of exocytosis. Here, whole-cell currents of the TRPM7 channel heterologously expressed in HEK293T cells were found to be augmented not only by perfusion of bath solution but also by osmotic swelling even under the conditions where exocytotic events can hardly take place in the cytosol dialyzed with ATP-free, Ca²⁺-free and EGTA-containing pipette solution. In addition, shear stress-induced augmentation was not affected by a blocker of vesicular protein traffic, brefeldin A. Furthermore, in cell-free patches, membrane stretch directly augmented single-channel activity of TRPM7 by increasing Po value at ? 20mV. We thus conclude that the TRPM7 channel can be directly activated by mechano-stress in a manner independent of exocytosis-mediated incorporation of this channel protein into the plasma membrane.

Protein phosphatase 2A and Ca 2+-activated K + channels contribute to 11,12-epoxyeicosatrienoic acid analog mediated mesenteric arterial relaxation

Epoxyeicosatrienoic acids (EETs) are considered to be endothelium-derived hyperpolarizing factors, and are potent activators of the large-conductance, Ca 2+-activated K + (BK Ca) channel in vascular smooth muscle. Here, we investigate the signal transduction pathway involved in the activation of BK Ca channels by 11,12-EET and 11,12-EET stable analogs in rat mesenteric vascular smooth muscle cells. 11,12-EET and the 11,12-EET analogs, 11-nonyloxy-undec-8( Z)-enoic acid (11,12-ether-EET-8-ZE), 11-(9-hydroxy-nonyloxy)-undec-8( Z)-enoic acid (11,12-ether-EET-8-ZE-OH) and 11,12- trans-oxidoeicosa-8( Z)-enoic acid (11,12-tetra-EET-8-ZE), caused vasorelaxation of mesenteric resistance arteries. Mesenteric myocyte whole-cell (perforated-patch) currents were substantially (∼150%) increased by 11,12-EET and 11,12-EET analogs. Single-channel recordings were conducted to identify the target for 11,12-EET. 11,12-EET and 11,12-EET analogs also increased mesenteric myocyte BK Ca channel activity in cell-attached patches. Similar results were obtained in cell-free patches. Baseline mesenteric myocyte BK Ca channel activity (NPo) in cell-free patches averaged less than 0.001 at +50 mV and 11,12-EET (1 μmol/L) increased NPo to 0.03 ± 0.02 and 11,12-EET analogs (1 μmol/L) increased NPo to 0.09 ± 0.006. Inhibition of protein phosphatase 2A (PP2A) activity with okadaic acid (10 nmol/L) completely reversed 11,12-EET stimulated BK Ca channel activity and greatly attenuated 11,12-ether-EET-8-ZE mesenteric resistance artery vasorelaxation. 11,12-EET and 11,12-EET analogs increased mesenteric myocyte PP2A activity by 3.5-fold. Okadaic acid and the EET inhibitor, 14,15-epoxyeicosa-5( Z)-enoic acid (14,15-EEZE) inhibited the 11,12-EET mediated increase in PP2A activity. These findings provide initial evidence that PP2A activity contributes to 11,12-EET and 11,12-EET analog activation of mesenteric resistant artery BK Ca channels and vasorelaxation.

Rapid signaling at the plasma membrane by a nuclear receptor for thyroid hormone

Many nuclear hormones have physiological effects that are too rapid to be explained by changes in gene expression and are often attributed to unidentified or novel G protein-coupled receptors. Thyroid hormone is essential for normal human brain development, but the molecular mechanisms responsible for its effects remain to be identified. Here, we present direct molecular evidence for potassium channel stimulation in a rat pituitary cell line (GH(4)C(1)) by a nuclear receptor for thyroid hormone, TRbeta, acting rapidly at the plasma membrane through phosphatidylinositol 3-kinase (PI3K) to slow the deactivation of KCNH2 channels already in the membrane. Signaling was disrupted by heterologous expression of TRbeta receptors with mutations in the ligand-binding domain that are associated with neurological disorders in humans, but not by mutations that disrupt DNA binding. More importantly, PI3K-dependent signaling was reconstituted in cell-free patches of membrane from CHO cells by heterologous expression of human KCNH2 channels and TRbeta, but not TRalpha, receptors. TRbeta signaling through PI3K provides a molecular explanation for the essential role of thyroid hormone in human brain development and adult lipid metabolism.

