islets were isolated in the diabetes research & wellness foundation human islet isolation facility by collagenase digestion ( serva , heidelberg , germany ) , using modified versions of published procedures ( 17,18 ) . islets were cultured overnight in cmrl medium containing 5.5 mmol / l glucose and 2 mmol / l l - glutamine . batches of 1020 size - matched islets ( in triplicates ) were preincubated in krebs - ringer buffer ( krb ) containing 2 mg / ml bsa and 1 mmol / l glucose for 1 h at 37c , followed by a 1-h test incubation in krb supplemented with glucose and ion channel blockers as indicated . the glucagon content of the supernatant was determined by radioimmunoassay ( millipore u.k . ltd . , freshly isolated islets were dispersed into single cells by trypsin digestion and plated onto plastic petri dishes . the cells were cultured in rpmi medium containing 10 mmol / l glucose prior to the experiments . patch - clamp experiments were performed by whole - cell measurements at 32c as previously described ( 19 ) . the extracellular solution for membrane potential measurements contained ( in mm ) 140 nacl , 3.6 kcl , 0.5 mgso4 , 1.5 cacl2 , 10 hepes , 0.5 nah2po4 , 5 nahco3 ( ph adjusted to 7.4 with naoh ) , and glucose as indicated . the extracellular solution for k - current measurements contained ( mm ) 138 nacl , 5.6 kcl , 2.6 cacl2 , 1.2 mgcl2 , 5 hepes , and 5 glucose ( ph 7.4 , with naoh ) . for all other experiments , na- or ca - currents were recorded in the presence of 1 mmol / l cocl2 or 0.1 g / ml ttx , respectively . the intracellular solution for recording voltage - gated k - currents was composed of ( mm ) 120 kcl , 1 mgcl2 , 10 egta , 1 cacl2 , 10 hepes , and 3 mgatp ( ph 7.2 , with koh ) . to measure voltage - gated na- or ca - currents , the pipette solution was composed of ( mm ) 125 cs - glutamate , 10 cscl , 10 nacl , 1 mgcl2 , 5 hepes , 0.05 egta , 3 mgatp , and 0.1 camp ( ph 7.15 with csoh ) . the intracellular solution contained ( mm ) 76 k2so4 , 10 kcl , 10 nacl , 1 mgcl2 , 5 hepes ( ph 7.35 with koh ) , and 0.24 mg / ml amphotericin b ( 21 ) . immunocytochemical identification of patch - clamped cells was performed as described previously ( 20,22 ) . intact islets were loaded with the ca - indicator fluo-4 am ( 2.5 mol / l ; invitrogen , carlsbad , ca ) in cmrl medium for 18 h at room temperature . the islets were immobilized in the recording chamber using a wide - bore holding pipette and continuously perifused with krb containing 1 mmol / l glucose . laser scanning confocal microscopy was performed using an lsm 510 meta laser scanning module ( zeiss ) mounted on an axioskop 2fs microscope . 1 ) been normalized to secretion measured at 1 mmol / l glucose in the absence of blockers . islets were isolated in the diabetes research & wellness foundation human islet isolation facility by collagenase digestion ( serva , heidelberg , germany ) , using modified versions of published procedures ( 17,18 ) . islets were cultured overnight in cmrl medium containing 5.5 mmol / l glucose and 2 mmol / l l - glutamine . batches of 1020 size - matched islets ( in triplicates ) were preincubated in krebs - ringer buffer ( krb ) containing 2 mg / ml bsa and 1 mmol / l glucose for 1 h at 37c , followed by a 1-h test incubation in krb supplemented with glucose and ion channel blockers as indicated . the glucagon content of the supernatant was determined by radioimmunoassay ( millipore u.k . ltd . , livingston , u.k . ) . insulin and somatostatin were determined as described previously ( 19,20 ) . freshly isolated islets were dispersed into single cells by trypsin digestion and plated onto plastic petri dishes . the cells were cultured in rpmi medium containing 10 mmol / l glucose prior to the experiments . patch - clamp experiments were performed by whole - cell measurements at 32c as previously described ( 19 ) . the extracellular solution for membrane potential measurements contained ( in mm ) 140 nacl , 3.6 kcl , 0.5 mgso4 , 1.5 cacl2 , 10 hepes , 0.5 nah2po4 , 5 nahco3 ( ph adjusted to 7.4 with naoh ) , and glucose as indicated . the extracellular solution for k - current measurements contained ( mm ) 138 nacl , 5.6 kcl , 2.6 cacl2 , 1.2 mgcl2 , 5 hepes , and 5 glucose ( ph 7.4 , with naoh ) . for all other experiments , 20 mmol / l teacl was added and nacl was reduced correspondingly . na- or ca - currents were recorded in the presence of 1 mmol / l cocl2 or 0.1 g / ml ttx , respectively . the intracellular solution for recording voltage - gated k - currents was composed of ( mm ) 120 kcl , 1 mgcl2 , 10 egta , 1 cacl2 , 10 hepes , and 3 mgatp ( ph 7.2 , with koh ) . to measure voltage - gated na- or ca - currents , the pipette solution was composed of ( mm ) 125 cs - glutamate , 10 cscl , 10 nacl , 1 mgcl2 , 5 hepes , 0.05 egta , 3 mgatp , and 0.1 camp ( ph 7.15 with csoh ) . the intracellular solution contained ( mm ) 76 k2so4 , 10 kcl , 10 nacl , 1 mgcl2 , 5 hepes ( ph 7.35 with koh ) , and 0.24 mg / ml amphotericin b ( 21 ) . immunocytochemical identification of patch - clamped cells was performed as described previously ( 20,22 ) . intact islets were loaded with the ca - indicator fluo-4 am ( 2.5 mol / l ; invitrogen , carlsbad , ca ) in cmrl medium for 18 h at room temperature . the islets were immobilized in the recording chamber using a wide - bore holding pipette and continuously perifused with krb containing 1 mmol / l glucose . laser scanning confocal microscopy was performed using an lsm 510 meta laser scanning module ( zeiss ) mounted on an axioskop 2fs microscope . 1 ) been normalized to secretion measured at 1 mmol / l glucose in the absence of blockers . glucagon secretion was measured from intact human islets exposed to 1 , 6 , and 20 mmol / l glucose . increasing the glucose concentration inhibited glucagon secretion ; maximal inhibition ( 62% ) was observed at 6 mmol mmol / l was associated with reduced inhibition , as previously observed in mouse islets ( 23 ) . the effects of glucose on glucagon secretion were compared with those on insulin and somatostatin secretion . at 6 mmol / l glucose , stimulation of insulin and somatostatin secretion was only 37 and 21% , respectively , of that produced by 20 mmol / l glucose ( fig . c : secretion of glucagon ( a ) , insulin ( b ) , and somatostatin ( c ) measured at 1 , 6 , and 20 mmol / l glucose . data are from 50 donors ( glucagon ) or 33 donors ( insulin and somatostatin ) . