Ectopic expression of cone-specific G-protein-coupled receptor kinase GRK7 in zebrafish rods leads to lower photosensitivity and altered responses.

To investigate the roles of G-protein receptor kinases (GRKs) in the light responses of vertebrate photoreceptors, we generated transgenic zebrafish lines, the rods of which express either cone GRK (GRK7) or rod GRK (GRK1) in addition to the endogenous GRK1, and we then measured the electrophysiological characteristics of single-cell responses and the behavioural responses of intact animals. Our study establishes the zebrafish expression system as a convenient platform for the investigation of specific components of the phototransduction cascade. The addition of GRK1 led to minor changes in rod responses. However, exogenous GRK7 in GRK7-tg animals led to lowered rod sensitivity, as occurs in cones, but surprisingly to slower response kinetics. Examination of responses to long series of very dim flashes suggested the possibility that the GRK7-tg rods generated two classes of single-photon response, perhaps corresponding to the interaction of activated rhodopsin with GRK1 (giving a standard response) or with GRK7(giving a very small response). Behavioural measurement of optokinetic responses (OKR) in intact GRK7-tg zebrafish larvae showed that the overall rod visual pathway was less sensitive, in accord with the lowered sensitivity of the rods. These results help provide an understanding for the molecular basis of the electrophysiological differences between cones and rods.

Suppression of a slow post-spike afterhyperpolarization by calcineurin inhibitors.

A subset of myenteric neurons in the intestine (AH neurons) generate prolonged (>5 s) post-spike afterhyperpolarizations (slow AHPs) that are insensitive to apamin and tetraethylammonium. Generation of slow AHPs depends critically on Ca(2+) entry and intracellular release of Ca(2+) from stores, which then leads to the activation of a K(+) conductance that underlies the slow AHP (g(sAHP)). Slow AHPs are inhibited by stimulation of the cAMP/protein kinase A (PKA) pathway, suggesting that phosphorylation of the K(+)-channels that mediate the g(sAHP) (K(sAHP)-channels) is responsible for suppression of slow AHPs and possibly for the repolarization phase of slow AHPs. In the present study, we investigated the possibility that the rising phase of the slow AHP is mediated by dephosphorylation of K(sAHP)-channels by calcineurin (CaN), a Ca(2+)-calmodulin-dependent protein phosphatase, leading to an increase in g(sAHP) and activation of the associated current I(sAHP). Slow AHPs and I(sAHP) were recorded using conventional recording techniques, and we tested the actions of two inhibitors of CaN, FK506 and cyclosporin A, and also the effect of the CaN autoinhibitory peptide applied intracellularly, on these events. We report here that all three treatments inhibited the slow AHP and I(sAHP) (>70%) without significantly affecting the ability of neurons to fire action potentials. In addition, the slow AHP and I(sAHP) were suppressed by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. Our results indicate that activation of the g(sAHP) that underlies the post-depolarization slow AHPs in AH neurons is mediated by the actions CaN and non-Ca(2+)-dependent phosphatases.

Action potential afterdepolarization mediated by a Ca2+-activated cation conductance in myenteric AH neurons.

We investigated the nature of afterdepolarizing potentials in AH neurons from the guinea-pig duodenum using whole-cell patch-clamp recordings in intact myenteric ganglia. Afterdepolarizing potentials were minimally activated following action-potential firing under normal conditions, but after application of charybdotoxin (40 nM) or tetraethyl ammonium (TEA; 10-20 mM) to the bathing solution, prominent afterdepolarizing potentials followed action potentials. The whole-cell current underlying afterdepolarizing potentials (I(ADP)) in the presence of TEA (10-20 mM) reversed at -38 mV and was not voltage-dependent. Reduction of NaCl in the bathing (Krebs) solution to 58 mM shifted the reversal potential of the I(ADP) to -58 mV, suggesting that the current underlying the afterdepolarizing potential was carried by a mixture of cations. The relative contributions of Na(+) and K(+) to this current were estimated to be about 1:5. Substitution of external Na(+) with N-methyl D-glucamine blocked the current while replacement of internal Cl(-) with gluconate did not block the I(ADP). The I(ADP) was also inhibited when CsCl-filled patch pipettes were used. The I(ADP) was blocked or substantially decreased in amplitude in the presence of N-type Ca(2+) channel antagonists, omega-conotoxin GVIA and omega-conotoxin MVIIC, respectively, and was eliminated by external Cd(2+), indicating that it was dependent on Ca(2+) entry. The I(ADP) was also inhibited by ryanodine (10-20 microM), indicating that Ca(2+)-induced Ca(2+) release was involved in its activation. Niflumic acid consistently inhibited the I(ADP) with an IC(50) of 63 microM. Using antibodies against the pore-forming subunits of L-, N- and P/Q-type voltage-gated Ca(2+) channels, we have demonstrated that myenteric AH neurons express N- and P/Q, but not L-type voltage-gated Ca(2+) channels. We conclude that the ADP in myenteric AH neurons, in the presence of an L-type Ca(2+)-channel blocker, is generated by the opening of Ca(2+)-activated non-selective cation channels following action potential-mediated Ca(2+) entry mainly through N-type Ca(2+) channels. Ca(2+) release from ryanodine-sensitive stores triggered by Ca(2+) entry contributes significantly to the activation of this current.

