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Initial characterization of a potassium delayed rectifier in canine and human isolated jejunal circular smooth muscle
1390
ABSTRACTS OF PAPERS
October 1992
REGULATION OF PHASIC ACTIVITY IN GASTRIC ANTRUM BY A Ca2+- ACTIVATED K+ CONDUCTANCE. J.B. Picou and N.G. Publicover. Dept. of Physiology, Univ. of Nevada School of Medicine, Reno, NV 89557. It has been proposed that phosphatidylinositol (PI) hydrolysis provides negative-feedback control of Ca2+ influx, limiting the amplitude of phasic contractile activity in gastric tissues (Ozaki et al. AJP: in pressz). One possible mechanism for this effect is stimulation of Ca +-activated K+ channels by Ins(l,4,5)P3-induced release of Ca2+ from internal stores. An increase in the open probability of Ca2+-activated K+ channels generated by a rise in [Ca2’li could limit the amplitude and duration of slow waves. In the present study, experiments were performed to examine the effects of Ca2+ release from internal stores on Ca 2+-activated K+ channel activity in gastric antrum. Smooth muscle cells were enzymatically dispersed from the circular muscle layer of canine antrum. Single-channel outward currents were recorded at 35-37’C using the cell-attached, patch clamp technique. Membrane potential was nulled by bathing cells in 140 mM KCl. [Ca”], was buffered to 1 uM. Channel openings were observed when patches were held at positive potentials and openings were more frequent as membrane potential was made more positive. Single channel conductance was approximately 250 pS. Caffeine (2.5 mM) applied to the bathing solution produced an increase in the frequency of single-channel openings. Peak channel activity occurred approximately 6 seconds following application of caffeine. For the next minute following peak activity and in the continued presence of caffeine, there was a slow decay in channel activity, suggesting depletion of Ca2+ stores. Wash-out of caffeine restored control activity. These data suggest that release of Ca2’ from I$releasable stores may increase the open probability of Ca activated K+ channels, lead to a decrease in slow wave amplitude and duration, reduce Ca2’ influx during the slow wave, and limit phasic contractions. (Supported by NIH grant DK 32176.)
INITIAL CHARACTERIZATION OF A POTASSIUM DELAYED RECTIFIER IN CANINEANDHUMAN ISOLATED JEJUNAL CIRCULAR SMOOTH MUSCLE G. Farruaia, J.L. Rae. H.G. Sarr. J.H. Szurszewski. Mayo Medical School, Rochester, MN. A whole cell potassium delayed rectifier current was characterized in canine and human jejunal circular smooth muscle cells using an amphotericin-based perforated patch clamp technique. Final access resistances were 5-15MD and partition times varied from 5-20 min. Dissociated cells (n-58) were whole cell voltage clamped and held at -60mV between pulse protocols. The mean (tSD) resting potential and cell capacitance of canine jejunal cells were -40+15mV and 64.2t16.5pF respectively. A delayed rectifier was found in all cells. The activation time was voltage dependant with a 10-90X rise time of 100*15ms at 20mV. The channels which gave rise to this current increased their open probability at about -55mV and reached a unit open probability at about t10mV. Selectivity was measured in various external potassium ion concentrations. There was a 59mV shift for a ten-fold increase in the potassium concentration, compared to 58mV for a perfectly potassium selective current. The current was very sensitive to quinidine blockade with full blockade occurring at .2mM. TEA block was less effective with about 7D% of the current blocked at 50mM. Blockade was accompanied by marked shifts in the reversal potential suggesting that this current was a major contributor to the resting cell voltage. The current was carried by rubidium to about the same degree as potassium and was not barium sensitive. It was not blocked by diltiazem nor by a calcium free bath solution. Unlike most delayed rectifiers it was only weakly holding voltage sensitive (~10% decrease in the current at OmV). In cells from normal human jejunum (n-6), resting voltage and capacitance were -43+12mV and 47.&16pF respectively. As in canine jejunum a delayed rectifier was found in all cells studied. It had a lo-90% rise time of 102f50ms and was blocked by quinidine. These studies are the first to report the occurrence of a highly selective delayed rectifier in circular muscle of the canine and human jejunum. The data suggest that this current may be a major contributor to the cell resting voltage. Supported by DK17238, DK39337, EY03282 and EY06005.