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 versus the previous lower glucose concentration . glucagon secretion was significantly lower at 20 mmol / l compared with 1 mmol / l glucose ( p < 0.05 ) . d : glucagon secretion measured at 1 and 6 mmol / l glucose under control conditions and in the presence of 100 nmol / l of cyn-154806 ( cyn ) . e : as in d but in the absence and presence of 100 nmol / l insulin . f : as in d but in the absence and presence of 30 mol / l zncl2 f : * p < 0.05 , * * p < 0.01 , * * * p < 0.001 versus 1 mmol / l glucose ( control ) or as indicated by brackets . the somatostatin - receptor subtype 2 specific antagonist cyn-154806 ( 0.1 mol / l ) did not affect the ability of 6 mmol / l glucose to inhibit glucagon secretion ( fig . it was ascertained that the cyn-154806 antagonized the effects of somatostatin on -cell membrane potential and [ ca]i ( supplementary fig . when applied at 1 mmol / l glucose , insulin ( 100 nmol / l ) inhibited glucagon secretion by 74% , similar to the inhibition produced by 6 mmol / l glucose . addition of insulin to islets exposed to 6 mmol / l glucose stimulated glucagon secretion ( fig . zncl2 ( 30 mol / l ) increased glucagon secretion at 1 and 6 mmol / l glucose by 50% but did not affect the suppression of glucagon release by glucose ( fig . neither insulin nor zncl2 affected the spontaneous [ ca]i - oscillations observed at 1 mmol / l glucose ( supplementary fig . the -cells had a membrane capacitance of 3.3  0.1 pf ( n = 197 ) . outward voltage - activated k - currents became detectable during membrane depolarizations from 70 to 30 mv and above ( fig . the k - current inactivated completely within 20 ms , whereas a sustained component was observed during depolarizations to 20 mv and above ( fig . the peak amplitude recorded during depolarizations to 0 mv averaged 349  61 pa ( n = 8) . the inactivation of the current could be described as the sum of two exponentials with time constants ( at + 30 mv ) of 18  2 and 567  147 ms ( n = 7 ) . the sustained current inactivated by > 90% over 15 s ( fig . 2a , inset ) . analysis of voltage - gated k - currents . a : family of voltage - activated k - currents ( lower ) evoked by depolarizing pulses from 70 mv to membrane potentials between 40 and + 80 mv . inset shows inactivation of current during a 15-s depolarization from 70 to + 20 mv . b : as in a but showing the initial part of the current responses during pulses to 40 , 30 , 20 , and 10 mv on an expanded time base ( sections highlighted in gray in a ) . c : i  v relationship for voltage - gated k - current ( n = 8) . d : example of a cell showing a clear shoulder on the i  v at voltages between + 30 and + 50 mv . data are shown for the peak current ( squares ) , the sustained current measured at the end of the 500-ms pulse ( circles ) , and the difference ( triangles ) . v relationship recorded from eight cells . in three of the eight cells , a prominent shoulder on the i figure 2d shows the peak current , the sustained current measured at the end of the 500 ms depolarization , and the difference between the peak and the sustained currents in one of these cells . similar current responses are seen in human -cells ( 20 ) and reflect activation of large - conductance ca - activated k - channels ( bk - channels ; compare with ref ) . the broad - spectrum k - channel blocker tetraethylammonium ( tea ) ( 10 mmol / l ) inhibited 74  2% ( n = 6 ; p < 0.01 ) of the peak current and 87  4% ( n = 6 ; p < 0.01 ) of the sustained current evoked by depolarizations to + 30 mv ( fig these pharmacological properties are those expected for a - type k - currents ( a - current ) ( 25 ) . the selective kv2.1/2.2 channel blocker stromatoxin ( 26 ) reduced the sustained current by 88  5% ( p < 0.01 , n = 4 ) but decreased the peak current by only 33  15% ( p = 0.05 ; fig . in seven different cells , the time constants of activation ( n ) and inactivation ( h ) averaged 0.26  0.06 and 12  3 ms , respectively ( n and h were estimated assuming nh kinetics ) . n decreased with increasing voltages ( reflecting more rapid activation ) , whereas no clear voltage dependence of h was observed ( not shown ) . the a - current was sensitive to the selective kv4.x - antagonist heteropodatoxin-2 ( 27 ) ( fig . n = 3 ) . pharmacological characterization of voltage - gated k - currents . a : current responses recorded during depolarizations to + 20 mv under control conditions , after addition of 10 mmol / l tea ( gray trace ) and after addition of 5 mmol / l 4-aminopyridine in the continued presence of tea ( n = 4 ) . b : as in a but pulse went to zero and currents were recorded in the absence and presence of stromatoxin ( 100 nmol / l , n = 4 ) . c : as in a but pulse went to 10 mv and currents were recorded in the presence of 10 mmol / l tea before and after application of heteropodatoxin-2 ( 0.5 mol / l ) . d : steady - state inactivation of the a - current analyzed by a two - pulse protocol consisting of a 200-ms conditioning pulse to membrane potentials between 90 and 20 mv followed by a 100-ms test pulse to + 30 mv after an interval of 10 ms . experiments were performed using the perforated - patch technique in the presence of tea . e : steady - state inactivation of the delayed - rectifying k - current was measured by applying 15-s conditioning pulses to membrane potentials between 60 and 20 mv followed by a 500-ms test pulse to + 20 mv ( interval 10 ms ) . f : voltage dependence of inactivation of a - current ( closed circles ) and delayed - rectifier current ( open circles ) . the responses after conditioning pulses to 90 and 60 mv , respectively , were taken as unity , and data are presented as a fraction of the maximal current displayed against the voltage during the conditioning pulse . a boltzmann function has been fit to the data points ( n = 57 ) . g : glucagon secretion measured in the absence ( open bars ) and presence ( filled bars ) of 0.5 mol / l heteropodatoxin-2 at 1 or 6 mmol / l glucose . * p < 0.01 versus 1 mmol / l glucose alone . 100% = 6.5  0.8 pg / islet / h ( n = 12 ; 4 donors ) . / l stromatoxin on glucagon secretion at 1 or 20 mmol / l glucose . * p < 0.05 versus 1 mmol / l glucose . 100% = 7.5  1.5 pg / islet / h ( n = 9 ; 3 donors ) . steady - state inactivation of voltage - gated k - currents was examined using two - pulse protocols in which conditioning pulses to between 90 and 20 mv preceded an activating test pulse ( figs . both the a - type and the sustained k - current underwent voltage - dependent inactivation that could be described by boltzmann functions ( fig . 3f ) . half - maximal inactivation ( vh ) of the a - current ( studied in the presence of tea ) was observed at 49  2 mv ; the slope factor ( nh ) averaged 4.8  0.4 mv the delayed - rectifier current ( ignoring the initial 50 ms to avoid contamination by the a - current ) inactivated at more positive potentials with vh and nh amounting to 37  2 and 5  1 mv , respectively ( n = 5 ; fig . the recovery of a - current from inactivation was measured by application of two 50-ms test pulses to + 30 mv separated by a conditioning pulse to 70 mv of increasing duration . the current recovered rapidly with a time constant of 45  7 ms ( n = 4 ; supplementary fig . 