Regulation of the Ca2+ Sensitivity of the Nonselective Cation Channel TRPM4

TRPM4, a Ca(2+)-activated cation channel of the transient receptor potential superfamily, undergoes a fast desensitization to Ca(2+). The mechanisms underlying the alterations in Ca(2+) sensitivity are unknown. Here we show that cytoplasmic ATP reversed Ca(2+) sensitivity after desensitization, whereas mutations to putative ATP binding sites resulted in faster and more complete desensitization. Phorbol ester-induced activation of protein kinase C (PKC) increased the Ca(2+) sensitivity of wild-type TRPM4 but not of two mutants mutated at putative PKC phosphorylation sites. Overexpression of a calmodulin mutant unable to bind Ca(2+) dramatically reduced TRPM4 activation. We identified five Ca(2+)-calmodulin binding sites in TRPM4 and showed that deletion of any of the three C-terminal sites strongly impaired current activation by reducing Ca(2+) sensitivity and shifting the voltage dependence of activation to very positive potentials. Thus, the Ca(2+) sensitivity of TRPM4 is regulated by ATP, PKC-dependent phosphorylation, and calmodulin binding at the C terminus.

Pharmacological Properties and Functional Role of a TRP-Related Ion Channel in Lobster Olfactory Receptor Neurons

Odors activate lobster olfactory receptor neurons (ORNs) through phosphoinositide signaling that appears to target a Na(+)-gated nonselective cation channel. The Na(+)-gated channel is a potential member of the growing family of transient receptor potential (TRP) channels. Here, we test the effect of potential antagonists on the channel in cell-free patches from cultured lobster ORNs. We show that the channel is antagonized by H+ and the TRP channel blockers 2-aminoethoxydiphenyl borate, SKF96365, ruthenium red, Al3+, Gd3+, and La3+. We then use this enhanced antagonist profile together with the agonists Na+ and Ca2+ to implicate the channel in signal amplification in the cells.

Voltage Dependence of the Ca2+-activated Cation Channel TRPM4

TRPM4 is a Ca2+-activated but Ca2+-impermeable cation channel. An increase of [Ca2+]i induces activation and subsequent reduction of currents through TRPM4 channels. This inactivation is strikingly decreased in cell-free patches. In whole cell and cell-free configuration, currents through TRPM4 deactivate rapidly at negative potentials. At positive potentials, currents are much larger and activate slowly. This voltage-dependent behavior induces a striking outward rectification of the steady state currents. The instantaneous current-voltage relationship, derived from the amplitude of tail currents following a prepulse to positive potentials, is linear. Currents show a Boltzmann type of activation; the fraction of open channels increases at positive potentials and is low at negative potentials. Voltage dependence is not due to block by divalent cations or to voltage-dependent binding of intracellular Ca2+ to an activator site, indicating that TRPM4 is a transient receptor potential channel with an intrinsic voltage-sensing mechanism. Voltage dependence of TRPM4 may be functionally important, especially in excitable tissues generating plateau-like or bursting action potentials.

Activation of TRPV1 by the Satiety Factor Oleoylethanolamide

The fatty acid oleoylethanolamide (OEA) is a satiety factor that excites peripheral vagal sensory nerves, but the mechanism by which this occurs and the molecular targets of OEA are unclear. In this study the ability of OEA to modulate the capsaicin receptor (TRPV1) was explored. OEA alone did not activate TRPV1 expressed in Xenopus oocytes under control conditions, but produced a differential modulation of agonist-evoked responses. OEA enhanced proton-gated TRPV1 currents, inhibited anandamide-evoked currents and had no effect on capsaicin-evoked responses. Following stimulation of protein kinase C (PKC), OEA alone directly activated TRPV1 channel with an EC50 of approximately 2 microm at room temperature. This effect was due to direct phosphorylation of TRPV1 because no responses to OEA were observed with mutant channels lacking critical PKC phosphorylation sites, S502A/S800A. In sensory neurons, OEA-induced Ca2+ rises that were selective for capsaicin-sensitive cells, inhibited by the TRPV1 blocker, capsazepine, and occurred in a PKC-dependent manner. Further, after PKC stimulation, OEA activated TRPV1 channels in cell-free patches suggesting a direct mode of action. Thus, TRPV1 represents a potential target for OEA and may contribute to the excitatory action of OEA on sensory nerves.