G-PROTEIN MODULATION OF Ca”-ACTIVATED K+ CHANNELS FROM COLON CIRCULAR SMOOTH MUSCLE C.L. A.L. Percival, KM. Sanders, A. Carl, and J.L. Kenyon, Department of Physiology, University of Nevada School of Medicine, Reno, NV 89557 Regulation of the large conductance Ca*‘-activated K+ channel (BK channel) may contribute to the control of GI motility. For example, Cole and Sanders (Am. J.Physiol. 257C5%, 1989) found that muscarinic stimulation, which enhances smooth muscle contraction, suppressed Ca*‘-activated K’ currents in colonic myocytes. Further, this inhibition was enhanced by intracellular GTP analogues. We studied BK channels from canine colonic circular smooth muscle reconstituted into artificial bilayers. In symmetrical 140 mM K+, BK channels have an average conductance of 347~s and are active between +80 and -80 mV with 1 to 10 PM free Ca*’ (10 mM HE?DTA). A lo-fold change in Ca*’ shifts the voltage at which open probability (P,,) is 0.5 by 134 mV, consistent with data collected in patch clamp experiments (Carl and Sanders, Am.J.Physiol. 257:C470, 1989). We tested the possibility that G-proteins interact directly with BK channels by examining the effect of nonhydrolyzable GTPyS on the gating of reconstituted channels. Control activity was recorded for 5 min., then GTPyS was added for 5 - 10 min. 100 PM GTP$ reduced N-P0 (N=the number of channels) in 6 of 8 experiments. The figure is a cumulative plot of N% from one of the 6 experiments. The reduction in N.P, ranged from 11.8% to 86.9% (average 44.7% n=6). Our results indicate that at least some BK channels from canine color& smooth muscle are inhibited by activation of a GTF’-binding urotein that is incomeor 1 iated with the chGe1 0 100 zoo 300 100 500 SECONDS into the artificial bilayer. Supported by DK 41315.
DIFFERENTIAL EFFECT OF SUBSTANCE P ON Ca-ACTIVATED K ( CHANNELS IN COLONIC MUSCLE MAY BE MEDIATED BY Ina 1,4,SP3 KQ! A PROTBIN KINASE C (PKC). w, S. Supplisson, EA. Mayer. Depts. d Physiology and Medlclne, UCLA and Center for Ulcer Research and Education (CURE), VA Wadsworth Medkzal Canter, Los Angeles, Ca. 90073. The Inhibition d channels by agonists resutting In d-ion has ral mechanism d contractlcn coupling In circular been suggested as a cdonic muscle. To characterize the effect d substance P (SP) on in longkudlnal colonlc muscle, and to determlns the channels k, the cellincreases In Ins l,4.5P3 and PKC, we attached recording ccnfiguratlon In myocytes fmm the longkudlnal muscle layer d the distal rabbk colon. ActMUon thresh&l and opening probabllty (P,J d channels were determined from ramp and step changes in pipette potential (V Whenbath and plpette contalned a physlologicalsalt solutbn (PSS) a Ca*+ pathways in the patch were blocked by La3+ (lOAh4 In pipette sdutlon). depolarlzatbn resulted in activationd b channels at a threshold V of -98~8 mV (n=‘l;meawS.E.M). Under these recording conditions, Ins 1 ,%,5P3-Independent Ca*+ release was stimulated by a,caffeine (10 mM); b. lowering free cytceollc [&+I (addition d lo* ionomycyin at bath [C$ + 1d 10%4) ; cadditbn d L& (104M) to the bath). All three Interventions (a-c) resulted In a leftward shift d thevokage-acthtkm curve.A similar leflward shift was observed when SP (10~10-108M) was added to the bath at low [Ca*+] ccnditcns b,c). or In cells pmtreated with the PKC inhlbkor staurcsporine (lO-i7(M). In contrast, In normal [tit], additiond SP (10-3.IO-7M) to the bath resUned first In a dosedependent rigL shllt d the vdtage-actlvatlon cutve Mowed by wmplete channel Inhlbltbn. This InhibItcry effect was mlmkked by addition d the PKC adlvatcr l-deyl-29cety((lo-5 M) to the bath. Channel InhIbitionwas also present when omitted from the pipette solution,and could not be reversed by the addltbn d caffeine.Similarresultswere obtained ln chemicallydepdarf& cells @osmakr replacement of NaCl by KU In the PSS). Together with recently publishsd data on SP effects on sub@asmalemrnsl [&+I. we ccnclude: 1. Resting [Ca2t]i exerts an lnhlbitory effect on lntrawllular Ca*+ release. 2. Under ccnditbns preventingactivation d PKC, SP ktduces w t release from subpksmalammai
ABSTRACTS OF PAPERS
October 1992
