Large-conductance voltage- and Ca²⁺-activated K⁺ (BK) channels are a key player in vasoregulation and control of neurotransmitter release. The channels are activated by depolarization and an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_in). Ca²⁺ binding is presumed to change the diameter of the cytosolic RCK gating ring, leading to opening of the gate in the pore module via the S6-RCK linkers. The length of the linkers is known to be important in the channel gating (Niu et al., 2004, Neuron 42:745) but whether other physicochemical characteristics are also critical is unclear. Recently, we identified a neuronal splice variant (Soom et al., 2008, Channels 2:278), termed "e9alt", because of alternative splicing of exon 9. e9 and e9alt channels differ in both, the S6 segment and the linker region. The most striking feature of the e9alt channel is its greater basal activation at -150 mV in elevated [Ca²⁺]_in. To delineate the contributions of S6 and linker effects on the gating characteristics of e9alt, we constructed chimeras comprising e9alt sequence of either S6 or the linker region in an e9 background. Channel constructs were expressed in HEK293 cells and assayed in the inside-out configuration of the patch-clamp method. Analysis of channel activity at -50 mV in nominally Ca²⁺-free internal solutions yielded open probabilities (P_open) of 0.070.02% (S6 chimera, n=3), and 0.080.03% (linker chimera, n=4). Thus, with respect to P_open, both chimeras are similar to e9 channels (P_open=0.130.04%, n=11) but different from e9alt channels (P_open=0.970.36%, n=9). In contrast, single-channel conductance of S6 chimera (110 pS) as well as linker chimera (128 pS), were similar to e9alt (135 pS) compared with e9 channels (204 pS). Whereas upon increasing [Ca²⁺]_in from 0 to 100 mM e9 channels exhibited a strong shift in the voltage for half-maximal activation by 2207 mV (n=7), e9alt channels showed only a shift by -9911 mV (n=6), as estimated from tail-current analysis. Corresponding shifts of -1788 and -1349 mV obtained for S6 and linker chimera (n=6, each), respectively, were intermediate. Neither chimeric nor e9alt channels exhibited an increase in apparent gating charge, as observed for e9 channels (1.6-fold) by elevating [Ca²⁺]_in. In 100 mM [Ca²⁺]_in, relative conductance at -150 mV of neither S6 (2.10.7%, n=6) nor linker (6.10.8%, n=6) chimera reached values as high as observed for e9alt channels (17.21.8%, n=6); the value for e9 channels was 1.00.3% (n=7). Therefore, the exchange of neither S6 nor the linker region alone is able to completely reconstitute an e9alt phenotype. This implicates that, in addition to the length of the S6-RCK linker, the exact sequence of the S6 region and the linker is important for BK channel gating.