Afterhyperpolarization current in myenteric neurons of the guinea pig duodenum.

Whole cell patch and cell-attached recordings were obtained from neurons in intact ganglia of the myenteric plexus of the guinea pig duodenum. Two classes of neuron were identified electrophysiologically: phasically firing AH neurons that had a pronounced slow afterhyperpolarization (AHP) and tonically firing S neurons that lacked a slow AHP. We investigated the properties of the slow AHP and the underlying current (I(AHP)) to address the roles of Ca(2+) entry and Ca(2+) release in the AHP and the characteristics of the K(+) channels that are activated. AH neurons had a resting potential of -54 mV and the AHP, which followed a volley of three suprathreshold depolarizing current pulses delivered at 50 Hz through the pipette, averaged 11 mV at its peak, which occurred 0.5-1 s following the stimulus. The duration of these AHPs averaged 7 s. Under voltage-clamp conditions, I(AHP)'s were recorded at holding potentials of -50 to -65 mV, following brief depolarization of AH neurons (20-100 ms) to positive potentials (+35 to +50 mV). The null potential of the I(AHP) at its peak was -89 mV. The AHP and I(AHP) were largely blocked by omega-conotoxin GVIA (0.6-1 microM). Both events were markedly decreased by caffeine (2-5 mM) and by ryanodine (10-20 microM) added to the bathing solution. Pharmacological suppression of the I(AHP) with TEA (20 mM) or charybdotoxin (50-100 nM) unmasked an early transient inward current at -55 mV following step depolarization that reversed at -34 mV and was inhibited by niflumic acid (50-100 microM). Mean-variance analysis performed on the decay of the I(AHP) revealed that the AHP K(+) channels have a mean chord conductance of ~10 pS, and there are ~4,000 per AH neuron. Spectral analysis showed that the AHP channels have a mean open dwell time of 2.8 ms. Cell-attached patch recordings from AH neurons confirmed that the channels that open following action currents have a small unitary conductance (10-17 pS) and open with a high probability (

Recording ionic events from cultured, DiI-labelled myenteric neurons in the guinea-pig proximal colon.

To date investigations of enteric neurons by patch clamping/calcium imaging have been limited by studying unidentified heterogeneous populations of neurons. In DiI-labelled colonic myenteric neurons, the feasibility of recording ionic events was determined by applying DiI either to the mucosa or the circular muscle, dispersing neurons after 48 h organotypic culture, and patch-clamping/calcium imaging labeled neurons after 3-7 days in culture. Myenteric neurons with diffuse DiI fluorescence were typically smooth and agranular. Neurons labeled after DiI was applied to circular muscle, fired in either a phasic or a tonic manner, and exhibited fast afterhyperpolarizations (100-300 ms duration) at the end of a depolarizing pulse. They expressed a fast inward current and at least three different outward currents. Action potentials elicited in DiI-labeled sensory neurons were followed by a prolonged afterhyperpolarization (AH, 4-6 s). The offset of a suprathreshold depolarizing step elicited a prolonged outward tail current that approximated the timecourse of the prolonged AH. In addition, in response to membrane depolarization in DiI-labeled neurons loaded with fura-2, robust Ca(2+) transients were recorded using the perforated patch technique. These results demonstrate that DiI labeling of cultured myenteric neurons is feasible, and patch clamp/Ca(2+) fluorescence recordings can be made from specific populations of cultured DiI-labeled colonic myenteric neurons.