Leucine Zipper Domain Targets cAMP-dependent Protein Kinase to Mammalian BK Channels

Large conductance, calcium- and voltage-activated potassium (BK) channels control excitability in many tissues and are regulated by several protein kinases and phosphatases that remain associated with the channels in cell-free patches of membrane. Here, we report the identification of a highly conserved, non-canonical, leucine zipper (LZ1) in the C terminus of mammalian BK channels that is required for cAMP-dependent protein kinase (PKA) to associate with the channel and regulate its activity. A synthetic polypeptide encompassing the central d position leucine residues in LZ1 blocks the regulation of recombinant mouse BK channels by endogenous PKA in HEK293 cells. In contrast, neither an alanine-substituted LZ1 peptide nor a peptide corresponding to another, more C-terminal putative leucine zipper, LZ2, had any effect on regulation of the channels by endogenous PKA. Mutagenesis of the central two LZ1 d position leucines to alanine in the BK channel also eliminated regulation by endogenous PKA in HEK293 cells without altering the channel sensitivity to activation by voltage or by exogenous purified PKA. Inclusion of the STREX splice insert in the BK channel protein, which switches channel regulation by PKA from stimulation to inhibition, did not alter the requirement for an intact LZ1. Although PKA does not bind directly to the channel protein in vitro, mutation of LZ1 abolished co-immunoprecipitation of PKA and the respective BK channel splice variant from HEK293 cells. Furthermore, a 127-amino acid fusion protein encompassing the functional LZ1 domain co-immunoprecipitates a PKA-signaling complex from rat brain. Thus LZ1 is required for the association and regulation of mammalian BK channels by PKA, and other putative leucine zippers in the BK channel protein may provide anchoring for other regulatory enzyme complexes.

PGE2 Action in Human Coronary Artery Smooth Muscle: Role of Potassium Channels and Signaling Cross-Talk

Cyclic AMP-stimulating agents are powerful vasodilators, but our knowledge of the signal transduction mechanisms of these agents, particularly in human arteries, is limited. We now report direct molecular effects of prostaglandin E₂ (PGE₂) on cultured human coronary artery smooth muscle cells (HCASMC). Patch-clamp studies revealed that 10 µM PGE₂ opens a high-conductance (∼200 pS), calcium-stimulated potassium (BK_Ca) channel in intact HCASMC. In contrast, PGE₂ had no direct effect on channels in cell-free patches, indicating involvement of a soluble second messenger. Enzyme immunoassay demonstrated that PGE₂ enhances production of cAMP in HCASMC, but does not increase [cGMP]. Furthermore, forskolin, CPT-cAMP, or CPT-cGMP mimicked the stimulatory effect of PGE₂ on BK_Ca channel activity. Interestingly, the response to PGE₂ was unaffected by inhibiting the cAMP-dependent protein kinase, but was antagonized by inhibitors of the cGMP-dependent protein kinase (PKG). Furthermore, cAMP-stimulated PKG activity mimicked the effect of PGE₂. These studies suggest a novel PGE₂ action in human arteries: opening of BK_Ca channels via cAMP cross-activation of PKG in HCASMC. It is proposed that this signaling mechanism may mediate the vasodilatory response to cAMP-dependent agents in the human coronary and other vascular beds.