3a and b , available in an online appendix ) . the delayed - rectifier k - current recovered from inactivation ( induced by a 15-s depolarization to 20 mv ) with a  of 2.9  0.7 s ( n = 4 , supplementary fig . the inhibitory effect of the k - channel blockers was comparable to that produced by 6 or 20 mmol / l glucose . mmol / l ) inhibited glucagon secretion as strongly as heteropodatoxin-2 ( not shown ) . voltage - gated inward currents were studied using cs - containing pipette solution and tea - containing bath solution to block k - currents . figure 4a shows membrane currents elicited by 5-ms depolarizations from 70 to 0 mv . under control conditions , the sustained current was inhibited by the broad - spectrum ca - channel blocker co ( 1 a rapidly activating and inactivating current was observed that was inhibited by the na - channel blocker ttx . experiments were performed in the presence of tea ( 10 mmol / l ) in the extracellular solution and after replacing k with cs in the pipette solution . a : currents recorded under control conditions , after addition of 1 mmol / l co and after addition of ttx ( 0.1 g / ml ) in the continued presence of co. b : voltage dependence of na - currents . the responses recorded in the presence of co during depolarizations to 40 , 30 , 20 , and 10 mv are shown . c : i  v relationship for na - currents ( n = 5 ) . a test pulse to + 10 mv was preceded by 50-ms conditioning pulses to membrane potentials between 150 and 0 mv ( 60 to 30 shown ) . the response after a conditioning pulse to 150 mv was taken as unity ( n = 6 ) . f : glucagon secretion measured in the absence ( open bars ) and presence ( filled bars ) of ttx ( 0.1 g / ml ) at 1 or 20 mmol / l glucose as indicated . 100% = 12.2  3.8 pg / islet / h ( n = 15 ; 4 donors ) . the na - current became detectable during depolarizations to 30 mv and above ( fig . v relationship exhibited a u - shaped voltage dependence with a maximal amplitude of 38  3 pa at 0 mv ( n = 5 ; fig . both the activation and deactivation of the na - current became faster with increasing voltages ( not shown ) . 4e ; vh and nh averaged 40  2 and 5  1 mv ( n = 6 ) , respectively . the effect of the na - channel blocker ttx on glucagon secretion from intact human islets is shown in fig . the inclusion of ttx in the extracellular medium reduced glucagon secretion as strongly as 20 mmol / l glucose , and glucose lacked further inhibitory action in the presence of the blocker . voltage - gated ca - currents ( responsible for the sustained current component in fig . figure 5a shows a family of voltage - clamp currents elicited by membrane depolarization from 70 mv to voltages between 60 and 0 mv . the current became detectable at voltages above 50 mv and peaked at 0 mv , where the amplitude averaged 67  7 pa ( n = 19 ) . a : family of voltage - gated ca - currents recorded in the presence of ttx during 100-ms depolarizations to between 60 and 0 mv as indicated . b : current voltage relationship of whole - cell ca - currents ( n = 14 ) . c : ca - current recorded under control conditions and after addition of 10 mol / l isradipine and -conotoxin ( 100 nmol / l ) and -agatoxin ( 200 nmol / l ) in the continued presence of isradipine as indicated . d : ca - currents elicited by voltage ramps ( speed , 3 v / s ) under control conditions and after addition of isradipine and -agatoxin in the continued presence of isradipine ( n = 7 , 7 , 4 under control conditions , in the presence of isradipine , and after addition of -agatoxin , respectively ) . e : isradipine- ( top ) and -agatoxin - sensitive components ( lower ) from d. f : ca - currents elicited by voltage ramps in the presence of isradipine alone ( 10 mol / l ) and after addition of nnc 55 - 0396 ( 3 g : inactivation of the t - type ca - current . a test pulse to 30 mv was preceded by 500-ms conditioning pulses to membrane potentials between 90 and 50 mv ( in the presence of 10 mol / l isradipine ) . a boltzmann fit has been superimposed on the data points ( n = 6 , experiments performed in the presence of isradipine ) . the ca - currents in human -cells are sensitive to the l - type ca - channel blocker isradipine and the p / q - type ca - channel antagonist -agatoxin iva . the isradipine- and -agatoxin - sensitive currents accounted for 21  4% ( p < 0.01 ; n = 9 ) and 70  6% ( p < 0.01 ; n = 5 ) of the integrated ca - current ( qca ) , respectively . in addition , we observed a small current component blocked by the n - type channel blocker -conotoxin gvia ( 11  3% reduction of qca ; p < 0.01 ; n = 13 ) . voltage ramps between 80 and + 50 mv were used to determine the voltage dependence of the different ca - current components ( fig . inward currents became detectable above 50 mv and the current amplitude then showed a triphasic dependence on membrane potential : a shoulder between 40 and 20 mv , a secondary acceleration between 20 and 0 mv , and a subsequent decline at more positive voltages ( reflecting the reduced electrochemical driving force ) . also shown in fig . 5d are the responses recorded after addition of isradipine alone and the combination of isradipine and -agatoxin , respectively . figure 5e shows the net isradipine- ( l - type ; upper panel ) and -agatoxin - sensitive ( p / q - type ; lower panel ) ca - currents . whereas the activation of the p / q - type current was monophasic , that of the l - type current was biphasic . the latter feature might reflect the expression of two l - type ca - channel isoforms in human islets ( 1c and 1d ; ) . that a small current component activating at negative voltages ( below 50 mv ) is resistant to all these ca - channel blockers . figure 5f shows current responses elicited by ramps in the presence of isradipine alone and in the combined presence of the t - type ca - channel antagonist nnc 55 - 0396 . the gray trace in fig . the low - threshold t - type current was seen in 10 of 14 cells and gave rise to a rapidly inactivating current during depolarization to 30 mv ( fig . the t - type current undergoes voltage - dependent inactivation with values for vh and nh of 71  2 and 8  1 mv , respectively ( n = 6 ; fig . 5 g and h ) . the t - type ca - current was not observed during depolarizations to 0 mv ( fig . 5c ) because it is obscured by a transient outward current that reflects efflux of cs via a - type k - channels ( not shown ) . the l - type ca - channel blocker isradipine inhibited glucagon secretion from human islets at 1 mmol / l glucose by 25% ( fig . glucose retained an inhibitory action in the presence of isradipine ( p < 0.05 ) . blocking the p / q - type ca - channels exerted a stronger suppressor effect and was as inhibitory as 20 mmol / l glucose ( fig . the t - type ca - channel blocker 55 - 0396 paradoxically increased secretion at 1 mmol / l glucose by 190  30% ( n = 3 ; p < 0.05 ) but did not affect the inhibitory action of 20 mmol / l glucose ( 65  5% ; n = 3 ; p < 0.05 ; not shown ) . the stimulation of glucagon secretion by the t - type ca - channel blocker can not be attributed to inhibition of somatostatin or insulin secretion ( 19,20 ) and may therefore reflect an unspecific action of the compound . a : glucagon secretion measured in the absence ( open bars ) and presence ( filled bars ) of 10 mol / l isradipine . * p < 0.01 versus 1 mmol / l glucose alone , p < 0.05 versus 1 mmol / l glucose and 10 mol / l isradipine . 