Diverse ionic currents and electrical activity of cultured myenteric neurons from the guinea pig proximal colon.

The aim of this study was to perform a patch-clamp analysis of myenteric neurons from the guinea pig proximal colon. Neurons were enzymatically dispersed, cultured for 2-7 days, and recorded from using whole cell patch clamp. The majority of cells fired phasically, whereas about one-quarter of the neurons fired in a tonic manner. Neurons were divided into three types based on the currents activated. The majority of tonically firing neurons lacked an A-type current, but generated a large fast transient outward current that was associated with the rapid repolarizing phase of an action potential. The fast transient outward current was dependent on calcium entry and was blocked by tetraethylammonium. Cells that expressed both an A-type current and a fast transient outward current were mostly phasic. Depolarization of these cells to suprathreshold potentials from less than -60 mV failed to trigger action potentials, or action potentials were only triggered after a delay of >50 ms. However, depolarizations from more positive potentials triggered action potentials with minimal latency. Neurons that expressed neither the A-type current or the fast transient outward current were all phasic. Sixteen percent of neurons were similar to AH/type II neurons in that they generated a prolonged afterhyperpolarization following an action potential. The current underlying the prolonged afterhyperpolarization showed weak inward rectification and had a reversal potential near the potassium equilibrium potential. Thus cultured isolated myenteric neurons of the guinea pig proximal colon retain many of the diverse properties of intact neurons. This preparation is suitable for further biophysical and molecular characterization of channels expressed in colonic myenteric neurons.

Potassium channels in gastrointestinal smooth muscle.

1. Electromechanical coupling in smooth muscle serves to coordinate the contractile activity of the syncytium. Electrical activity of smooth muscle of the gut is generated by ionic conductances that regulate and in turn are regulated by the membrane potential of smooth muscle cells. This activity determines the extent of Ca2+ entry into smooth muscle cells, and thus, the timing and intensity of contractions. 2. Potassium channels play an important role in regulating the excitability of the syncytium. The different types of K+ channel are characterized by different sensitivities to membrane potential, to intracellular Ca2+ levels and to modulation by agonists. 3. This review highlights the different types of K+ channels found in gut smooth muscle and describes their possible roles in regulating the electrical activity of the muscle.

Functional innervation of the biliary sphincter of the guinea-pig revealed by anti-autonomic drugs.

1. The roles of excitatory and inhibitory intrinsic motor nerves on contractions reflexly evoked by wall distension were investigated in the isolated sphincter of Oddi of the guinea-pig (SO-GP). 2. Distension of the terminal bile duct for 30-60 s time periods increased the frequency of contractions from about 2 to 12 min(-1) (n = 16). 3. Hexamethonium (HEX; 300 microM) largely prevented the distension-evoked increase in contraction frequency (4.5 min(-1), n = 8) as did atropine (ATR; 1 microM) (0.8 min(-1), n = 6), while tetrodotoxin (TTX; 1 microM) blocked the contractions triggered during distension. 4. L-nitroarginine (L-NA; 100 microM) significantly increased the frequency of contractions during and in the absence distension while apamin (APAM; 0.5 microM) significantly increased their frequency and doubled their mean amplitude during distension. 5. These results suggest that distension activates excitatory cholinergic motor nerves to increase the frequency of contractions in the SO-GP. These actions are modulated by the concomitant activation of intrinsic nitrergic and non-nitrergic inhibitory motor nerves.

Voltage-dependent large conductance channels in visceral smooth muscle.

The ionic conductances that underlie the resting membrane potential of visceral smooth muscle are not fully understood. Using the patch-clamp technique in the whole-cell configuration, single large conductance channels (LCCs) with unitary conductances of up to 400 pS were recorded in isolated smooth muscle cells of the opossum esophagus. These channels were active at physiological potentials (-100 to -40 mV) and opened with increasing frequency as the membrane potential was hyperpolarized. This voltage dependence gave rise to an inwardly rectifying macroscopic current which was half-maximally activated at -65 mV. The current through LCCs was carried by cations because reduction of external [NaCl] shifted the reversal potential of the LCC current towards the predicted Nernst potential for a nonselective cation current. These results suggest that LCCs may contribute to resting membrane potential in the circular muscle of the opossum esophagus.