Changes in cAMP Affinity Modulate HCN Channel Activity

Wang et al. describe a mechanism whereby activity-dependent changes in adenosine 3′,5′-monophosphate (cAMP) binding affinity, rather than changes in cAMP concentration, can modulate activity of the hyperpolarization-activated, cyclic nucleotide-gated, cation nonselective (HCN) channel and can help regulate rhythmic firing. HCN channels are involved in pace-making and are modulated by cAMP, which facilitates channel opening. This has been modeled as an allosteric interaction in which channel opening is coupled with a transition to a conformation that enhances cAMP binding. Wang et al. used inside-out patch-clamp and voltage-clamp analyses of oocytes expressing HCN2 channel mRNA to test this model. Concentrations of cAMP ≥10 μM accelerated current activation and shifted the voltage dependence to more positive potentials, whereas 10 nM cAMP produced a slow phase of activation, consistent with preferential binding of cAMP to the open channel, shifting the equilibrium between closed and open unbound channels and eliciting a delayed phase of channel opening. Activation kinetics of channels in inside-out patches without cAMP fit a single exponential time course; a second exponential was apparent when cAMP was present. Activation kinetics of channels in intact oocytes resembled those of cell-free patches in low concentrations of cAMP; mutant channels that were unable to bind cAMP, however, showed only a single exponential component. Normal--but not mutant--channels showed "memory" and responded to a train of hyperpolarizations with a prolonged tail of channel opening that lasted for tens of seconds. Modeling studies indicated that cAMP modulation of neuronal HCN channels could play an important role in regulating rhythmic activity. J. Wang, S. Chan, M. F. Nolan, S. A. Siegelbaum, Activity-dependent regulation of HCN pacemaker channels by cyclic AMP: Signaling through dynamic allosteric coupling. Neuron 36 , 451-461 (2002). [Online Journal]

A mechanogated nonselective cation channel in proximal tubule that is ATP sensitive.

Ion channels that are gated in response to membrane deformation or "stretch" are empirically designated stretch-activated channels. Here we describe a stretch-activated nonselective cation channel in the basolateral membrane (BLM) of the proximal tubule (PT) that is nucleotide sensitive. Single channels were studied in cell-intact and cell-free patches from the BLM of PT cells that maintain their epithelial polarity. The limiting inward Cs+ conductance is ~28 pS, and channel activity persists after excision into a Ca2+- and ATP-free bath. The stretch-dose response is sigmoidal, with half-maximal activation of about -19 mmHg at -40 mV, and the channel is activated by depolarization. The inward conductance sequence is: NH ~ Cs+ ~ Rb+ > K+ ~ Na+ ~ Li+ > Ca2+ ~ Ba2+ > N-methyl-D-glucamine ~ tetraethylammonium. The venom of the common Chilean tarantula, Grammostola spatulata, completely blocks channel activity in cell-attached patches. Hypotonic swelling reversibly activates the channel. Intracellular ATP concentration ([ATP]i) reversibly blocks the channel (inhibitory constant approximately 0.48 mM), suggesting that channel function is coupled to the metabolic state of the cell. We conclude that this channel may function as a Ca2+ entry pathway and/or be involved in regulation of cell volume. We speculate this channel may be important when [ATP]i is depleted, as occurs during periods of increased transepithelial transport or with ischemic injury.

(Xeno)estrogen Sensitivity of Smooth Muscle BK Channels Conferred by the Regulatory β1 Subunit: A STUDY OF β1 KNOCKOUT MICE

Estrogen and xenoestrogens (i.e. agents that are not steroids but possess estrogenic activity) increase the open probability (P(o)) of large conductance Ca(2+)-activated K(+) (BK) channels in smooth muscle. The mechanism of action may involve the regulatory beta1 subunit. We used beta1 subunit knockout (beta1-/-) mice to test the hypothesis that the regulatory beta1 subunit is essential for the activation of BK channels by tamoxifen, 4-OH tamoxifen (a major biologically active metabolite), and 17beta-estradiol in native myocytes. Patch clamp recordings demonstrate BK channels from beta1-/- mice were similar to wild type with the exception of markedly reduced Ca(2+)/voltage sensitivity and faster activation kinetics. In wild type myocytes, (xeno)estrogens increased NP(o) (P(o) x the number of channels, N), shifted the voltage of half-activation (V(12)) to more negative potentials, and decreased unitary conductance. These effects were non-genomic and direct, because they were rapid, reversible, and observed in cell-free patches. None of the (xeno)estrogens increased the NP(o) of BK channels from beta1-/- mice, but all three agents decreased single channel conductance. Thus, (xeno)estrogens increase BK NP(o) through a mechanism involving the beta1 subunit. The decrease in conductance did not require the beta1 subunit and probably reflects an interaction with the pore-forming alpha subunit. We demonstrate regulation of smooth muscle BK channels by physiological (steroid hormones) and pharmacological (chemotherapeutic) agents and reveal the critical role of the beta1 subunit in these responses in native myocytes.