100% = 10.5  0.6 pg / islet / h ( n = 9 ; 3 donors ) . b : same as in a but effects of 200 nmol / l -agatoxin were tested . * p < 0.01 versus 1 mmol / l glucose alone , 100% = 21.1  3.7 pg / islet / h ( n = 9 ; 3 donors ) . the background membrane conductance of -cells was measured in the perforated - patch whole - cell configuration by applying 10 mv depolarizing pulses from 70 mv . the membrane conductance at 1 mmol / l glucose averaged 0.12  0.01 ns ( n = 27 ) . it was not reduced by elevation of glucose to 610 mmol / l ( 0.12  0.04 ns ; n = 13 ) or after addition of 100 mol / l tolbutamide ( 0.11  0.04 ns , n = 13 ) . however , diazoxide ( 100 mol / l ) increased the membrane conductance ( from 0.14  0.05 to 0.32  0.12 ns ; p = 0.05 , n = 7 ) . 7a ) . of these , 76% were continuously active , whereas the action potentials tended to be grouped in bursts in the remaining 24% of the cells . action potentials were initiated from an average threshold potential of 42  1 mv and peaked at 2  1 mv ( n = 26 ) . the membrane potential of -cells was unaffected by increasing the glucose concentration ( 39  5 mv at 1 mmol / l glucose and 37  4 mv at 610 mmol / l , n = 15 ) , but tolbutamide ( 100400 mol / l ) depolarized the cells by 10 mv , from 42  3 to 32  2 mv ( p < 0.001 , n = 9 ) . this was associated with a reduction of action potential peak voltage from 5  3 to 12  2 mv ( p < 0.05 ; fig . 7b ) . diazoxide ( 100 mol / l ) hyperpolarized the -cell membrane potential by 19  4 mv ( p < 0.05 , n = 5 ; not shown ) . for unknown reasons , regenerative electrical activity subsided rapidly in most cells and was replaced by small and irregular membrane potential oscillations . this precluded a detailed analysis of the effects of different ion channel blockers on -cell electrical activity . electrical activity and [ ca]i oscillations . a : membrane potential recording from an -cell exposed to 1 mmol / l glucose . c : spontaneous [ ca]i oscillations in an -cell within an intact islet exposed to 1 mmol / l glucose before and during addition of 5 mol / l adrenaline . d : as in c but testing the effects of -agatoxin ( 200 nmol / l ) , isradipine ( 10 mol / l ) , bay k8644 ( 10 mol / l ) , and k ( 70 mmol / l ) at 1 mmol / l glucose . the inset shows the segment of the recording highlighted in gray on an expanded timebase . e : histogram summarizing the average amplitude of the [ ca]i oscillations under the indicated experimental conditions ( 13 cells in four islets obtained from two donors ; * p < 0.01 , * * p < 0.001 vs. 1 mmol g : histogram summarizing results obtained as described in f from 14 cells in four islets from two donors ( * * p < 0.001 vs. 1 mmol / l glucose ) . many cells in intact human islets exposed to 1 mmol / l glucose exhibited spontaneous oscillations in cytosolic ca - concentration ( [ ca]i ) . of these cells , 71% responded with a large elevation of [ ca]i when exposed to 5 mol / l adrenaline ( fig . experiments on isolated human islet cells revealed that 77% of the cells that responded to adrenaline with an elevation of [ ca]i were -cells ( as determined by immunocytochemistry ) . addition of 200 nmol / l -agatoxin had a small inhibitory effect on [ ca]i , whereas subsequent addition of 10 mol / l isradipine was strongly inhibitory , an effect that could not be antagonized by bayk8644 in islets that had been exposed to -agatoxin ( fig . application of isradipine to islets that had not been exposed to -agatoxin also reduced [ ca]i , but under these conditions , the effect was partially antagonized by bayk8644 ( fig . the experiments were concluded by depolarizing the cells with 70 mmol / l k. the amplitude of the response to high [ k]o was much larger when the cells had not been exposed to -agatoxin ( p < 0.001 ) . high - resolution capacitance measurements were applied to examine the exocytotic properties of individual -cells . the time course of exocytosis was monitored by applying progressively longer depolarizing pulses ( 20500 ms ) from 70 to 0 mv ( fig . the amplitude of the responses was typically fairly small and limited to 40  5 ff ( n = 32 ) for a 500-ms depolarization . however , pulses as short as 20 ms elicited good responses in 50% of the cells . figure 8b summarizes the relationship between the duration of the depolarizing command and the exocytotic response . the average responses tended to plateau within 50100 ms with a secondary acceleration becoming apparent for pulse durations 200 ms . a : increase in membrane capacitance evoked by 20500-ms depolarizations from 70 to 0 mv . the circles above the capacitance traces indicate the percentage of responding cells ( black part , n = 23 ) . b : relationship between pulse duration and exocytotic response ( cm ; n = 23 ) . c : change in cell capacitance ( cm ) evoked by 500-ms depolarizations from 70 mv to membrane potentials between 40 and + 40 mv ( n = 8) . d : change in cell capacitance evoked by 500-ms depolarization from 70 to 0 mv under control conditions and after addition of -agatoxin ( 200 nmol / l ) . the maximum responses were observed during depolarizations to 0 mv . as shown in fig . 8d , exocytosis elicited by a 500-ms depolarization from 70 to 0 mv was inhibited by 97  3% ( p < 0.05 ; n = 4 ) by -agatoxin . exocytosis was not affected by stromatoxin , heteropodatoxin-2 , and ttx ( 90% of control ) but was strongly reduced ( > 75% ) by 4-aminopyridine . thus , the strong inhibitory effect of 4-aminopyridine on glucagon secretion may result from direct interference with -cell exocytosis . glucagon secretion was measured from intact human islets exposed to 1 , 6 , and 20 mmol / l glucose . increasing the glucose concentration inhibited glucagon secretion ; maximal inhibition ( 62% ) was observed at 6 mmol mmol / l was associated with reduced inhibition , as previously observed in mouse islets ( 23 ) . the effects of glucose on glucagon secretion were compared with those on insulin and somatostatin secretion . at 6 mmol / l glucose , stimulation of insulin and somatostatin secretion was only 37 and 21% , respectively , of that produced by 20 mmol / l glucose ( fig . c : secretion of glucagon ( a ) , insulin ( b ) , and somatostatin ( c ) measured at 1 , 6 , and 20 mmol / l glucose . data are from 50 donors ( glucagon ) or 33 donors ( insulin and somatostatin ) . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 versus the previous lower glucose concentration . glucagon secretion was significantly lower at 20 mmol / l compared with 1 mmol / l glucose ( p < 0.05 ) . d : glucagon secretion measured at 1 and 6 mmol / l glucose under control conditions and in the presence of 100 nmol / l of cyn-154806 ( cyn ) . e : as in d but in the absence and presence of 100 nmol / l insulin . f : as in d but in the absence and presence of 30 mol / l zncl2 f : * p < 0.05 , * * p < 0.01 , * * * p < 0.001 versus 1 mmol / l glucose ( control ) or as indicated by brackets . the somatostatin - receptor subtype 2 specific antagonist cyn-154806 ( 0.1 mol / l ) did not affect the ability of 6 mmol / l glucose to inhibit glucagon secretion ( fig . it was ascertained that the cyn-154806 antagonized the effects of somatostatin on -cell membrane potential and [ ca]i ( supplementary fig . when applied at 1 mmol / l glucose , insulin ( 100 nmol / l ) inhibited glucagon secretion by 74% , similar to the inhibition produced by 6 mmol / l glucose . addition of insulin to islets exposed to 6 mmol / l glucose stimulated glucagon secretion ( fig . zncl2 ( 30 mol / l ) increased glucagon secretion at 1 and 6 mmol / l glucose by 50% but did not affect the suppression of glucagon release by glucose ( fig . neither insulin nor zncl2 affected the spontaneous [ ca]i - oscillations observed at 1 mmol / l glucose ( supplementary fig . all electrophysiological recordings were conducted in individual human -cells subsequently identified by immunocytochemistry . the -cells had a membrane capacitance of 3.3  0.1 pf ( n = 197 ) . outward voltage - activated k - currents became detectable during membrane depolarizations from 70 to 30 mv and above ( fig . the k - current inactivated completely within 20 ms , whereas a sustained component was observed during depolarizations to 20 mv and above ( fig . the peak amplitude recorded during depolarizations to 0 mv averaged 349  61 pa ( n = 8) . the inactivation of the current could be described as the sum of two exponentials with time constants ( at + 30 mv ) of 18  2 and 567  147 ms ( n = 7 ) . the sustained current inactivated by > 90% over 15 s ( fig . 2a , inset ) . . a : family of voltage - activated k - currents ( lower ) evoked by depolarizing pulses from 70 mv to membrane potentials between 40 and + 80 mv . inset shows inactivation of current during a 15-s depolarization from 70 to + 20 mv . b : as in a but showing the initial part of the current responses during pulses to 40 , 30 , 20 , and 10 mv on an expanded time base ( sections highlighted in gray in a ) . c : i  v relationship for voltage - gated k - current ( n = 8) . d : example of a cell showing a clear shoulder on the i  v at voltages between + 30 and + 50 mv . data are shown for the peak current ( squares ) , the sustained current measured at the end of the 500-ms pulse ( circles ) , and the difference ( triangles ) . v relationship recorded from eight cells . in three of the eight cells , a prominent shoulder on the i figure 2d shows the peak current , the sustained current measured at the end of the 500 ms depolarization , and the difference between the peak and the sustained currents in one of these cells . similar current responses are seen in human -cells ( 20 ) and reflect activation of large - conductance ca - activated k - channels ( bk - channels ; compare with ref ) . the broad - spectrum k - channel blocker tetraethylammonium ( tea ) ( 10 mmol / l ) inhibited 74  2% ( n = 6 ; p < 0.01 ) of the peak current and 87  4% ( n = 6 ; p < 0.01 ) of the sustained current evoked by depolarizations to + 30 mv ( fig . these pharmacological properties are those expected for a - type k - currents ( a - current ) ( 25 ) . the selective kv2.1/2.2 channel blocker stromatoxin ( 26 ) reduced the sustained current by 88  5% ( p < 0.01 , n = 4 ) but decreased the peak current by only 33  15% ( p = 0.05 ; fig . the tea- and stromatoxin - resistant a - current underwent rapid activation and inactivation . in seven different cells , the time constants of activation ( n ) and inactivation ( h ) averaged 0.26  0.06 and 12  3 ms , respectively ( n and h were estimated assuming nh kinetics ) . n decreased with increasing voltages ( reflecting more rapid activation ) , whereas no clear voltage dependence of h was observed ( not shown ) . the a - current was sensitive to the selective kv4.x - antagonist heteropodatoxin-2 ( 27 ) ( fig . 3c ; n = 3 ) . pharmacological characterization of voltage - gated k - currents . a : current responses recorded during depolarizations to + 20 mv under control conditions , after addition of 10 mmol / l tea ( gray trace ) and after addition of 5 mmol / l 4-aminopyridine in the continued presence of tea ( n = 4 ) . b : as in a but pulse went to zero and currents were recorded in the absence and presence of stromatoxin ( 100 nmol / l , n = 4 ) . c : as in a but pulse went to 10 mv and currents were recorded in the presence of 10 mmol / l tea before and after application of heteropodatoxin-2 ( 0.5 mol / l ) . d : steady - state inactivation of the a - current analyzed by a two - pulse protocol consisting of a 200-ms conditioning pulse to membrane potentials between 90 and 20 mv followed by a 100-ms test pulse to + 30 mv after an interval of 10 ms . e : steady - state inactivation of the delayed - rectifying k - current was measured by applying 15-s conditioning pulses to membrane potentials between 60 and 20 mv followed by a 500-ms test pulse to + 20 mv ( interval 10 ms ) . f : voltage dependence of inactivation of a - current ( closed circles ) and delayed - rectifier current ( open circles ) . the responses after conditioning pulses to 90 and 60 mv , respectively , were taken as unity , and data are presented as a fraction of the maximal current displayed against the voltage during the conditioning pulse . a boltzmann function has been fit to the data points ( n = 57 ) . g : glucagon secretion measured in the absence ( open bars ) and presence ( filled bars ) of 0.5 mol / l heteropodatoxin-2 at 1 or 6 mmol / l glucose . * p < 0.01 versus 1 mmol / l glucose alone . 100% = 6.5  0.8 pg / islet / h ( n = 12 ; 4 donors ) . h : effects of 100 nmol / l stromatoxin on glucagon secretion at 1 or 20 mmol / l glucose . * p < 0.05 versus 1 mmol / l glucose . 100% = 7.5  1.5 pg / islet / h ( n = 9 ; 3 donors ) . steady - state inactivation of voltage - gated k - currents was examined using two - pulse protocols in which conditioning pulses to between 90 and 20 mv preceded an activating test pulse ( figs . both the a - type and the sustained k - current underwent voltage - dependent inactivation that could be described by boltzmann functions ( fig . half - maximal inactivation ( vh ) of the a - current ( studied in the presence of tea ) was observed at 49  2 mv ; the slope factor ( nh ) averaged 4.8  0.4 mv the delayed - rectifier current ( ignoring the initial 50 ms to avoid contamination by the a - current ) inactivated at more positive potentials with vh and nh amounting to 37  2 and 5  1 mv , respectively ( n = 5 ; fig . the recovery of a - current from inactivation was measured by application of two 50-ms test pulses to + 30 mv separated by a conditioning pulse to 70 mv of increasing duration . the current recovered rapidly with a time constant of 45  7 ms ( n = 4 ; supplementary fig . 