Inhibition of voltage-activated K+ currents in smooth muscle cells of the guinea pig proximal colon by noradrenergic agonists.

1. The effects of noradrenaline and isoprenaline on the Ca2+i-insensitive, voltage-activated K+ current in smooth muscle cells from the circular muscle layer of the guinea pig proximal colon were investigated by using standard whole-cell patch-clamp techniques at room temperature (22-24 degrees C). 2. The Ca2+-activated K+ current was eliminated by bathing cells in tetraethylammonium (TEA;2-5 mM) and a Ca2+-entry blocker (Cd2+, 0.1 mM) or nifedipine, 2-10 microM) and by internally perfusing cells with 3 mM EGTA. 3. Two Ca2+i-insensitive, voltage-activated K+ currents were recorded at potentials positive to -50 mV: (a) a transient K+ current (IKto) that was blocked by 4-aminopyridine (5 mM) and (b) a delayed rectifier-type K+ current (IKdel) that was blocked by TEA (>10 mM). 4. Both noradrenaline (10-50 microM) and isoprenaline (5-50 microM) reduced the amplitudes of IKto and IKdel irreversibly after a slow onset (2-5 min). This reduction was mimicked by forskolin (50-100 microM) and by 8 bromo-c-AMP (500 microM). 5. The voltage of half-maximal availability (V0.5) of IKto (-74.6+/-2.3 mV) was unaffected by isoprenaline (10 microM) (-76.7+/-3.6 mV, n=4), but the background "leak" current (Ileak) was increased from -48+/-9 to -70+/-20 pA. 6. Our data suggest that stimulation of beta-adrenoceptors in the circular muscle layer of the guinea pig proximal colon inhibits voltage-activated Ca2+i-insensitive K+ currents.

Nitric oxide suppresses a Ca(2+)-stimulated Cl- current in smooth muscle cells of opossum esophagus.

Nitric oxide (NO) hyperpolarizes visceral smooth muscles. Using the patch-clamp technique, we investigated the possibility that NO-mediated hyperpolarization in the circular muscle of opossum esophagus results from the suppression of a Ca(2+)-stimulated Cl- current. Smooth muscle cells were dissociated from the circular layer and bathed in high-K+ Ca(2+)-EGTA-buffered solution. Macroscopic ramp currents were recorded from cell-attached patches. Contaminating K(+)-channel currents were blocked with tetrapentylammonium chloride (200 microM) added to all solutions. Raising bath Ca2+ concentration above 150 nM in the presence of A-23187 (10 microM) activated a leak current (IL-Ca) with an EC50 of 1.2 microM at -100 mV. The reversal potential (Erev) of IL-Ca (-8.5 +/- 1.8 mV, n = 8) was significantly different (P < 0.05) from Erev of the background current (+4.2 +/- 1.2 mV, n = 8). Equimolar substitution of 135 mM Cl- in the pipette solution with gluconate significantly shifted Erev of IL-Ca to +16.6 +/- 3.4 mV (n = 4) (P < 0.05 compared with background), whereas replacement of total Na+ with Tris+ suppressed IL-Ca but did not affect Erev (-15 +/- 3 mV, n = 3; P > 0.05). IL-Ca was inhibited by DIDS (500 microM). Diethylenetriamine-NO adduct (200 microM), a NO donor, and 8-bromo-cGMP (200 microM) suppressed IL-Ca by 59 +/- 15% (n = 5) and 62 +/- 21% (n = 4) at -100 mV, respectively. We conclude that in opossum esophageal smooth muscle NO-mediated hyperpolarization may be produced by suppression of a Ca(2+)-stimulated Cl(-)-permeable conductance via formation of cGMP.

Molecular identification of a component of delayed rectifier current in gastrointestinal smooth muscles.

