ࡱ > M O L R [H bjbjVV 8J < < n n , $ c N 2 9 2 k Mt v L 0 S S S 2 2 S n { : Table 1. PCR primers. PCR primers for human detrusor sample were designed by using the conserved sequences between humans and mice. (p), (h) (m) represent pig, human and mouse, respectively. ClonesPrimer SequencePrimer siteProduct Length Accession No (+): Sense, (-): Antisense(bp) SUR1(+):5'- ACCTGGTGATTGTCCTGAAG -3'(p)174(p)CD572197(-):5'- AGTTAGAAAACCCAGGCAGG -3'(p)(p)CD572197SUR2A(+):5'- CAGCTGAAGAATATGGTCAAATC -3'(h)=(m)376(h)NM_005691(-):5'- CTACTTGTTGGTCATCACCAAAG -3'(h)=(m)(m)D86037SUR2B(+):5'- CAGCTGAAGAATATGGTCAAATC -3'(h)=(m)285(h)NM_020297(-):5'- TTCATCACAATAACCAGGTCTGC -3'(h)=(m)(m)D86038Kir 6.1(+):5'- CCTGTTGATAACCCGCTTGAG -3' (p)403(p)CF181244(-):5'- GCTTCCTCAGAGAGTTCTGGT -3' (p)(p)CF181244Kir 6.2(+):5'- GTGTTCACCACGCTGGTGGAC -3' (h)=(m)386(h)NM_000525(-):5'- GCATGCTTGCTGAAGATGAGGGT -3' (h)=(m)(m)NM_010602The reaction condition The reaction condition was: 1) in Kir6.1, after 95C for 3 min., 35 cycles of 95C for 35 sec, 58C for 35 sec and 72C for 40 sec 2) in Kir6.2, after 95C for 3 min., 35 cycles of 95C for 35 sec, 61C for 35 sec and 72C for 40 sec 3) in SUR2A, after 95C for 3 min., 3 cycles of 51C for 40 sec and 72C for 40 sec, and subsequently 33 cycles of 95C for 35 sec, 56C for 35 sec and 72C for 40 sec 4) in SUR2B, after 95C for 3 min., 3 cycles of 49C for 40 sec and 72C for 40 sec, and subsequently 33 cycles of 95C for 35 sec, 54C for 35 sec and 72C for 40 sec. Supplementary Methods The whole-cell current data was low-passed at 1 kHz (-3 dB) by a 3-pole Bessel filter, sampled at 10 ms (continuous traces) or 1 ms (ramp currents) and analyzed on a computer (Macintosh IIci) using the commercial software Mac Lab 3.5.6 (ADInstruments Pty Ltd, Castle Hill, Australia). The traces were illustrated by Kaleidagraph v3.04 (Synergy Software, Reading, PA, USA) or Corel.For the single channel study, current signals were low-pass filtered at 1 kHz and digitized at 5 kHz before being stored on a computer hard disk. The all representative traces of single channel currents filtered at 300 Hz using an 8-pole Bessel filter were made by WinEDR v3.0.0 (kindly provided by Dr John Dempster, Universtiy of Strathclyde, Scotland)and Kaleidagraph v3.04 (Fig. 1, 2, 3 & 4). In the cell-attached patch configuration, single channel currents were induced by 100 mM l e v c r o m a k a l i m a n d i n h i b i t e d b y 1 0 mM g l i b e n c l a m i d e a t a h o l d i n g p o t e n t i a l o f 0 m V ( B ) . S i n c e , i n t h e p r e s e n t e x p e r i m e n t s , w e d i d n o t d e f i n e t h e t o t a l n u m b e r o f c h a n n e l s p r e s e n t i n e a c h p a t c h m e m b r a n e , o p e n s t a t e p r o b a b i l i t y v a l u e s [ ( n u m b e r o f c h a n n e l s x (mean open probability of the single channels): NPo)] were calculated using the following formula: Channel activity = (a1 + 2a2+...+nan) / (a1 + a2+...+an), where a1, a2 and an are the areas under each peak of the amplitude histogram with the channel closed, one channel open and simultaneous openings of 2 and n channels, respectively. Single channel amplitudes and channel open times were determined by the free web software WinEDR v.3.0.0. The graphs were obtained from 2 min. recordings. 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