REGULATION OF PHASIC ACTIVITY IN GASTRIC ANTRUM BY A Ca2+- ACTIVATED K+ CONDUCTANCE. J.B. Picou and N.G. Publicover. Dept. of Physiology, Univ. of Nevada School of Medicine, Reno, NV 89557. It has been proposed that phosphatidylinositol (PI) hydrolysis provides negative-feedback control of Ca2+ influx, limiting the amplitude of phasic contractile activity in gastric tissues (Ozaki et al. AJP: in pressz). One possible mechanism for this effect is stimulation of Ca +-activated K+ channels by Ins(l,4,5)P3-induced release of Ca2+ from internal stores. An increase in the open probability of Ca2+-activated K+ channels generated by a rise in [Ca2’li could limit the amplitude and duration of slow waves. In the present study, experiments were performed to examine the effects of Ca2+ release from internal stores on Ca 2+-activated K+ channel activity in gastric antrum. Smooth muscle cells were enzymatically dispersed from the circular muscle layer of canine antrum. Single-channel outward currents were recorded at 35-37’C using the cell-attached, patch clamp technique. Membrane potential was nulled by bathing cells in 140 mM KCl. [Ca”], was buffered to 1 uM. Channel openings were observed when patches were held at positive potentials and openings were more frequent as membrane potential was made more positive. Single channel conductance was approximately 250 pS. Caffeine (2.5 mM) applied to the bathing solution produced an increase in the frequency of single-channel openings. Peak channel activity occurred approximately 6 seconds following application of caffeine. For the next minute following peak activity and in the continued presence of caffeine, there was a slow decay in channel activity, suggesting depletion of Ca2+ stores. Wash-out of caffeine restored control activity. These data suggest that release of Ca2’ from I$releasable stores may increase the open probability of Ca activated K+ channels, lead to a decrease in slow wave amplitude and duration, reduce Ca2’ influx during the slow wave, and limit phasic contractions. (Supported by NIH grant DK 32176.)
INITIAL CHARACTERIZATION OF A POTASSIUM DELAYED RECTIFIER IN CANINEANDHUMAN ISOLATED JEJUNAL CIRCULAR SMOOTH MUSCLE G. Farruaia, J.L. Rae. H.G. Sarr. J.H. Szurszewski. Mayo Medical School, Rochester, MN. A whole cell potassium delayed rectifier current was characterized in canine and human jejunal circular smooth muscle cells using an amphotericin-based perforated patch clamp technique. Final access resistances were 5-15MD and partition times varied from 5-20 min. Dissociated cells (n-58) were whole cell voltage clamped and held at -60mV between pulse protocols. The mean (tSD) resting potential and cell capacitance of canine jejunal cells were -40+15mV and 64.2t16.5pF respectively. A delayed rectifier was found in all cells. The activation time was voltage dependant with a 10-90X rise time of 100*15ms at 20mV. The channels which gave rise to this current increased their open probability at about -55mV and reached a unit open probability at about t10mV. Selectivity was measured in various external potassium ion concentrations. There was a 59mV shift for a ten-fold increase in the potassium concentration, compared to 58mV for a perfectly potassium selective current. The current was very sensitive to quinidine blockade with full blockade occurring at .2mM. TEA block was less effective with about 7D% of the current blocked at 50mM. Blockade was accompanied by marked shifts in the reversal potential suggesting that this current was a major contributor to the resting cell voltage. The current was carried by rubidium to about the same degree as potassium and was not barium sensitive. It was not blocked by diltiazem nor by a calcium free bath solution. Unlike most delayed rectifiers it was only weakly holding voltage sensitive (~10% decrease in the current at OmV). In cells from normal human jejunum (n-6), resting voltage and capacitance were -43+12mV and 47.&16pF respectively. As in canine jejunum a delayed rectifier was found in all cells studied. It had a lo-90% rise time of 102f50ms and was blocked by quinidine. These studies are the first to report the occurrence of a highly selective delayed rectifier in circular muscle of the canine and human jejunum. The data suggest that this current may be a major contributor to the cell resting voltage. Supported by DK17238, DK39337, EY03282 and EY06005.