Kv2.2, homologous to the shab family of Drosophila voltage-gated K+ channels, was isolated from human and canine colonic circular smooth muscle-derived mRNA. Northern hybridization analysis performed on RNA prepared from tissues and RT-PCR performed on RNA isolated from dispersed and selected smooth muscle cells demonstrate that Kv2.2 is expressed in smooth muscle cells found in all regions of the canine gastrointestinal (GI) tract and in several vascular tissues. Injection of Kv2.2 mRNA into Xenopus oocytes resulted in the expression of a slowly activating K+ current (time to half maximum current, 97 +/- 8.6 ms) mediated by 15 pS (symmetrical K+) single channels. The current was inhibited by tetraethylammonium (IC50 = 2.6 mM), 4-aminopyridine (IC50 = 1.5 mM at +20 mV), and quinine (IC50 = 13.7 microM) and was insensitive to charybdotoxin. Low concentrations of quinine (1 microM) were used to preferentially block the slow component of the delayed rectifier current in native colonic myocytes. These data suggest that Kv2.2 may contribute to this current in native GI smooth muscle cells.

An intermediate conductance K+ channel in the cell membrane of mouse intestinal smooth muscle.

Single channel currents were recorded from cell-attached and inside-out patches in smooth muscle cells of the mouse ileum in order to identify TEA-sensitive Ca2+-dependent K+ channels. Cells were bathed in high-K+ (150 mM) solution with [Ca2+] buffered to 80-150 nM with EGTA and patch pipettes were filled with low-K+ (2.5 mM) physiological solution. Two distinct TEA-sensitive unitary outward current levels were identified at a holding potential (Vh) of 0 mV, corresponding to intermediate conductance (IK, approximately 40 pS) and large conductance (BK, >200 pS) K+ channels. The open probability (Po) of IK channels increased with depolarization, the voltage for half-maximal activation averaging +12 mV in 80 nM Cabath2+. Raising the [Ca2+] in the high-K+ solution from 80 nM to 150 nM increased the Po of IK channels at Vh=0 mV from 0.078 to 0.21. Likewise, the open probability of BK channels at 0 mV was increased from 0.003 to 0.026. Unlike BK channels, IK channels inactivated with maintained depolarization with a voltage for half-maximal inactivation of -66 mV. IK channels were blocked by 2-5 mM external TEA and were sensitive to both charybdotoxin (100 nM) and apamin (500 nM). Our results suggest that IK channels contribute significantly to the Ca2+-dependent K+ conductance in visceral smooth muscle.

Activation of small conductance Ca(2+)-dependent K+ channels by purinergic agonists in smooth muscle cells of the mouse ileum.

1. Whole-cell and single-channel K+ currents were recorded at room temperature (22-24 degrees C), from smooth muscle cells enzymatically dispersed from the mouse ileum, using variations of the patch-clamp technique. 2. Net outward K+ currents recorded through amphotericin-B-perforated patches in response to step depolarizations positive to -50 mV from a holding potential of -80 mV were decreased by up to 70% by external apamin (0.5 microM). Apamin-sensitive whole-cell currents were also recorded from cells perfused internally with 150 nM Ca2+ but not from cells perfused internally with 85 nM Ca2+. 3. Three types of non-inactivating Ca(2+)-sensitive K+ channels were identified in cell-attached and excised patches under an asymmetrical K+ gradient: (i) large conductance (BKCa; approximately 200 pS) channels blocked by 2 mM external TEA; (ii) intermediate conductance (IKCa; approximately 39 pS) channels blocked by 2 mM external TEA and inhibited by external apamin (0.5 microM); and (iii) small conductance (SKCa; approximately 10 pS) channels that were not blocked by 5 mM external TEA but were sensitive to extracellular apamin (0.5 microM). 4. The TEA-resistant SKCa channels were activated by an increase in [Ca2+]i with an EC50 of 1.5 microM and a Hill coefficient of 1.3. 5. P2 purinoceptor agonists 2-methylthioATP (2-MeSATP), 2-chloroATP and ATP (10-50 microM) increased an apamin-sensitive whole-cell outward K+ current. Extrapatch application of 2-MeSATP (20-100 microM) stimulated the apamin-sensitive IKCa and SKCa channels and activated an apamin-sensitive steady outward current at 0 mV. 6. Smooth muscle cells from the mouse ileum possess two apamin-sensitive K+ channels (IKCa and SKCa); of these, the IKCa channels are TEA sensitive while the SKCa channels are TEA resistant. These channels, along with an apamin-sensitive but TEA-resistant steady outward current, may mediate membrane hyperpolarization elicited by purinergic agonists.