3a and b , available in an online appendix ) . the delayed - rectifier k - current recovered from inactivation ( induced by a 15-s depolarization to 20 mv ) with a  of 2.9  0.7 s ( n = 4 , supplementary fig . the inhibitory effect of the k - channel blockers was comparable to that produced by 6 or 20 mmol / l glucose . mmol / l ) inhibited glucagon secretion as strongly as heteropodatoxin-2 ( not shown ) . voltage - gated inward currents were studied using cs - containing pipette solution and tea - containing bath solution to block k - currents . figure 4a shows membrane currents elicited by 5-ms depolarizations from 70 to 0 mv . under control conditions , the sustained current was inhibited by the broad - spectrum ca - channel blocker co ( 1 mmol / l ) . in the presence of co , a rapidly activating and inactivating current was observed that was inhibited by the na - channel blocker ttx . experiments were performed in the presence of tea ( 10 mmol / l ) in the extracellular solution and after replacing k with cs in the pipette solution . a : currents recorded under control conditions , after addition of 1 mmol / l co and after addition of ttx ( 0.1 g / ml ) in the continued presence of co. b : voltage dependence of na - currents . the responses recorded in the presence of co during depolarizations to 40 , 30 , 20 , and 10 mv are shown . c : i  v relationship for na - currents ( n = 5 ) . a test pulse to + 10 mv was preceded by 50-ms conditioning pulses to membrane potentials between 150 and 0 mv ( 60 to 30 shown ) . the response after a conditioning pulse to 150 mv was taken as unity ( n = 6 ) . f : glucagon secretion measured in the absence ( open bars ) and presence ( filled bars ) of ttx ( 0.1 g / ml ) at 1 or 20 mmol / l glucose as indicated . 100% = 12.2  3.8 pg / islet / h ( n = 15 ; 4 donors ) . the na - current became detectable during depolarizations to 30 mv and above ( fig . v relationship exhibited a u - shaped voltage dependence with a maximal amplitude of 38  3 pa at 0 mv ( n = 5 ; fig . both the activation and deactivation of the na - current became faster with increasing voltages ( not shown ) . 4e ; vh and nh averaged 40  2 and 5  1 mv ( n = 6 ) , respectively . the effect of the na - channel blocker ttx on glucagon secretion from intact human islets is shown in fig . the inclusion of ttx in the extracellular medium reduced glucagon secretion as strongly as 20 mmol / l glucose , and glucose lacked further inhibitory action in the presence of the blocker . voltage - gated ca - currents ( responsible for the sustained current component in fig . figure 5a shows a family of voltage - clamp currents elicited by membrane depolarization from 70 mv to voltages between 60 and 0 mv . the current became detectable at voltages above 50 mv and peaked at 0 mv , where the amplitude averaged 67  7 pa ( n = 19 ) . a : family of voltage - gated ca - currents recorded in the presence of ttx during 100-ms depolarizations to between 60 and 0 mv as indicated . b : current voltage relationship of whole - cell ca - currents ( n = 14 ) . c : ca - current recorded under control conditions and after addition of 10 mol / l isradipine and -conotoxin ( 100 nmol / l ) and -agatoxin ( 200 nmol / l ) in the continued presence of isradipine as indicated . d : ca - currents elicited by voltage ramps ( speed , 3 v / s ) under control conditions and after addition of isradipine and -agatoxin in the continued presence of isradipine ( n = 7 , 7 , 4 under control conditions , in the presence of isradipine , and after addition of -agatoxin , respectively ) . e : isradipine- ( top ) and -agatoxin - sensitive components ( lower ) from d. f : ca - currents elicited by voltage ramps in the presence of isradipine alone ( 10 mol / l ) and after addition of nnc 55 - 0396 ( 3 g : inactivation of the t - type ca - current . a test pulse to 30 mv was preceded by 500-ms conditioning pulses to membrane potentials between 90 and 50 mv ( in the presence of 10 mol / l isradipine ) . a boltzmann fit has been superimposed on the data points ( n = 6 , experiments performed in the presence of isradipine ) . the ca - currents in human -cells are sensitive to the l - type ca - channel blocker isradipine and the p / q - type ca - channel antagonist -agatoxin iva . the isradipine- and -agatoxin - sensitive currents accounted for 21  4% ( p < 0.01 ; n = 9 ) and 70  6% ( p < 0.01 ; n = 5 ) of the integrated ca - current ( qca ) , respectively . in addition , we observed a small current component blocked by the n - type channel blocker -conotoxin gvia ( 11  3% reduction of qca ; p < 0.01 ; n = 13 ) . voltage ramps between 80 and + 50 mv were used to determine the voltage dependence of the different ca - current components ( fig . inward currents became detectable above 50 mv and the current amplitude then showed a triphasic dependence on membrane potential : a shoulder between 40 and 20 mv , a secondary acceleration between 20 and 0 mv , and a subsequent decline at more positive voltages ( reflecting the reduced electrochemical driving force ) . also shown in fig 5d are the responses recorded after addition of isradipine alone and the combination of isradipine and -agatoxin , respectively . figure 5e shows the net isradipine- ( l - type ; upper panel ) and -agatoxin - sensitive ( p / q - type ; lower panel ) ca - currents . whereas the activation of the p / q - type current was monophasic , that of the l - type current was biphasic . the latter feature might reflect the expression of two l - type ca - channel isoforms in human islets ( 1c and 1d ; ) . that a small current component activating at negative voltages ( below 50 mv ) is resistant to all these ca - channel blockers . figure 5f shows current responses elicited by ramps in the presence of isradipine alone and in the combined presence of the t - type ca - channel antagonist nnc 55 - 0396 . the gray trace in fig . the low - threshold t - type current was seen in 10 of 14 cells and gave rise to a rapidly inactivating current during depolarization to 30 mv ( fig . the t - type current undergoes voltage - dependent inactivation with values for vh and nh of 71  2 and 8  1 mv , respectively ( n = 6 ; fig . the t - type ca - current was not observed during depolarizations to 0 mv ( fig . 5c ) because it is obscured by a transient outward current that reflects efflux of cs via a - type k - channels ( not shown ) . the l - type ca - channel blocker isradipine inhibited glucagon secretion from human islets at 1 mmol / l glucose by 25% ( fig . glucose retained an inhibitory action in the presence of isradipine ( p < 0.05 ) . blocking the p / q - type ca - channels exerted a stronger suppressor effect and was as inhibitory as 20 mmol / l glucose ( fig . the t - type ca - channel blocker 55 - 0396 paradoxically increased secretion at 1 mmol / l glucose by 190  30% ( n = 3 ; p < 0.05 ) but did not affect the inhibitory action of 20 mmol / l glucose ( 65  5% ; n = 3 ; p < 0.05 ; not shown ) . the stimulation of glucagon secretion by the t - type ca - channel blocker can not be attributed to inhibition of somatostatin or insulin secretion ( 19,20 ) and may therefore reflect an unspecific action of the compound . a : glucagon secretion measured in the absence ( open bars ) and presence ( filled bars ) of 10 mol / l isradipine . * p < 0.01 versus 1 mmol / l glucose alone , p < 0.05 versus 1 mmol / l glucose and 10 mol / l isradipine . 