G-PROTEIN MODULATION OF Ca”-ACTIVATED K+ CHANNELS FROM COLON CIRCULAR SMOOTH MUSCLE C.L. A.L. Percival, KM. Sanders, A. Carl, and J.L. Kenyon, Department of Physiology, University of Nevada School of Medicine, Reno, NV 89557 Regulation of the large conductance Ca*‘-activated K+ channel (BK channel) may contribute to the control of GI motility. For example, Cole and Sanders (Am. J.Physiol. 257C5%, 1989) found that muscarinic stimulation, which enhances smooth muscle contraction, suppressed Ca*‘-activated K’ currents in colonic myocytes. Further, this inhibition was enhanced by intracellular GTP analogues. We studied BK channels from canine colonic circular smooth muscle reconstituted into artificial bilayers. In symmetrical 140 mM K+, BK channels have an average conductance of 347~s and are active between +80 and -80 mV with 1 to 10 PM free Ca*’ (10 mM HE?DTA). A lo-fold change in Ca*’ shifts the voltage at which open probability (P,,) is 0.5 by 134 mV, consistent with data collected in patch clamp experiments (Carl and Sanders, Am.J.Physiol. 257:C470, 1989). We tested the possibility that G-proteins interact directly with BK channels by examining the effect of nonhydrolyzable GTPyS on the gating of reconstituted channels. Control activity was recorded for 5 min., then GTPyS was added for 5 - 10 min. 100 PM GTP$ reduced N-P0 (N=the number of channels) in 6 of 8 experiments. The figure is a cumulative plot of N% from one of the 6 experiments. The reduction in N.P, ranged from 11.8% to 86.9% (average 44.7% n=6). Our results indicate that at least some BK channels from canine color& smooth muscle are inhibited by activation of a GTF’-binding urotein that is incomeor 1 iated with the chGe1 0 100 zoo 300 100 500 SECONDS into the artificial bilayer. Supported by DK 41315.
DIFFERENTIAL EFFECT OF SUBSTANCE P ON Ca-ACTIVATED K ( CHANNELS IN COLONIC MUSCLE MAY BE MEDIATED BY Ina 1,4,SP3 KQ! A PROTBIN KINASE C (PKC). w, S. Supplisson, EA. Mayer. Depts. d Physiology and Medlclne, UCLA and Center for Ulcer Research and Education (CURE), VA Wadsworth Medkzal Canter, Los Angeles, Ca. 90073. The Inhibition d channels by agonists resutting In d-ion has ral mechanism d contractlcn coupling In circular been suggested as a cdonic muscle. To characterize the effect d substance P (SP) on in longkudlnal colonlc muscle, and to determlns the channels k, the cellincreases In Ins l,4.5P3 and PKC, we attached recording ccnfiguratlon In myocytes fmm the longkudlnal muscle layer d the distal rabbk colon. ActMUon thresh&l and opening probabllty (P,J d channels were determined from ramp and step changes in pipette potential (V Whenbath and plpette contalned a physlologicalsalt solutbn (PSS) a Ca*+ pathways in the patch were blocked by La3+ (lOAh4 In pipette sdutlon). depolarlzatbn resulted in activationd b channels at a threshold V of -98~8 mV (n=‘l;meawS.E.M). Under these recording conditions, Ins 1 ,%,5P3-Independent Ca*+ release was stimulated by a,caffeine (10 mM); b. lowering free cytceollc [&+I (addition d lo* ionomycyin at bath [C$ + 1d 10%4) ; cadditbn d L& (104M) to the bath). All three Interventions (a-c) resulted In a leftward shift d thevokage-acthtkm curve.A similar leflward shift was observed when SP (10~10-108M) was added to the bath at low [Ca*+] ccnditcns b,c). or In cells pmtreated with the PKC inhlbkor staurcsporine (lO-i7(M). In contrast, In normal [tit], additiond SP (10-3.IO-7M) to the bath resUned first In a dosedependent rigL shllt d the vdtage-actlvatlon cutve Mowed by wmplete channel Inhlbltbn. This InhibItcry effect was mlmkked by addition d the PKC adlvatcr l-deyl-29cety((lo-5 M) to the bath. Channel InhIbitionwas also present when omitted from the pipette solution,and could not be reversed by the addltbn d caffeine.Similarresultswere obtained ln chemicallydepdarf& cells @osmakr replacement of NaCl by KU In the PSS). Together with recently publishsd data on SP effects on sub@asmalemrnsl [&+I. we ccnclude: 1. Resting [Ca2t]i exerts an lnhlbitory effect on lntrawllular Ca*+ release. 2. Under ccnditbns preventingactivation d PKC, SP ktduces w t release from subpksmalammai