Regulation of hyperpolarization-activated HCN channel gating and cAMP modulation due to interactions of COOH terminus and core transmembrane regions.

Members of the hyperpolarization-activated cation (HCN) channel family generate HCN currents (I(h)) that are directly regulated by cAMP and contribute to pacemaking activity in heart and brain. The four different HCN isoforms show distinct biophysical properties. In cell-free patches from Xenopus oocytes, the steady-state activation curve of HCN2 channels is 20 mV more hyperpolarized compared with HCN1. Whereas the binding of cAMP to a COOH-terminal cyclic nucleotide binding domain (CNBD) markedly shifts the activation curve of HCN2 by 17 mV to more positive potentials, the response of HCN1 is much less pronounced (4 mV shift). A previous deletion mutant study suggested that the CNBD inhibits hyperpolarization-gating in the absence of cAMP; the binding of cAMP shifts gating to more positive voltages by relieving this inhibition. The differences in basal gating and cAMP responsiveness between HCN1 and HCN2 were proposed to result from a greater inhibitory effect of the CNBD in HCN2 compared with HCN1. Here, we use a series of chimeras between HCN1 and HCN2, in which we exchange the NH(2) terminus, the transmembrane domain, or distinct domains of the COOH terminus, to investigate further the molecular bases for the modulatory action of cAMP and for the differences in the functional properties of the two channels. Differences in cAMP regulation between HCN1 and HCN2 are localized to sequence differences within the COOH terminus of the two channels. Surprisingly, exchange of the CNBDs between HCN1 and HCN2 has little effect on basal gating and has only a modest one on cAMP modulation. Rather, differences in cAMP modulation depend on the interaction between the CNBD and the C-linker, a conserved 80-amino acid region that connects the last (S6) transmembrane segment to the CNBD. Differences in basal gating depend on both the core transmembrane domain and the COOH terminus. These data, taken in the context of the previous data on deletion mutants, suggest that the inhibitory effect of the CNBD on basal gating depends on its interactions with both the C-linker and core transmembrane domain of the channel. The extent to which cAMP binding is able to relieve this inhibition is dependent on the interaction between the C-linker and the CNBD.

Regulation of a Human Chloride Channel: A PARADIGM FOR INTEGRATING INPUT FROM CALCIUM, TYPE II CALMODULIN-DEPENDENT PROTEIN KINASE, AND INOSITOL 3,4,5,6-TETRAKISPHOSPHATE

We have studied the regulation of Ca(2+)-dependent chloride (Cl(Ca)) channels in a human pancreatoma epithelial cell line (CFPAC-1), which does not express functional cAMP-dependent cystic fibrosis transmembrane conductance regulator chloride channels. In cell-free patches from these cells, physiological Ca(2+) concentrations activated a single class of 1-picosiemens Cl(-)-selective channels. The same channels were also stimulated by a purified type II calmodulin-dependent protein kinase (CaMKII), and in cell-attached patches by purinergic agonists. In whole-cell recordings, both Ca(2+)- and CaMKII-dependent mechanisms contributed to chloride channel stimulation by Ca(2+), but the CaMKII-dependent pathway was selectively inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P(4)). This inhibitory effect of Ins(3,4,5,6)P(4) on Cl(Ca) channel stimulation by CaMKII was reduced by raising [Ca(2+)] and prevented by inhibition of protein phosphatase activity with 100 nm okadaic acid. These data provide a new context for understanding the physiological relevance of Ins(3,4,5,6)P(4) in the longer term regulation of Ca(2+)-dependent Cl(-) fluxes in epithelial cells.

Properties of hyperpolarization-activated pacemaker current defined by coassembly of HCN1 and HCN2 subunits and basal modulation by cyclic nucleotide.