100% = 10.5  0.6 pg / islet / h ( n = 9 ; 3 donors ) . b : same as in a but effects of 200 nmol / l -agatoxin were tested . * p < 0.01 versus 1 mmol / l glucose alone , 100% = 21.1  3.7 pg / islet / h ( n = 9 ; 3 donors ) . the background membrane conductance of -cells was measured in the perforated - patch whole - cell configuration by applying 10 mv depolarizing pulses from 70 mv . the membrane conductance at 1 mmol / l glucose averaged 0.12  0.01 ns ( n = 27 ) . it was not reduced by elevation of glucose to 610 mmol / l ( 0.12  0.04 ns ; n = 13 ) or after addition of 100 mol / l tolbutamide ( 0.11  0.04 ns , n = 13 ) . however , diazoxide ( 100 mol / l ) increased the membrane conductance ( from 0.14  0.05 to 0.32  0.12 ns ; p = 0.05 , n = 7 ) . 7a ) . of these , 76% were continuously active , whereas the action potentials tended to be grouped in bursts in the remaining 24% of the cells . action potentials were initiated from an average threshold potential of 42  1 mv and peaked at 2  1 mv ( n = 26 ) . the membrane potential of -cells was unaffected by increasing the glucose concentration ( 39  5 mv at 1 mmol / l glucose and 37  4 mv at 610 mmol / l , n = 15 ) , but tolbutamide ( 100400 mol / l ) depolarized the cells by 10 mv , from 42  3 to 32  2 mv ( p < 0.001 , n = 9 ) . this was associated with a reduction of action potential peak voltage from 5  3 to 12  2 mv ( p < 0.05 ; fig . 7b ) . diazoxide ( 100 mol / l ) hyperpolarized the -cell membrane potential by 19  4 mv ( p < 0.05 , n = 5 ; not shown ) . for unknown reasons , regenerative electrical activity subsided rapidly in most cells and was replaced by small and irregular membrane potential oscillations . this precluded a detailed analysis of the effects of different ion channel blockers on -cell electrical activity . electrical activity and [ ca]i oscillations . a : membrane potential recording from an -cell exposed to 1 mmol / l glucose . c : spontaneous [ ca]i oscillations in an -cell within an intact islet exposed to 1 mmol d : as in c but testing the effects of -agatoxin ( 200 nmol / l ) , isradipine ( 10 mol / l ) , bay k8644 ( 10 mol / l ) , and k ( 70 mmol / l ) at 1 mmol / l glucose . the inset shows the segment of the recording highlighted in gray on an expanded timebase . e : histogram summarizing the average amplitude of the [ ca]i oscillations under the indicated experimental conditions ( 13 cells in four islets obtained from two donors ; * p < 0.01 , * * p < 0.001 vs. 1 mmol g : histogram summarizing results obtained as described in f from 14 cells in four islets from two donors ( * * p < 0.001 vs. 1 mmol / l glucose ) . many cells in intact human islets exposed to 1 mmol / l glucose exhibited spontaneous oscillations in cytosolic ca - concentration ( [ ca]i ) . of these cells , 71% responded with a large elevation of [ ca]i when exposed to 5 mol / l adrenaline ( fig . experiments on isolated human islet cells revealed that 77% of the cells that responded to adrenaline with an elevation of [ ca]i were -cells ( as determined by immunocytochemistry ) . addition of 200 nmol / l -agatoxin had a small inhibitory effect on [ ca]i , whereas subsequent addition of 10 mol / l isradipine was strongly inhibitory , an effect that could not be antagonized by bayk8644 in islets that had been exposed to -agatoxin ( fig . application of isradipine to islets that had not been exposed to -agatoxin also reduced [ ca]i , but under these conditions , the effect was partially antagonized by bayk8644 ( fig . the experiments were concluded by depolarizing the cells with 70 mmol / l k. the amplitude of the response to high [ k]o was much larger when the cells had not been exposed to -agatoxin ( p < 0.001 ) . high - resolution capacitance measurements were applied to examine the exocytotic properties of individual -cells . the time course of exocytosis was monitored by applying progressively longer depolarizing pulses ( 20500 ms ) from 70 to 0 mv ( fig . the amplitude of the responses was typically fairly small and limited to 40  5 ff ( n = 32 ) for a 500-ms depolarization . however , pulses as short as 20 ms elicited good responses in 50% of the cells . figure 8b summarizes the relationship between the duration of the depolarizing command and the exocytotic response . the average responses tended to plateau within 50100 ms with a secondary acceleration becoming apparent for pulse durations 200 ms . a : increase in membrane capacitance evoked by 20500-ms depolarizations from 70 to 0 mv . the circles above the capacitance traces indicate the percentage of responding cells ( black part , n = 23 ) . b : relationship between pulse duration and exocytotic response ( cm ; n = 23 ) . c : change in cell capacitance ( cm ) evoked by 500-ms depolarizations from 70 mv to membrane potentials between 40 and + 40 mv ( n = 8) . d : change in cell capacitance evoked by 500-ms depolarization from 70 to 0 mv under control conditions and after addition of -agatoxin ( 200 nmol / l ) . the maximum responses were observed during depolarizations to 0 mv . as shown in fig . 8d , exocytosis elicited by a 500-ms depolarization from 70 to 0 mv was inhibited by 97  3% ( p < 0.05 ; n = 4 ) by -agatoxin . exocytosis was not affected by stromatoxin , heteropodatoxin-2 , and ttx ( 90% of control ) but was strongly reduced ( > 75% ) by 4-aminopyridine . thus , the strong inhibitory effect of 4-aminopyridine on glucagon secretion may result from direct interference with -cell exocytosis . we have conducted the first electrophysiological characterization of the ion channels involved in human -cell action potential firing and glucagon secretion . we demonstrate that human -cells , like their rodent counterparts ( 13 ) , are electrically active at low glucose concentrations and generate spontaneous oscillations in [ ca]i . the spontaneous [ ca]i oscillations observed at 1 mmol / l glucose were reduced ( but not abolished ) by the l - type ca - channel blocker isradipine , an effect that could be antagonized by the l - type ca - channel agonist bayk8644 . the isradipine - resistant [ ca]i oscillations are likely to reflect ca - influx via p / q - type and/or t - type ca - channels . in agreement with this interpretation , the [ ca]i - oscillations were completely suppressed when isradipine was applied to islets exposed to the p / q - type ca - channel blocker -agatoxin . collectively , these findings make clear that the [ ca]i oscillations depend on influx of extracellular ca . it is surprising that -agatoxin alone only had a minor effect on [ ca]i although it strongly suppressed glucagon secretion and p / q - type channels account for 70% of the whole - cell ca - currents . this indicates that these channels activate very briefly during the peak of the action potentials and therefore mediate only a fraction of the ca - entry during electrical activity . these observations suggest that p / q - type ca - channels are tightly linked to the exocytosis of the glucagon granules . indeed , the capacitance measurements revealed that depolarization - evoked exocytosis was completely dependent on ca - influx through p / q - type ca - channels . we addressed the potential roles of somatostatin and insulin as paracrine regulators of glucagon secretion . blocking somatostatin - receptor subtype 2 ( the subtype expressed