Cloning and expression of the large-conductance Ca(2+)-activated K+ channel from colonic smooth muscle.

We have cloned cDNAs encoding the alpha- and beta-subunits of a large-conductance Ca(2+)-activated K+ channel (BK channel) from canine colonic smooth muscle (cslo-alpha and cslo-beta). Nucleotide sequence homology of cslo-alpha with mslo and dslo suggests that it is the canine homologue of these genes. The carboxy-terminal end of the protein is the most diverse between species, and we have also found alternative exons in cslo-alpha in this region. We have identified a unique splice site in the carboxy-terminal region of cslo-alpha, which we term site 5. Northern analysis demonstrates expression of both alpha- and beta-subunits in all canine vascular and visceral smooth muscles tested. Expression of alpha-1 alone and alpha + beta-subunit cRNA in Xenopus oocytes results in a Ca(2+)- and voltage-dependent conductance. The activity of alpha/beta-channels, measured as either changes in the voltage of half-maximal activation (V0.5) in open probability (NP0) or in the normalized conductance (G/Cmax), was more sensitive to [Ca2+]free than channels composed of the alpha-subunit alone. Neither alpha- nor alpha/beta-channels expressed in membrane patches of Xenopus oocytes were found to be regulated by protein kinase G.

Suppression of two cloned smooth muscle-derived delayed rectifier potassium channels by cholinergic agonists and phorbol esters.

Functional coupling between muscarinic (m3) receptors and two voltage-gated K+ (Kv) channels (Kv1.2 and Kv1.5) cloned originally from canine colonic smooth muscle was studied using the Xenopus oocytes expression system and a mammalian cell line (COS cells). Oocytes were coinjected with cRNAs encoding the human m3 receptor and the Kv channel clones. COS cells were stably transfected with the hm3 cDNA and the cDNA encoding Kv1.5 channels. In oocytes coexpressing hm3 receptors and Kv channels, acetylcholine (ACh, 100 microM) decreased the whole-oocyte Kv channel current (IKv) by 72% over 20 min. ACh was equally effective at suppressing IKv1.2 as IKv1.5. In oocytes expressing only Kv channels phorbol esters (phorboldibutyrate) and phorbol dideconoate (10-30 nM) mimicked the action of ACh on IKv in oocytes coexpressing hm3 receptors. At the single-channel level, both ACh and phorbol dibutyrate applied to the extra-patch membrane reduced the open probability of Kv channels in the cell-attached patches without affecting single-channel conductance. In cotransfected COS cells, over a similar time course as in oocytes ACh suppressed whole-cell IKv1.5, but only by 30% and the effect was not reversible. These data indicate that stimulation of m3 receptors in cells that express Kv1.2 and Kv1.5 channels causes a poorly reversible decrease in the open probability of these channels.

Effects of nerve stimulation on the spontaneous action potentials recorded in the proximal renal pelvis of the guinea-pig.

The effects of nerve stimulation on the electrical and mechanical activity of the smooth muscle of the proximal renal pelvis of the guinea-pig were investigated using standard tension and microelectrode recording techniques. Spontaneous action potentials were deemed to have been recorded from three cell types: (1) "pacemaker" cells (9 of > 120) had membrane potentials (MPs) of -42.1 +/- 2.9 mV and fired action potentials of a simple waveform; (2) "driven" cells (> 100) had more stable MPs of -56.1 +/- 1.2 mV (n = 36) and more complex "ureter-like" action potentials; (3) the remaining cells had MPs of -45.5 +/- 1.7 mV (n = 15) and action potentials with a waveform "intermediate" to groups (1) and (2). Nifedipine (0.1-1 microM) and Cd2+ (0.1-1 mM) blocked all spontaneous action potential discharge and depolarized the membrane to near -40 mV. Intramural nerve stimulation (10-50 Hz for 1-10 s) increased both the amplitude and frequency of the spontaneous contractile activity, this increase peaked in about 30 s and decayed slowly over several minutes. Nerve stimulation depolarized pacemaker and driven cells 9.1 +/- 3.5 (n = 3) and 1.6 +/- 0.7 (n = 6) mV, respectively; the frequency of their action potential discharge increased from 7.6 +/- 2.7 and 9.9 +/- 1.1/min to 17.3 +/- 0.5 and 11.1 +/- 1.4/min, respectively. The duration of the action potentials in driven cells also increased significantly for several minutes. All these effects were blocked by tetrodotoxin (TTX) (1.6 microM). It was concluded that the positive chronotropic and inotropic effects of nerve stimulation on renal pelvis contractility can be correlated with the changes in the frequency and duration of the action potentials recorded in driven cells.

Identification of single transiently opening ("A-type") K channels in guinea-pig colonic myocytes.

Two K+ channel populations were identified in depolarized cell-attached membrane patches of myocytes freshly dispersed from the circular smooth muscle of guinea-pig proximal colon. First, a large-conductance (150 pS) Ca(2+)-activated K+ channel which was non-inactivating and sensitive to blockade by tetraethylammonium (TEA, 0.5-5 mM); and second, a smaller conductance K+ channel which opened and closed within 100 ms, was insensitive to TEA (0.5-5 mM), but was blocked by 5 mM 4-aminopyridine (4-AP) or maintained depolarization, and which had a unitary conductance of 12-13 pS. The averaged time course of these smaller conductance K+ channels closely resembled the time course of the 4-AP-sensitive, Ca(2+)-insensitive transient outward K+ current recorded in the whole-cell recording mode.

Cloning and characterization of a Kv1.5 delayed rectifier K+ channel from vascular and visceral smooth muscles.

We have cloned and characterized the expression of a Kv1.5 K+ channel (cKv1.5) from canine colonic smooth muscle. The amino acid sequence displayed a high level of identity to other K+ channels of the Kv1.5 class in the core region between transmembrane segments S1-S6; however, identity decreased to between 74 and 82% in the NH2 and COOH terminal segments, suggesting that cKv1.5 is a distinct isoform of the Kv1.5 class. Functional expression of cKv1.5 in oocytes demonstrated a channel highly selective for K+, which activates in a voltage-dependent manner on depolarization to membrane potentials positive to -40 mV. At room temperature the channel showed fast activation (time to half of peak current, 5.5 ms) and slow inactivation that was incomplete after 20-s depolarizations. Single channel analysis of the channel expressed in oocytes displayed a linear current-voltage curve and had a slope conductance of 9.8 +/- 1.1 pS. Northern blot analysis demonstrated differential expression of cKv1.5 in smooth muscles of the gastrointestinal tract and abundant expression in several vascular smooth muscles. We propose that cKv1.5 represents a component of the delayed rectifier current in both vascular and visceral smooth muscles.

Cholinergic nerve-mediated excitation in the guinea pig choledochoduodenal junction activated by distension.

The electrical basis of propulsive contractions in the guinea pig choledochoduodenal junction (CDJ), which are triggered by distension, was investigated using intracellular microelectrode recording techniques. The isolated CDJ was placed in a continuously perfused tissue chamber at 37 degrees C. Membrane potential was recorded from smooth muscle cells in either the ampulla or in the upper CDJ (upper junction) regions, which were immobilized by pinning. Distension of the upper junction (20-30 s) by increasing intraductal hydrostatic pressure (mean elevation: 2.0 +/- 0.3 kPa, n = 13) triggered "transient depolarizations" (TDs: < 5 mV in amplitude and 2-5 s in duration) and action potentials in the circular muscle layer of the ampulla. The frequency of TDs in the ampulla was increased from 2.2 +/- 0.2 to 15.9 +/- 2.2 min-1 (n = 13) during distension. Simultaneous impalements of cells in the longitudinal and circular muscle layers in the ampulla revealed that subthreshold TDs in the circular layer were associated with an increased rate of action potential discharge in the longitudinal layer. Atropine (Atr; 1.4 x 10(-6) M) and tetrodotoxin (TTX; 3.1 x 10(-6) M blocked the distension-evoked increase in TD frequency, without affecting the frequency of ongoing TDs. The sulfated octapeptide of cholecystokinin (1-5 x 10(-8) M) increased the amplitude of TDs recorded in the circular muscle layer of the ampulla and increased action potential discharge rate. In separate recordings, radial stretch of the ampulla region increased the rate of discharge of action potentials in the smooth muscle of the upper junction.(ABSTRACT TRUNCATED AT 250 WORDS)