Title

 

Authors

M. M. Dinardo¹, G. M. Camerino¹, A. Mele¹, G. Cannone¹, R. Latorre², D. Conte Camerino¹, D. Tricarico^1
 
1Dept. of Pharmacobiology, Faculty of Pharmacy, University of Bari, Italy
²Universidad de Valparaiso, Centro de Neurociencia, Valparaiso , Chile

 

Abstract

Calcium-activated K⁺-channels (BK) of slow-twitch and fast-twitch skeletal muscles differ in their activity and response to Ca²⁺ and acetazolamide (ACTZ). Fast-twitch flexor digitorum brevis (FDB) muscle is more sensitive to Ca²⁺ and ACTZ than slow-twitch soleus (SOL) (Tricarico et al., 2005). However, the molecular composition of BK channels in different muscle phenotypes is unknown. Patch-clamp experiments in native fibers in parallel with transcript scanning of SOL and FDB rat skeletal muscles were performed to investigate on the different biophysical properties and drug responses of BK channels in both muscular phenotypes and to isolate the splice variants. BK of FDB showed low channel activity and was activated by NS1619, quercetine (QUERC), bendroflumethiazide (BFT), dichorphenamide (DCP), ethoxzolamide (ETX) and resveratrol (RESV), but not by metazolamide (MTZ) and hydrochlorthiazide (HCT) (10-100 μM). BK of SOL had high channel activity and was not sensitive to QUERC, MTZ and BFT, less sensitive to DCP, HCT and NS1619,  while high sensitive to RESV and ETX.  Experiments of drug responses of BK in function of their Ca²⁺ sensitivity showed that a high Ca²⁺ sensitivity is correlated with enhanced drug responses except for RESV. Total RNA was isolated from FDB and SOL and RT-PCR was performed. PCR amplicons of all 7 known splicing sites were cloned, characterized and sequenced; C1, C2, C3, C4, C5 and C6 sites for the C-terminal region and the N1 site for the N-terminal region of the Kcnma1 cDNA, codifying the α -subunit of the BK channel. Transcript scanning of FDB and SOL cDNA showed two isoforms with distinct inserts at sites C1 and C2. At C1, only the insertless “null” e17 transcript was found in SOL and FDB. At C2, two variants were isolated: the e22 resulting from exclusion of exons 20 and 21;and a variant with 29amino acid insert resulting from inclusion of part of intron 22 between exons 19 and 22. Experiments of real-time PCR, performed using TaqMan MGB probes (Applied Biosystems), showed that the +29 was the most abundant and equally expressed in the FDB and SOL muscles, e22 was significantly more expressed in FDB. The e22 splicing isoform is responsible of the  increase in Ca²⁺ and voltage sensitivity and inhibition by PKA phosphorylationof the channel (Shipston et al., 2001). At C4 site, we isolated two products for Sol and FDB, represented by the +27 variant (Slo27) and the insertless variant (Slo0). The Slo27 variant is expressed equally in Sol and in FDB, while Slo0 showed a significant abundance in FBD respect to Sol,  as confirmed by real-time experiments. For N1, C5 and C6 sites,  we found no splicing of the analyzed regions in SOL and FDB. PCR of C3 site region had a different behaviour, because it showed no splicing in SOL, but was not amplified in FDB, probably for a change in the sequence composition of the annealing sites of the used primers. Real time PCR showed that the expression of the total BK channel was more abundant in SOL than in FDB according to the recorded channel activity in both muscle types. As it is known that theproperties of BK channels are influenced by the accessory β -subunits (beta1-4) (Gribkoff et al.2001), we analyzed cDNA of the four β -subunits of BK channel in Sol and FDB to test their presence in the rat skeletal muscles. None of the genes observed was amplified by this method, excluding their expression in the samples analyzed. On the basis of these results we can conclude that the muscle-phenotype dependent properties and drug-responses of BK are related to the observed quantitative isoforms distribution; e22 appears to be the drug target. Supported by Telethon GGP10101.
 
Tricarico et al. (2005). Neurobiology of Disease20, 296–302
Shipston et al. (2001). TRENDS in Cell Biology11, 353-358
Gribkoff et al. (2001). Neuroscientist 7, 166–177