Members of the HCN channel family generate hyperpolarization-activated cation currents (Ih) that are directly regulated by cAMP and contribute to pacemaker activity in heart and brain. The four HCN isoforms show distinct but overlapping patterns of expression in different tissues. Here, we report that HCN1 and HCN2, isoforms coexpressed in neocortex and hippocampus that differ markedly in their biophysical properties, coassemble to generate heteromultimeric channels with novel properties. When expressed in Xenopus oocytes, HCN1 channels activate 5-10-fold more rapidly than HCN2 channels. HCN1 channels also activate at voltages that are 10-20 mV more positive than those required to activate HCN2. In cell-free patches, the steady-state activation curve of HCN1 channels shows a minimal shift in response to cAMP (+4 mV), whereas that of HCN2 channels shows a pronounced shift (+17 mV). Coexpression of HCN1 and HCN2 yields Ih currents that activate with kinetics and a voltage dependence that tend to be intermediate between those of HCN1 and HCN2 homomers, although the coexpressed channels do show a relatively large shift by cAMP (+14 mV). Neither the kinetics, steady-state voltage dependence, nor cAMP dose-response curve for the coexpressed Ih can be reproduced by the linear sum of independent populations of HCN1 and HCN2 homomers. These results are most simply explained by the formation of heteromeric channels with novel properties. The properties of these heteromeric channels closely resemble the properties of I(h) in hippocampal CA1 pyramidal neurons, cells that coexpress HCN1 and HCN2. Finally, differences in Ih channel properties recorded in cell-free patches versus intact oocytes are shown to be due, in part, to modulation of Ih by basal levels of cAMP in intact cells.

Calpastatin Domain L Is Involved in the Regulation of L-Type Ca2+ Channels in Guinea Pig Cardiac Myocytes

We have found previously that L-type Ca2+ channel run-down in cell-free patches is partially (10–28%) reversed by calpastatin (CS) and have suggested that CS, an endogenous inhibitor of calpain, has a Ca2+-channel-regulating function. CS is composed of repetitive domains 1–4 (calpain-inhibitory domain) and domain L (a domain whose function is unknown). We therefore investigated which domain of CS was involved in the regulation of Ca2+ channel activity in guinea pig cardiac myocytes using the patch-clamp technique. After the patches were excised into inside-out mode in basic internal solution, the Ca2+ channel activity ran down to 0.45% of the control level recorded in the cell-attached mode. Application of human recombinant full-length CS (25 μM) and domain L (25 μM) restored the Ca2+ channel activity to 13 and 19% of the control level, respectively, while the channel activity was not restored by CS domain 1 (25 μM) (0.66%). Mouse CS domain XLL (25 μM), a complex of domain XL and domain L, restored the calcium channel activity to 11% of the control level. These results suggested that the Ca2+-channel-regulating function of CS is located in domain L. This study is the first description of the function of CS domain L.

Tetraethylammonium potentiates the activity of muscarinic potassium channels in guinea-pig atrial myocytes.

The modulation of native muscarinic potassium channels (KACh) by tetraethylammonium (TEA) was studied at 35 degrees C in cell-free patches from acutely dissociated guinea-pig atrial myocytes. The channels were identified unambiguously by their conductance, inward rectification, rapid gating kinetics and pharmacological responses to muscarinic agonists and GTPgammaS. Addition of 5 mM TEA to the cytoplasmic side of the patches almost doubled the open probability of KACh channels that had been activated maximally by GTPgammaS. In contrast even 30 mM TEA did not significantly potentiate the response to carbachol in whole-cell recordings. Unlike GTPgammaS, TEA alone did not activate KACh channels de novo, but in patches that showed spontaneous KACh activity, 5 mM TEA increased channel open probability fourfold in the absence of added sodium, ATP or guanine nucleotides. Furthermore, the effect of TEA was not blocked by 10 uM atropine or by 1 mM GDPbetaS, and subsequent addition of 0.1 mM GTPgammaS did not stimulate channel activity further in the presence of TEA. Phosphatidylinositol 4,5-bisphosphate (PIP2) also stimulates KACh channels under these conditions, but the kinetics of gating differ from channels stimulated by either TEA or GTP, which are very similar to one another. The effects of TEA were not mimicked by tetramethyl- or tetrapentylammonium or by sodium or spermine, and TEA did not potentiate the activity of other inwardly rectifying potassium (KATP) channels in patches from cardiac myocytes. We consider the possibility that TEA is mimicking the effect of an unidentified cellular factor, not sodium or PIP2, which normally occupies the TEA site on KACh channel proteins but which diffuses away when the patch is excised.