in human -cells ; supplementary fig . s1 and ref ) does not interfere with the ability of glucose to inhibit glucagon secretion . in accordance with previous in vivo observations in human ( 31 ) and isolated mouse islets ( 32 ) , insulin inhibited glucagon secretion from human islets exposed to 1 mmol / l glucose . although it would be tempting to interpret this finding in terms of insulin mediating the inhibitory effect of glucose on glucagon secretion , it should be noted that glucagon secretion is inhibited over a range of glucose concentrations with relatively small effects on insulin secretion . in fact , the stronger stimulation of insulin secretion occurring at glucose concentrations above 6 mmol / l coincides with stimulation ( not inhibition ) of glucagon secretion . unlike what is seen in rat islets ( 33 ) the reason for this difference is unclear , but it is pertinent that several voltage - gated channels expressed in human -cells are modulated by zn ( a - currents , t - type ca - channels ) . the stimulatory effects of insulin and zn may underlie the reduced inhibition of glucagon secretion seen above 6 mmol it has been proposed that glucose inhibits glucagon secretion by inhibition of katp - channels , leading to membrane depolarization and voltage - dependent inactivation of voltage - gated ion channels involved in action potential firing ( 14 ) . we have so far not observed consistent effects of glucose on membrane potential and conductance of isolated human -cells . this may reflect technical difficulties because electrical activity in these cells is very rarely stable enough to assess glucose effects , which develop over several minutes . however , tolbutamide promptly depolarized the -cells by 10 mv and decreased the peak voltage of the action potential , although it was without detectable effect on the resting membrane conductance . thus , minute changes in katp - channel activity have strong effects on -cell electrical activity . importantly , several of the voltage - gated membrane currents in human -cells exhibit voltage - dependent inactivation , and this may account for the observed reduction of the action potentials peak voltage . a decrease in peak voltage of the action potential can therefore be expected to exert a strong inhibitory effect on glucagon secretion . a role of membrane depolarization mediated by closure of katp - channels in the glucose - induced suppression of glucagon secretion is suggested by our previous finding that micromolar concentrations of the katp - channel activator diazoxide antagonize the effect of the sugar ( 7 ) . the ion channel complements of human - and -cells show great similarities , and the inactivation and activation properties are very similar ( 20 ) . why then does membrane depolarization produced by tolbutamide stimulate insulin and somatostatin secretion ( 19,20 ) but inhibit glucagon secretion ( 7 ) ? key electrophysiological characteristics of human islet cells are summarized in supplementary table 1 , available in an online appendix . it can be seen that the -cell differs from the other islet cells in having a low resting membrane conductance . perhaps as a result of this , the interspike membrane potential of the -cell is more depolarized than that of the - and -cells . another difference is that the na - current , which contributes to the upstroke of the action potential , is smaller in the -cells than in - and -cells . the more depolarized interspike potential exacerbates this difference , and the na - current that remains available for action potential firing is only 1 pa / pf in -cells compared with 12 pa / pf in - and -cells . in addition , -cells can be distinguished from -cells by the presence of a prominent a - type k - current . the functional significance of this current is underscored by the observation that heteropodatoxin-2 , like the kv2.1/2.2 antagonist stromatoxin , is a strong inhibitor of glucagon secretion . 7a ) after each action potential that results from the activation of these channels may be particularly important for reactivation of the na - channels in -cells . both types of k - currents undergo partial voltage - dependent inactivation at physiological membrane potentials , and the a - current is almost completely inactivated at the interspike potential attained in the presence of tolbutamide . it is possible that the -cells have a greater capacity to repolarize the membrane voltage in the presence of tolbutamide ( and glucose ) because the downstroke of the action potential is due to opening of ca - activated bk - channels ( 20 ) . these k - channels may be less susceptible to voltage - dependent inactivation , so that action potential height is maintained also during sustained membrane depolarization . more work is clearly needed to establish precisely how glucose regulates glucagon secretion . the findings reported here illustrate , however , that -cell electrical activity depends on a complex balance between depolarizing and repolarizing conductances and that any process that perturbs this balance has dramatic effects on glucagon secretion .
objectiveto document the properties of the voltage - gated ion channels in human pancreatic -cells and their role in glucagon release.research design and methodsglucagon release was measured from intact islets . [ ca2+]i was recorded in cells showing spontaneous activity at 1 mmol / l glucose . 
 membrane currents and potential were measured by whole - cell patch - clamping in isolated -cells identified by immunocytochemistry.resultsglucose inhibited glucagon secretion from human islets ; maximal inhibition was observed at 6 mmol / l glucose . 
 glucagon secretion at 1 mmol / l glucose was inhibited by insulin but not by zncl2 . 
 glucose remained inhibitory in the presence of zncl2 and after blockade of type-2 somatostatin receptors . 
 human -cells are electrically active at 1 mmol / l glucose . 
 inhibition of katp - channels with tolbutamide depolarized -cells by 10 mv and reduced the action potential amplitude . 
 human -cells contain heteropodatoxin - sensitive a - type k+-channels , stromatoxin - sensitive delayed rectifying k+-channels , tetrodotoxin - sensitive na+-currents , and low - threshold t - type , isradipine - sensitive l - type , and -agatoxin - sensitive p / q - type ca2 + -channels . 
 glucagon secretion at 1 mmol / l glucose was inhibited by 4070% by tetrodotoxin , heteropodatoxin-2 , stromatoxin , -agatoxin , and isradipine . 
 the [ ca2+]i oscillations depend principally on ca2 + -influx via l - type ca2 + -channels . 
 capacitance measurements revealed a rapid ( < 50 ms ) component of exocytosis . 
 exocytosis was negligible at voltages below 20 mv and peaked at 0 mv . blocking p / q - type ca2 + -currents abolished depolarization - evoked exocytosis.conclusionshuman -cells are electrically excitable , and blockade of any ion channel involved in action potential depolarization or repolarization results in inhibition of glucagon secretion . 
 we propose that voltage - dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose .