A Novel Isoform of SK2 Assembles with Other SK Subunits in Mouse Brain

Abstract

The SK2 subtype of small conductance Ca 2؉ -activated K ؉ channels is widely distributed throughout the central nervous system and modulates neuronal excitability by contributing to the afterhyperpolarization that follows an action potential. Western blots of brain membrane proteins prepared from wild type and SK2-null mice reveal two isoforms of SK2, a 49-kDa band corresponding to the previously reported SK2 protein (SK2-S) and a novel 78-kDa form. Complementary DNA clones from brain and Western blots probed with an antibody specific for the longer form, SK2-L, identified the larger molecular weight isoform as an N-terminally extended SK2 protein. The N-terminal extension of SK2-L is cysteine-rich and mediates disulfide bond formation between SK2-L subunits or with heterologous proteins. Immunohistochemistry revealed that in brain SK2-L and SK2-S are expressed in similar but not identical patterns. Heterologous expression of SK2-L results in functional homomeric channels with Ca 2؉ sensitivity similar to that of SK2-S, consistent with their shared core and intracellular C-terminal domains. In contrast to the diffuse, uniform surface distribution of SK2-S, SK2-L channels cluster into sharply defined, distinct puncta suggesting that the extended cysteine-rich Nterminal domain mediates this process. Immunoprecipitations from transfected cells and mouse brain demonstrate that SK2-L co-assembles with the other SK subunits. Taken together, the results show that the SK2 gene encodes two subunit proteins and suggest that native SK2-L subunits may preferentially partition into heteromeric channel complexes with other SK subunits.SK channels are voltage-independent K ϩ channels that are activated by elevated levels of intracellular Ca 2ϩ , such as occur following an action potential (1). In many central neurons, SK channel activity dampens excitability by contributing to the afterhyperpolarization, affecting interspike intervals during a burst of action potentials as well as the length of the burst (2-5).SK1, SK2, and SK3 are expressed in overlapping yet distinct patterns throughout the central nervous system with SK2 being the most highly expressed (6 -8). These genes are also expressed in many peripheral tissues along with the fourth member of the SK gene family, IK1 (SK4), whose expression is limited to the periphery (9).1 Apamin specifically blocks SK1-3 channels, and the physiological consequences of apamin application likely reflect SK channel blockade. Apamin block of hippocampal SK channels facilitates the induction of synaptic plasticity at CA3 Schaffer collateral to CA1 synapses. Consistent with this result, systemic apamin injection facilitates the acquisition of hippocampal dependent learning (10). These effects depend upon SK2 channels as SK2-null mice lack apaminsensitive currents in CA1 neurons, but these currents are unchanged in SK1-null or SK3-null mice (11).SK channels share the six transmembrane domain topology of the Shaker prototype. Within their transmembrane core domains the different SK subunits ...

Organization and Regulation of Small Conductance Ca2+-activated K+Channel Multiprotein Complexes

“…Consistent with distinct roles, disruption of any one of the SK genes does not result in altered expression levels of the other, intact genes (Bond et al, 2004). Nevertheless, heteromeric channels have been detected (Strassmaier et al, 2005), and overlapping functions cannot be discounted. Their central role in modulating local Ca 2ϩ transients and Ca 2ϩ -dependent events suggested that Ca 2ϩ -independent signaling mechanisms might modulate SK channels, perhaps by tuning their Ca 2ϩ sensitivity.…”

Small-conductance, Ca2+-activated K+ channel 2 is the key functional component of SK channels in mouse urinary bladder

“…As a result of the multimeric assembly nature of SK channels, this leads to the possibility of heteromultimeric assembly in which more than one SK family member (SK1-3) may assemble into a given channel complex. This has been demonstrated in heterologous expression systems (1,17,20,26) and directly demonstrated in native tissue using immunoprecipitation (33). In addition, the transgenic expression of a dominant negative SK subunit supports the heteromultimeric assembly of SK channels in vivo (38).…”

K+ Channel Modulators for the Treatment of Neurological Disorders and Autoimmune Diseases

“…164 In expression systems, both homomeric and heteromeric K Ca 2 channels are formed, 165,166 but the existence of heteromeric K Ca 2 channels has so far not been confirmed in native tissues. Similar to K Ca 1.1 channels, K Ca 2 channels can be alternatively spliced to generate channels with modified calmodulin binding as in the case of K Ca 2.1, 167 with an extended N-terminus dubbed SK2-L as in the case of K Ca 2.2, 168 or without the N-terminus and first transmembrane segment as in the case of K Ca 2.3. 169,170 Alternative splicing of K Ca 2.3 resulting in the insertion of fifteen additional amino acids in the outer pore region can also produce K Ca 2.3 channels, which are insensitive to the peptide toxins apamin and leiurotoxin-I.…”